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Expression and purification of a cold-adapted group III trypsin in Escherichia coli
Protein Expression and PuriWcation 51 (2007) 243–252 www.elsevier.com/locate/yprep
Expression and puriWcation of a cold-adapted group III trypsin in Escherichia coli Helga Margrét Pálsdóttir, Ágústa Gudmundsdóttir ¤ Science Institute, University of Iceland, Laeknagardi, Vatnsmýrarvegi 16, Reykjavík IS-101, Iceland Received 6 June 2006 Available online 23 June 2006
Abstract The recently classiWed group III trypsins include members like Atlantic cod (Gadus morhua) trypsin Y as well as seven analogues from other cold-adapted Wsh species. The eight group III trypsins have been characterized from their cDNAs and deduced amino acid sequences but none of the enzymes have been isolated from their native sources. This study describes the successful expression and puriWcation of a recombinant HP-thioredoxin–trypsin Y fusion protein in the His-Patch ThioFusion Escherichia coli expression system and its puriWcation by chromatographic methods. The recombinant form of trypsin Y was previously expressed in Pichia pastoris making it the Wrst biochemically characterized group III trypsin. It has dual substrate speciWcity towards trypsin and chymotrypsin substrates and demonstrates an increasing activity at temperatures between 2 and 21 °C with a complete inactivation at 30 °C. The aim of the study was to facilitate further studies of recombinant trypsin Y by Wnding an expression system yielding higher amounts of the enzyme than possible in our hands in the P. pastoris system. Also, commercial production of trypsin Y will require an eYcient and inexpensive expression system like the His-Patch ThioFusion E. coli expression system described here as the enzyme is produced in very low amounts in the Atlantic cod. © 2006 Elsevier Inc. All rights reserved. Keywords: Trypsin Y; Group III trypsin; Atlantic cod (Gadus morhua); Escherichia coli; Expression
The cDNA of trypsin Y was previously isolated from an Atlantic cod cDNA library and characterized [1]. Sequence alignments of trypsin Y and seven other vertebrate trypsins indicated novel characteristics of these enzymes relative to the traditionally classiWed trypsin groups I and II. The novel Wsh trypsins have now been distinguished from other trypsins on the basis of amino acid sequence identities by their placement into the new group III [1,2]. Amino acid sequence identities between the group III and group I trypsins are only about 45–50%. However, the identities are in the order of 60–94% within each of the two trypsin groups. Despite the low sequence identities, the group III trypsins are more similar to the group I vertebrate trypsins than to any other known proteins. It has been postulated that the
*
Corresponding author. Fax: +354 525 4886. E-mail address: [email protected] (Á. Gudmundsdóttir).
1046-5928/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.pep.2006.06.008
group III trypsins have speciWc roles that are diVerent from those of the group I trypsins [2]. The group III trypsins have also been suggested to be adapted to colder environments than other traditionally classiWed cold-adapted trypsins such as cod trypsin I [2,3]. Cold-adapted enzymes appear to have a higher degree of conformational Xexibility than their mesophilic counterparts, presumably due to weaker intramolecular interactions [4–6]. This may facilitate the accommodation and transformation of substrates at low energy costs as shown for the cold-adapted alkaline protease from an Antarctic Pseudomonas species [7]. In general, cold-adapted enzymes have a decreased thermal stability compared to their mesophilic counterparts, most probably in association with their high catalytic eYciency [8–10]. However, recent Wndings suggest that there may not be a strict relationship between high activity of enzymes at low temperatures and a decreased thermal stability [11].
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The recombinant forms of various cold-adapted bacterial enzymes have been successfully expressed in microorganisms [12–15]. The cold-adapted uracil-DNA glycosylase enzyme from the Atlantic cod was recently expressed in an active form in Escherichia coli [16]. Furthermore, the precursor form of the recombinant trypsin I from Atlantic cod was successfully expressed in a His-Patch ThioFusion E. coli expression system and puriWed [17]. All of the group III trypsin cDNAs have been isolated from Wsh species spending at least part of their lives in extremely cold environments, at 0 °C or lower temperatures. Today, none of the group III trypsin polypeptides have been isolated from their native sources. Their characterization has therefore mainly been based on analysis of the cDNA sequences encoding the enzymes [1,2] as well as on biochemical analysis of recombinant Atlantic cod trypsin Y produced in P. pastoris [3,18]. The recombinant enzyme was shown to have dual substrate speciWcity, i.e. towards trypsin and chymotrypsin speciWc synthetic substrates. Furthermore, the recombinant trypsin Y showed increasing proteolytic activity at temperatures between 2 and 21 °C with a complete inactivation at 30 °C [3]. This narrow range of enzymatic activity with temperature is diVerent from the cold-adapted group I trypsins. Therefore, trypsin Y may be ideal for commercial use, for example, in food processing where low environmental temperatures are required for a short period of time to ensure quality. The dual substrate speciWcity of trypsin Y may also be an advantage for speciWc industrial or medical applications [18]. It appears that trypsin Y is produced in very low amounts in the Atlantic cod relative to trypsin I (results submitted for publication) and its isolation from the native source may not be feasible. Therefore, the industrial application of trypsin Y will probably depend on using the recombinant form produced in an eYcient and low-cost large-scale expression system like the His-Patch ThioFusion E. coli under study here. Materials and methods Host strains and vectors The ampicillin resistant E. coli TOP10 strain (recA, endA) (Invitrogen, CA, USA) was used for propagation of the recombinant plasmids as well as for the expression of the recombinant trypsin Y fusion constructs. The E. coli His-Patch ThioFusion expression system kit (Invitrogen), containing the pThioHis A expression vector, was used for cloning, expression, and partial puriWcation of the recombinant trypsin Y protein. The pThioHis A expression vector contains a trc promoter in addition to the gene encoding the Lac repressor (lacIq) that regulates expression from the trc promoter. IPTG1 (isopropyl--D-thiogalactopyrano-
1
Abbreviation used: IPTG, isopropyl--D-thiogalactopyranoside.
side) was used to induce production of the recombinant protein, as IPTG binds to the Lac repressor protein and blocks its action. The pThioHis A vector contains a histidine patch thioredoxin (HP-thioredoxin) gene mutated to create a metal-binding domain in the HP-thioredoxin protein to ease puriWcation of the recombinant fusion protein on a metal-chelating resin. The fusion to the HP-thioredoxin protein also confers solubility to insoluble heterologous proteins [19]. An enterokinase cleavage site is engineered into the pThioHis A vector between the HP-thioredoxin gene and the multiple cloning site. This enables cleavage of the desired recombinant protein from the HP-thioredoxin fusion part by enterokinase. Construction of expression vectors Two diVerent constructs of the pThioHis A vector and the trypsinogen Y cDNA were designed. This was done in order to determine if the cDNA sequence encoding the prepro part of trypsinogen Y was required for proper folding of the recombinant enzyme. The trypsinogen Y was cloned downstream from the trc promoter and the thioredoxin gene in the pThioHis A vectors. The Wrst construct contained the entire prepro sequence of the cDNA encoding trypsinogen Y. This included the signal sequence and activation peptide of the putative polypeptide starting at residue Ile(-6) according to the chymotrypsinogen numbering system. This cDNA construct was termed HP-thioredoxin–trypsin Y IGLA, where IGLA represents the Wrst four N-terminal amino acid residues of the putative trypsin Y polypeptide. Using the same nomenclature, the second construct, HP-thioredoxin–trypsin Y IIGG, was designed to encode the putative mature form of the enzyme starting at residue Ile16 in the recombinant trypsin Y polypeptide. The two diVerent trypsin Y cDNA constructs (IGLA and IIGG) were ampliWed in a PCR reaction according to standard protocols using a pBluescript vector containing the entire trypsinogen Y cDNA sequence. The PCR ampliWcations were performed in a PCR thermocycler 9700 system (Applied Biosystems, CA, USA). The following oligonucleotide primers (TAGCopenhagen, Denmark) were used to create the trypsin Y constructs: IGLA forward primer: 5⬘-TATATGGTACCTATCGGCCTGGCTCTG CTA-3⬘; IIGG forward primer: 5⬘-TATATGGTACCTAT CATCGGCGGACAGGAC-3⬘; IGLA and IIGG reverse primer: 5⬘-TATATTCTAGATCAGGGGTTGGCATCC AGTGA-3⬘. A KpnI restriction site was incorporated at the 5⬘-end of the trypsinogen Y cDNA and an XbaI site at its 3⬘-end. A stop codon was designed at the 3⬘-end between the trypsin Y construct and the XbaI restriction site. Transformation into E. coli TOP10 strain The two diVerent recombinant HP-thioredoxin–trypsin Y plasmids (IGLA and IIGG) were transformed into competent E. coli TOP10 cells using standard protocols [20]. A pThioHis A vector, without a trypsinogen Y insert, was
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also transformed into competent E. coli TOP10 cells to be used as a control in the expression and puriWcation experiments. The transformed cells were spread onto Luria-Bertani (LB) agar plates containing 100 g/mL ampicillin (LBamp) for selection of positive colonies. Isolation of plasmids from positive transformants was carried out using the QIAGEN miniprep kit (QIAGEN, Hilden, Germany). The presence of trypsinogen Y inserts in the plasmids was determined by agarose gel electrophoresis after digestion with KpnI (Fermentas, St. Leon-Rot, Germany) and XbaI (GE Healthcare, UK) restriction enzymes. The plasmid–insert boundaries were sequenced to conWrm a correct reading frame of the inserts. This was done in a Perkin Elmer ABIPrism 377 DNA sequencer (Applied Biosystems) using ABI-prism Big-Dye Terminator Cycle Sequencing Kit with AmpliTaq DNA Polymerase, FS (Applied Biosystems). Expression of the recombinant HP-thioredoxin–trypsin Y fusion protein The two diVerent HP-thioredoxin–trypsin Y fusion constructs (IGLA and IIGG) were expressed in E. coli Top 10 cells. To analyse the time and temperature conditions for optimal expression of each recombinant protein, several colonies were grown for pilot expression using small-scale volumes (10 mL) in an Innova 4000 incubator shaker (New Brunswick ScientiWc Co. Inc., NJ, USA). This was followed by growing the cells in large-scale volumes in an incubator shaker (1–2 L). For the pilot expression, a single colony of each construct, previously grown on an LBamp agar plate, was used to inoculate a 1 mL LBamp medium. The culture was incubated overnight at 37 °C with shaking at 200– 225 rpm. Five hundred microlitres of the overnight culture were used to inoculate 10 mL of fresh LBamp medium in a 50 mL culture Xask and grown under same condition until the OD550 reached 0.5. Then IPTG was added to a Wnal concentration of 1 mM to induce expression of the recombinant HP-thioredoxin–trypsin Y fusion protein. Prior to the addition of IPTG, the zero time sample was prepared by pelleting a 1 mL sample at a maximum speed for 3 min in a microcentrifuge. The pellet was stored at ¡20 °C until further processing. In order to determine the optimum time of expression, samples were collected after 1, 4, 7, 10, 12, 19, and 25 h of incubation and handled the same way as the zero point sample. The expression was performed at four diVerent temperatures, 18, 25, 30, and 37 °C in order to Wnd the optimal temperature for expression. For the large-scale expression experiments in an incubator shaker, a 5 mL seed culture was grown overnight at 37 °C with vigorous shaking (200–225 rpm) in an LBamp medium. The overnight culture was then used to inoculate 1–2 L of fresh LBamp medium and grown until the OD550 reached 0.6. Protein expression was then induced by adding IPTG to a Wnal concentration of 1 mM. The culture was transferred to an incubator at 25 °C, the optimum temperature for expression of the fusion protein. The culture was grown with shaking (200–225 rpm) for 12 h followed by
245
harvesting the cells by centrifugation at 3000g for 10 min at 4 °C. Preparation of cell-free extracts The E. coli cell pellets containing the two diVerent HPthioredoxin–trypsin Y fusion proteins were resuspended in an imidazole buVer (20 mM Tris–HCl, pH 8, 2.5 mM EDTA, 5 mM imidazole) according to manufacturer’s instructions (Invitrogen) for analysis of the expression. Samples that were loaded directly onto a ProBond chromatography column (Invitrogen) were resuspended in Binding buVer (20 mM sodium phosphate, 500 mM NaCl, pH 7.8) instead of imidazole buVer. The cell suspension was placed on ice and sonicated with three 10 s bursts using a handheld sonicator (Sonics, CT, USA) to release the HP-thioredoxin–trypsin Y fusion protein from the cells. The lysate was then Xash-frozen in liquid nitrogen followed by thawing at 37 °C. After completion of three sonication-freezethaw cycles, the lysate was centrifuged at 3000 rpm for 15 min at 4 °C to pellet the cell debris and insoluble matter. Recombinant trypsin Y identity conWrmation Expression of the HP-thioredoxin–trypsin Y fusion protein was conWrmed by SDS–PAGE gel electrophoresis (12% separating gel with 5% stacking gel) and Western blot analysis using polyclonal anti-thio antibodies (Santa Cruz Biotechnology, CA, USA) or monoclonal anti-thio antibodies (Invitrogen). The protein bands on the SDS–PAGE gels were also stained with a SYPRO Red protein dye according to manufacturer’s instructions (GE Healthcare). The SYPRO Red stained proteins were detected by Xuorometric scanning in a TYPHOON 8600 variable mode imager spectrophotometer (GE Healthcare). Prestained low range molecular weight standards (21.2–110 kDa) (Bio-Rad, CA, USA) and a prestained protein molecular weight marker (20–118 kDa) (Fermentas) were used as markers on the SDS–PAGE gels. PuriWcation of the HP-thioredoxin–trypsin Y fusion protein on a ProBond column The cell supernatants suspended in ProBond Binding buVer were applied to a ProBond resin equilibrated in the ProBond Binding buVer according to manufacturer’s protocol (Invitrogen). Supposedly, the HP-thioredoxin–trypsin Y fusion protein binds to the ProBond metal-chelating column, whereas the majority of the host cell proteins will not bind. After loading the sample, the column was washed twice with two column volumes (CV) of ProBond Binding buVer. This was done by resuspending the resin in a ProBond Binding buVer followed by rocking the column on a platform shaker for 2 min and removal of the supernatant. The column was then washed with two CV of ProBond Washing buVer at pH 6.0 (20 mM sodium phosphate, 500 mM NaCl, pH 6.0) using the same method
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as described for the ProBond Binding buVer. This was repeated until the absorbance of the supernatant (Xow through) measured at 280 nm was less than 0.01. The protein was then eluted by applying consecutively 2.5 CV of each of the four ProBond imidazole elution buVers with increasing imidazole concentrations (50, 200, 350, and 500 mM imidazole, each in 20 mM sodium phosphate, 500 mM NaCl, pH 6.0). All the Xow-through fractions were collected for analysis. The elution of the HP-thioredoxin–trypsin Y fusion protein was monitored by measuring the absorbance of the fractions at 280 nm and by Western blot analysis using anti-thio antibodies for detection. Fractions showing measurable absorbance and a band on the Western blot corresponding to the HP-thioredoxin–trypsin Y fusion protein were mixed together for further puriWcation. HPLC puriWcation of the HP-thioredoxin–trypsin Y fusion protein on a MonoQ anion exchange column The ProBond puriWed supernatant was run through a MonoQ 5/5 ion exchange column in an automated HPLC system (Äkta PuriWer, GE Healthcare). The ProBond puriWed sample was Wrst dialysed against 20 mM Tris, pH 9.3, 5 mM ethanolamine, 10 mM CaCl2 (buVer A), and 1 mL of the dialysed sample was applied onto the MonoQ column at a Xow rate of 1.0 mL/min. The protein was eluted with an increasing linear gradient of NaCl up to 1.0 M after washing with buVer A. The absorbance at 280 nm was analysed by the software equipped with the FPLC system during the entire run. Analysis of the HP-thioredoxin–trypsin Y fusion protein expression The sample containing the recombinant HP-thioredoxin–trypsin Y fusion protein was tested for purity on SDS–PAGE gels by Western blot analysis and by SYPRO Red staining. The samples were activated by cleaving the HP-thioredoxin part from the trypsin Y fusion protein by using native Atlantic cod trypsin I. Approximately 25 g of HPthioredoxin–trypsin Y samples, in a total volume of 200 L were treated with 0.01, 0.1, and 1.0 U native Atlantic cod trypsin at 4 °C for 6 h. The trypsin and chymotrypsin activities of the cell supernatants and the partially puriWed recombinant HPthioredoxin–trypsin Y were routinely assayed with synthetic substrates after activation with native cod trypsin I. The synthetic substrates used were N-CBZ-Gly-Pro-ArgpNA (Sigma, MO, USA) for trypsin activity and suc-AlaAla-Pro-Phe-pNA (Sigma) for chymotrypsin activity. The substrate solutions used were 25 mM dissolved in dimethylsulfoxide (Sigma). The activation assays contained 50 L of the sample previously activated by native cod trypsin I, 100 L of 0.1 M Tris buVer at pH 9.0 and 10 L 25 mM synthetic substrate. The reactions were monitored
at RT, at 410 nm in a Spectra Max 384 Microplate spectrophotometer (Molecular Devices, CA, USA). Results Vector construction and expression Two diVerent trypsinogen Y cDNA constructs were cloned into the pThioHis A expression vector. The constructs were termed HP-thioredoxin–trypsin Y IGLA and HP-thioredoxin–trypsin Y IIGG based on the Wrst four amino acid residues in their polypeptide sequences. The calculated molecular mass of the cod trypsin Y IGLA construct fused with the HP-thioredoxin protein is approximately 40 kDa where the HP-thioredoxin part adds approximately 13 to 27 kDa trypsin Y. The molecular mass of the HP-thioredoxin–trypsin Y IIGG construct is slightly smaller or about 38 kDa due to the lack of a presequence in the part of the construct encoding trypsin Y. Western blots using polyclonal anti-thio antibodies showed that the HP-thioredoxin–trypsin Y constructs (IGLA and IIGG) were produced in the E. coli cells. Two control samples were used, i.e. E. coli cells containing the pThioHis A expression vector without the trypsinogen Y insert and E. coli cells with no vector inserts. Fig. 1 shows the 40 kDa protein band of the HP-thioredoxin–trypsin Y IGLA construct (lane 1). This band was not seen in the control samples (lanes 2 and 3). The 16 kDa HP-thioredoxin protein band was only detected in the control sample containing the pThioHis A expression vector (Fig. 1, lane 2). The optimal temperature for induction of expression of the HP-thioredoxin–trypsin Y fusion protein, as determined in pilot expression experiments, was found to be 25 °C and the optimal time of induction was about 12 h. The Western blot seen in Fig. 2 demonstrates that the amount of the putative 40 kDa recombinant HP-thioredoxin–trypsin Y IGLA fusion protein increases during the induction period (t0 to t12). At the zero time point (t0) (lane 1), no protein is expressed but after only 1 h of induction (t1) (lane 2), a 40 kDa protein band is detected. This band increased during the induction period and the strongest band was detected after 12 h (t12) (lane 6). Other cellular protein bands were also shown to increase during the induction period from t0 to t12. When the cells were induced for a longer time period (19 and 25 h), the intensity of the 40 kDa HP-thioredoxin–trypsin Y IGLA protein band started to decrease (data not shown). A 16 kDa protein band corresponding to the HP-thioredoxin control was observed on the Western blot (Fig. 2, lane 7) after 12 h of expression. The 40 kDa HP-thioredoxin–trypsin Y IGLA protein band was not seen in this sample. Pilot expression experiments demonstrated that only the full length trypsin Y IGLA construct could be activated by proteolytic cleavage with native cod trypsin I (data not shown). Therefore, this construct was used in the experiments presented in this paper.
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Fig. 2. Western blot analysis showing the optimal time of expression for HP-thioredoxin–trypsin Y IGLA production. Anti-thio antibodies were used for detection on the blot. The induction time of expression was from t0 to t10 (0–10 h). Lane 1 shows a sample taken prior to induction (t0), whereas lanes 2–6 show samples after 1, 4, 7, 10, and 12 h of expression (t1 to t12), respectively. The intensity of the 40 kDa HP-thioredoxin–trypsin Y IGLA fusion protein band increases during the induction period, where the strongest band is detected after 12 h (lane 6). The 16 kDa HPthioredoxin protein, expressed in E. coli for 12 h was used as a control (lane 7).
Fig. 1. Western blot analysis of the HP-thioredoxin–trypsin Y IGLA fusion protein and control samples from pilot expression experiments using anti-thio antibodies for detection. The 40 kDa HP-thioredoxin– trypsin Y protein band was clearly seen in the HP-thioredoxin–trypsin Y IGLA sample (lane 1) but it was absent in the HP-thioredoxin (lane 2) and E. coli (lane 3) control samples. The HP-thioredoxin control was seen as a band of »16 kDa on the blot (lane 2).
PuriWcation of the HP-thioredoxin–trypsin Y IGLA fusion protein The HP-thioredoxin–trypsin Y IGLA fusion protein was expressed on a large-scale and the cell lysates were puriWed on a ProBond column. The elution proWle of the fusion protein measured as absorbance at 280 nm is seen in Fig. 3A. Protein bands (40 kDa) corresponding to the peaks seen in part A (tubes 11–25) were observed in Western blot analysis using polyclonal (part B) and monoclonal (part C) anti-thio antibodies. Notably, the putative HP-thioredoxin–trypsin Y IGLA protein is not present in tube 10, the last wash fraction prior the HP-thioredoxin–trypsin Y IGLA elution (Fig. 3B). A great diVerence was observed on the Western blots with polyclonal (Fig. 3B) versus monoclonal (Fig. 3C) anti-thio antibodies. The polyclonal antibody cross-reacts with numerous cellular proteins in the cell lysates.
The HP-thioredoxin control protein was also puriWed on a ProBond column in the same manner as the HP-thioredoxin–trypsin Y IGLA sample and used as a positive control in this experiment. A strong 16 kDa HP-thioredoxin protein band was detected in Western blot analysis using polyclonal anti-thio antibodies (data not shown). The ProBond puriWcation method does not achieve puriWcation of the HP-thioredoxin–trypsin Y fusion protein to homogeneity. Therefore, further puriWcation of the ProBond puriWed sample was performed on a MonoQ anion exchange column in an automatic HPLC system. Fig. 4A shows the elution proWles of all fractions from the MonoQ column measured at 280 nm. An absorbance peak was seen after approximately 13 mL of elution, corresponding to a salt concentration of 0.18 M. The samples giving the absorbance peaks (13–37 mL) were run on SDS–PAGE gels and protein bands were visualized on Western blots using polyclonal anti-thio antibodies (Fig. 4B). The 40 kDa HP-thioredoxin–trypsin Y IGLA protein band was Wrst detected after elution with 25 mL of buVer corresponding to a salt concentration of 0.4 M. The ProBond puriWed HP-thioredoxin control sample was also run through the MonoQ-HPLC column. The MonoQ elution proWle measured at 280 nm is seen in Fig. 5A. A very sharp absorbance peak at 280 nm is seen when eluted with 31% NaCl. The samples giving the absorbance peak also gave a strong 16 kDa protein band on a Western blot when using polyclonal anti-thio antibodies (Fig. 5B, volumes 18–28). It can be noted that the sample is puriWed to homogeneity when compared with the non-puriWed sample (Fig. 5B, control).
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Fig. 3. Elution proWle and Western blots of the HP-thioredoxin–trypsin Y IGLA fusion protein puriWed on a ProBond metal-chelating resin. (A) Elution proWle measured at 280 nm. Tubes 1–10 show fractions collected after washing with wash buVer. Tubes 11–15 show the fractions collected after elution with 50 mM imidazole buVer, tubes 16–20 show the fractions collected after elution with 200 mM imidazole buVer, tubes 21–25 show the fractions collected after elution with 350 mM imidazole buVer and tubes 26–32 show the fractions collected after elution with 500 mM imidazole buVer. (B) Western blot analysis of the puriWed fractions (tubes 10–25) using polyclonal anti-thio antibodies. The numbers below the Wgure indicate the collection tube numbers according to the elution proWle in part A. (C) Western blot analysis of the puriWed fractions (tubes 12–20) using monoclonal anti-thio antibodies. The numbers below the Wgure indicate the collection tube numbers according to the elution proWle in part A.
Cleavage of the HP-thioredoxin part and activity measurements During the entire puriWcation process, the enzymatic activity of the HP-thioredoxin–trypsin Y IGLA protein was routinely assayed. The samples were activated by cleaving the HP-thioredoxin part from the recombinant trypsin Y IGLA polypeptide (r-trypsin Y) using native cod trypsin I. Our experiments showed that 0.01 U of native cod trypsin I per 25 g sample was the optimum amount for the activation process. Fig. 6 shows the activity measurements of activated MonoQ puriWed samples when measured towards the synthetic substrates N-CBZGly-Pro-Arg-pNA for trypsin activity (part A) and sucAla-Ala-Pro-Phe-pNA for chymotrypsin activity (part B). Notably, the HP-thioredoxin–trypsin Y IGLA showed higher enzymatic activity towards the trypsin and chymo-
trypsin synthetic substrates than the HP-thioredoxin control sample treated in the same manner. The activated r-trypsin Y was run on a SYPRO Red stained SDS– PAGE gel (Fig. 6C) and also analysed on a Western blot using polyclonal anti-thio antibodies for detection (Fig. 6D). The SYPRO Red stained gel shows the activated r-trypsin Y (Fig. 6C, lane 2) in comparison to the non-activated r-trypsin Y containing the HP-thioredoxin part (lane 3). The activated r-trypsin Y protein band is approximately 25 kDa and the HP-thioredoxin–trypsin Y band is about 40 kDa. The gel also shows a native cod trypsin I sample (23.5 kDa) used to cleave the HP-thioredoxin part from the r-trypsin Y polypeptide (lane 1). As expected, only the non-activated HP-thioredoxin–trypsin Y protein band is detected in Western blot analysis using anti-thio antibodies (Fig. 6D, lane 3). Cleavage of the HPthioredoxin part from the r-trypsin Y polypeptide should
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Fig. 4. Elution proWle and Western blots of the HP-thioredoxin–trypsin Y IGLA fusion protein puriWed on a MonoQ anion exchange column. (A) Elution proWle measured at 280 nm using a linear NaCl gradient from 0 to 1 M. (B) Western blot analysis of the puriWed fractions using polyclonal anti-thio antibodies. The numbers below the Wgure indicate the collection volume (mL) according to the elution proWle in part A.
result in a polypeptide free of the anti-thio part of the fusion protein (Fig. 6D, lane 2). The native cod trypsin I was not detected on the Western blot (Fig. 6D, lane 1). The puriWcation steps provided a sevenfold and an 87-fold increase in speciWc activity of the r-trypsin Y polypeptide as measured towards the synthetic substrate N-CBZ-Gly-Pro-Arg-pNA (Table 1). The Wnal preparation of r-trypsin Y was puriWed 631-fold with an overall yield of 43%. Starting with a 221 mg of non-puriWed proteins, this puriWcation process yielded around 150 g of puriWed r-trypsin Y polypeptide (Table 1). Discussion
Fig. 5. Elution proWle and Western blots of the HP-thioredoxin control protein puriWed on a MonoQ anion exchange column. (A) Elution proWle measured at 280 nm using a linear NaCl gradient from 0 to 1 M. (B) Western blot analysis of the puriWed fractions using polyclonal anti-thio antibodies. The numbers below the Wgure indicate the collection volume (mL) according to the elution proWle in part A.
The precursor form of Atlantic cod trypsin Y was readily produced in a soluble form as a 40 kDa fusion protein with HP-thioredoxin in the His-Patch ThioFusion E. coli expression system. Cleavage of the HP-thioredoxin–trypsin Y IGLA fusion protein with a minute amount of native trypsin I generated an active r-trypsin Y. Seemingly, using the native cod trypsin I to activate the recombinant HP-thioredoxin–trypsin Y IGLA fusion protein is a disadvantage due to its contribution to the activity of the Wnal preparation. However, this was shown to be the best enzyme for the activation process. Its use did not cause any confusion of false positive results of activity as a HP-thioredoxin control sample was prepared and treated with native cod trypsin I in the same manner as the activated HP-thioredoxin–trypsin Y IGLA fusion protein in all the activation experiments performed. Higher-vertebrate trypsins usually have a cluster of four consecutive negatively charged residues that serve as the
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Fig. 6. Enzymatic activity, SDS–PAGE, and Western blot analysis of the activated r-trypsin Y from the MonoQ column. (A) Trypsin activity of the r-trypsin Y sample was measured towards the synthetic substrate N-CBZGly-Pro-Arg-pNA at 410 nm in 0.1 M Tris buVer at pH 9.0. (B) The chymotrypsin activity of the r-trypsin Y sample was measured towards the synthetic substrate suc-Ala-Ala-Pro-Phe-pNA at 410 nm in 0.1 M Tris buVer at pH 9.0. (C) SYPRO red stained SDS–PAGE gel of the activated r-trypsin Y sample (lane 2) in comparison to the non-activated r-trypsin Y containing the HP-thioredoxin part (lane 3). The native cod trypsin I sample used to cleave the HP-thioredoxin part from the trypsin Y polypeptide is shown in lane 1. (D) Western blot analysis using polyclonal anti-thio antibodies. The non-activated r-trypsin Y containing the HP-thioredoxin part was detected (lane 3). As expected, the activated r-trypsin Y (lane 2) and the native cod trypsin I (lane 1) were not detected with the anti-thio antibodies.
recognition site for enterokinase, the physiologic activator of trypsinogen [21]. Also, an enterokinase cleavage site is engineered into the pThioHis A plasmid between the HPthioredoxin protein and the multiple cloning site. However, enterokinase cleavage did not activate the HP-thioredoxin– trypsin Y fusion protein. This may be due to the fact that the trypsin Y activation peptide contains only two negatively charged residues (Glu-Asp) instead of four found in most group I trypsins and therefore, lacks an enterokinase recognition site (Asp-Asp-Asp-Asp) [1,3].
As expected, the activated r-trypsin Y showed enzymatic activity towards the synthetic substrates speciWc for trypsin (N-CBZ-Gly-Pro-Arg-pNA) and chymotrypsin (suc-AlaAla-Pro-Phe-pNA). No activity was detected in this fraction without proteolytic cleavage by the native cod trypsin I. The E. coli cell lysates were used as the starting point to create Table 1. The puriWcation steps provided a sevenfold (ProBond) and an 87-fold (MonoQ) increase in speciWc activity as measured towards the synthetic substrate N-CBZ-Gly-Pro-Arg-pNA (Table 1). The Wnal preparation of the r-trypsin Y was puriWed 631-fold with an overall yield of 43% as compared to the non-puriWed recombinant HP-thioredoxin–trypsin Y fusion protein. Starting with a 221 mg of non-puriWed HP-thioredoxin–trypsin Y fusion protein, this puriWcation process yielded around 150 g of homogenous r-trypsin Y. The activation step of the HP-thioredoxin–trypsin Y fusion protein is the most problematic one in the process. Thus, to improve the yields of the active r-trypsin Y, this step needs to be further studied with respect to stability of the active recombinant enzyme generated as well as other factors. The His-Patch ThioFusion E. coli expression system has many advantages when compared with the P. pastoris expression system. However, the P. pastoris system has been used with good results for the expression of r-trypsin Y [3,18] as well as for the expression of Antarctic krill euphauserase [22]. The E. coli expression system oVers milder environmental conditions and a shorter time of cell growth with an induction time of only 12 h at 25 °C for trypsin Y expression, compared to 3–5 days at 20 °C in P. pastoris [3]. Also, due to the high amount of HP-thioredoxin–trypsin Y fusion protein produced in E. coli, the protein is easily detected during the expression process using either antibodies raised to the thioredoxin part of the fusion protein or by SDS–PAGE gel staining. Recombinant proteins tend to be poorly soluble and prone to molecular aggregation [13]. The presence of the highly soluble HP-thioredoxin part of the fusion protein contained at the N-terminal part of the cod trypsin Y sequence increases its solubility [19]. It also seems to protect the N-terminal end of the r-cod trypsin Y from proteolytic cleavage during expression and handling. In addition, it enables puriWcation of the fusion protein on a metal chelating ProBond aYnity column. Cold-adapted proteolytic enzymes from bacteria [14,23– 25] and marine invertebrates [22] have been successfully expressed in microorganisms. However, the recombinant forms of cold-adapted proteolytic enzymes from Wsh are diYcult to produce in an active form in mesophilic expression systems. This conclusion is based on literature search [26] as well as on our own experience with the expression of Wsh peptidases. The main reason seems to be their high thermal sensitivity and susceptibility to autolysis. Also, as is the case for many mesophilic enzymes, cold-adapted enzymes are prone to aggregation during their expression [13]. Combined, these problems lead to degradation and inactivation of the recombinant enzymes. Atlantic cod
H.M. Pálsdóttir, Á. Gudmundsdóttir / Protein Expression and PuriWcation 51 (2007) 243–252
251
Table 1 PuriWcation of the r-trypsin Y PuriWcation step
Volume (mL)
Total activity (U)
Total protein (mg)
SpeciWc activity (U/mg)
Yield (%)
PuriWcation
Cell lysates ProBond MonoQ
100 35 10
0.42 0.32 0.18
221 23.5 0.15
0.0019 0.014 1.2
100 77 43
1.00 7.2 631
The HP-thioredoxin–trypsin Y IGLA fusion protein was isolated from a 1 L culture of E. coli containing approximately 9 g wet cell weights. The cells were resuspended in 100 mL of ProBond binding buVer. The enzymatic activity was measured at RT towards the synthetic substrate N-CBZ-Gly-Pro-ArgpNA. The activation assays contained 50 L of the sample activated with cod trypsin I, 100 L of 0.1 M Tris buVer, pH 9.0 and 10 L of 25 mM synthetic substrate. The measured values for HP-thioredoxin control samples treated in the same manner as the HP-thioredoxin–trypsin Y IGLA sample have been subtracted from the values presented.
trypsin I is exceptionally sensitive to autolysis, even at 18– 25 °C [27]. Isolation and puriWcation of cold-adapted proteases is also challenging for the same reasons as their expression [8,27–29]. Future experiments will focus on improvements of the expression, puriWcation, and activation of the recombinant cod trypsin Y. Site-speciWc mutagenesis of the cDNA encoding the enzyme may also be used to improve the stability of r-trypsin Y without sacriWcing the catalytic eYciency of the enzyme [30]. Such derivatives may broaden the commercial applicability of the r-trypsin Y. In conclusion, the recombinant form of Atlantic cod trypsin Y has been successfully expressed in the E. coli His-Patch ThioFusion expression system. Thus, an alternative method has been developed for cod trypsin Y production facilitating further studies and commercial use of this enzyme. Acknowledgments This work was supported by grants from the University of Iceland Research Fund, the Icelandic Research Fund, and the Icelandic Graduate Research Fund. References [1] R. Spilliaert, A. Gudmundsdottir, Atlantic cod trypsin Y-member of a novel trypsin group, Marine Biotechnol. 1 (1999) 598–607. [2] J.C. Roach, A clade of trypsins found in cold-adapted Wsh, Proteins 47 (2002) 31–44. [3] H.M. Palsdottir, Á. Gudmundsdottir, Recombinant trypsin Y from Atlantic cod—properties for commercial use, J. Aqua. Food Prod. Technol. 13 (2004) 85–100. [4] G. Feller, Molecular adaptations to cold in psychrophilic enzymes, Cell. Mol. Life Sci. 60 (2003) 648–662. [5] A.O. Smalås, H.K. Schrøder Leiros, V. Os, N.P. Willassen, Cold adapted enzymes, Biotechnol. Annu. Rev. 6 (2000) 1–57. [6] P. Zavodszky, J. Kardos, A. Svingor, G.A. Petsko, Adjustment of conformational Xexibility is a key event in the thermal adaptation of proteins, Proc. Natl Acad. Sci. USA 95 (1998) 7406–7411. [7] N. Aghajari, F. Van Petegem, V. Villeret, J.P. Chessa, C. Gerday, R. Haser, J. Van Beeumen, Crystal structures of a psychrophilic metalloprotease reveal new insights into catalysis by cold-adapted proteases, Proteins 50 (2003) 636–647. [8] B. Asgeirsson, J.W. Fox, J.B. Bjarnason, PuriWcation and characterization of trypsin from the poikilotherm Gadus morhua, Eur. J. Biochem. 180 (1989) 85–94. [9] S. D’Amico, J.C. Marx, C. Gerday, G. Feller, Activity-stability relationships in extremophilic enzymes, J. Biol. Chem. 278 (2003) 7891–7896.
[10] B.K. Shoichet, W.A. Baase, R. Kuroki, B.W. Matthews, A relationship between protein stability and protein function, Proc. Natl Acad. Sci. USA 92 (1995) 452–456. [11] K. Miyazaki, P.L. Wintrode, R.A. Grayling, D.N. Rubingh, F.H. Arnold, Directed evolution study of temperature adaptation in a psychrophilic enzyme, J. Mol. Biol. 297 (2000) 1015–1026. [12] L. Birolo, M.L. Tutino, B. Fontanella, C. Gerday, K. MainolW, S. Pascarella, G. Sannia, F. Vinci, G. Marino, Aspartate aminotransferase from the Antarctic bacterium Pseudoalteromonas haloplanktis TAC 125. Cloning, expression, properties, and molecular modelling, Eur. J. Biochem. 267 (2000) 2790–2802. [13] G. Feller, O. Le Bussy, C. Gerday, Expression of psychrophilic genes in mesophilic hosts: assessment of the folding state of a recombinant alpha-amylase, Environ. Microbiol. 64 (2000) 1163–1165. [14] J. Arnorsdottir, R.B. Smaradottir, O.T. Magnusson, S.H. Thorbjarnardottir, G. Eggertsson, M.M. Kristjansson, Characterization of a cloned subtilisin-like serine proteinase from a psychrotrophic Vibrio species, Eur. J. Biochem. 269 (2002) 5536–5546. [15] J.A. Irwin, S.V. Lynch, S. Coughlan, P.J. Baker, H.M. Gudmundsson, G.A. Alfredsson, D.W. Rice, P.C. Engel, Alanine dehydrogenase from the psychrophilic bacterium strain PA-43: overexpression, molecular characterization, and sequence analysis, Extremophiles 7 (2003) 135–143. [16] O. Lanes, I. Leiros, A.O. Smalas, N.P. Willassen, IdentiWcation, cloning, and expression of uracil-DNA glycosylase from Atlantic cod (Gadus morhua): characterization and homology modeling of the cold-active catalytic domain, Extremophiles 6 (2002) 73–86. [17] G. Jonsdottir, J.B. Bjarnason, A. Gudmundsdottir, Recombinant cold-adapted trypsin I from Atlantic cod-expression, puriWcation, and identiWcation, Protein Expr. Purif. 33 (2004) 110–122. [18] A. Gudmundsdottir, H.M. Palsdottir, Atlantic cod trypsins: from basic research to practical applications, Mar. Biotechnol. (NY) 7 (2005) 77–88. [19] E.R. LaVallie, E.A. DiBlasio, S. Kovacic, K.L. Grant, P.F. Schendel, J.M. McCoy, A thioredoxin gene fusion expression system that circumvents inclusion body formation in the E. coli cytoplasm, Biotechnol. (NY) 11 (1993) 187–193. [20] F.M. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, K. Struhl, Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley-Interscience, New York, 1994. [21] A. Light, H. Janska, Enterokinase (enteropeptidase): comparative aspects, Trends Biochem. Sci. 14 (1989) 110–112. [22] S. Kristjansdottir, A. Gudmundsdottir, Propeptide dependent activation of the Antarctic krill euphauserase precursor produced in yeast, Eur. J. Biochem. 267 (2000) 2632–2639. [23] C. Gerday, M. Aittaleb, M. Bentahir, J.P. Chessa, P. Claverie, T. Collins, S. D’Amico, J. Dumont, G. Garsoux, D. Georlette, A. Hoyoux, T. Lonhienne, M.A. Meuwis, G. Feller, Cold-adapted enzymes: from fundamentals to biotechnology, Trends Biotechnol. 18 (2000) 103–107. [24] E. Narinx, E. Baise, C. Gerday, Subtilisin from psychrophilic Antarctic bacteria: characterization and site-directed mutagenesis of residues possibly involved in the adaptation to cold, Protein Eng. 10 (1997) 1271–1279.
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[25] S. Taguchi, A. Ozaki, T. Nonaka, Y. Mitsui, H. Momose, A coldadapted protease engineered by experimental evolution system, J. Biochem. (Tokyo) 126 (1999) 689–693. [26] M. Macouzet, B.K. Simpson, B.H. Lee, Cloning of Wsh enzymes and other Wsh protein genes, Crit. Rev. Biotechnol. 19 (1999) 179–196. [27] L. Helgadottir, Trypsin I from Atlantic cod: puriWcation, speciWcity, stability and autolysis, MSc Thesis, Department of Chemistry, Faculty of Science, University of Iceland, Reykjavík, Iceland (2002).
[28] B. Asgeirsson, J.B. Bjarnason, Structural and kinetic properties of chymotrypsin from Atlantic cod (Gadus morhua). Comparison with bovine chymotrypsin, Comp. Biochem. Physiol. B 99 (1991) 327–335. [29] M.M. Kristjansson, S. Guthmundsdottir, J.W. Fox, J.B. Bjarnason, Characterization of a collagenolytic serine proteinase from the Atlantic cod (Gadus morhua), Comp. Biochem. Physiol. B 110 (1995) 707–717. [30] D.C. Benjamin, S. Kristjansdottir, A. Gudmundsdottir, Increasing the thermal stability of euphauserase. A cold-active and multifunctional serine protease from Antarctic krill, Eur. J. Biochem. 268 (2001) 127–131.
Expression and puriWcation of a cold-adapted group III trypsin in Escherichia coli Helga Margrét Pálsdóttir, Ágústa Gudmundsdóttir ¤ Science Institute, University of Iceland, Laeknagardi, Vatnsmýrarvegi 16, Reykjavík IS-101, Iceland Received 6 June 2006 Available online 23 June 2006
Abstract The recently classiWed group III trypsins include members like Atlantic cod (Gadus morhua) trypsin Y as well as seven analogues from other cold-adapted Wsh species. The eight group III trypsins have been characterized from their cDNAs and deduced amino acid sequences but none of the enzymes have been isolated from their native sources. This study describes the successful expression and puriWcation of a recombinant HP-thioredoxin–trypsin Y fusion protein in the His-Patch ThioFusion Escherichia coli expression system and its puriWcation by chromatographic methods. The recombinant form of trypsin Y was previously expressed in Pichia pastoris making it the Wrst biochemically characterized group III trypsin. It has dual substrate speciWcity towards trypsin and chymotrypsin substrates and demonstrates an increasing activity at temperatures between 2 and 21 °C with a complete inactivation at 30 °C. The aim of the study was to facilitate further studies of recombinant trypsin Y by Wnding an expression system yielding higher amounts of the enzyme than possible in our hands in the P. pastoris system. Also, commercial production of trypsin Y will require an eYcient and inexpensive expression system like the His-Patch ThioFusion E. coli expression system described here as the enzyme is produced in very low amounts in the Atlantic cod. © 2006 Elsevier Inc. All rights reserved. Keywords: Trypsin Y; Group III trypsin; Atlantic cod (Gadus morhua); Escherichia coli; Expression
The cDNA of trypsin Y was previously isolated from an Atlantic cod cDNA library and characterized [1]. Sequence alignments of trypsin Y and seven other vertebrate trypsins indicated novel characteristics of these enzymes relative to the traditionally classiWed trypsin groups I and II. The novel Wsh trypsins have now been distinguished from other trypsins on the basis of amino acid sequence identities by their placement into the new group III [1,2]. Amino acid sequence identities between the group III and group I trypsins are only about 45–50%. However, the identities are in the order of 60–94% within each of the two trypsin groups. Despite the low sequence identities, the group III trypsins are more similar to the group I vertebrate trypsins than to any other known proteins. It has been postulated that the
*
Corresponding author. Fax: +354 525 4886. E-mail address: [email protected] (Á. Gudmundsdóttir).
1046-5928/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.pep.2006.06.008
group III trypsins have speciWc roles that are diVerent from those of the group I trypsins [2]. The group III trypsins have also been suggested to be adapted to colder environments than other traditionally classiWed cold-adapted trypsins such as cod trypsin I [2,3]. Cold-adapted enzymes appear to have a higher degree of conformational Xexibility than their mesophilic counterparts, presumably due to weaker intramolecular interactions [4–6]. This may facilitate the accommodation and transformation of substrates at low energy costs as shown for the cold-adapted alkaline protease from an Antarctic Pseudomonas species [7]. In general, cold-adapted enzymes have a decreased thermal stability compared to their mesophilic counterparts, most probably in association with their high catalytic eYciency [8–10]. However, recent Wndings suggest that there may not be a strict relationship between high activity of enzymes at low temperatures and a decreased thermal stability [11].
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H.M. Pálsdóttir, Á. Gudmundsdóttir / Protein Expression and PuriWcation 51 (2007) 243–252
The recombinant forms of various cold-adapted bacterial enzymes have been successfully expressed in microorganisms [12–15]. The cold-adapted uracil-DNA glycosylase enzyme from the Atlantic cod was recently expressed in an active form in Escherichia coli [16]. Furthermore, the precursor form of the recombinant trypsin I from Atlantic cod was successfully expressed in a His-Patch ThioFusion E. coli expression system and puriWed [17]. All of the group III trypsin cDNAs have been isolated from Wsh species spending at least part of their lives in extremely cold environments, at 0 °C or lower temperatures. Today, none of the group III trypsin polypeptides have been isolated from their native sources. Their characterization has therefore mainly been based on analysis of the cDNA sequences encoding the enzymes [1,2] as well as on biochemical analysis of recombinant Atlantic cod trypsin Y produced in P. pastoris [3,18]. The recombinant enzyme was shown to have dual substrate speciWcity, i.e. towards trypsin and chymotrypsin speciWc synthetic substrates. Furthermore, the recombinant trypsin Y showed increasing proteolytic activity at temperatures between 2 and 21 °C with a complete inactivation at 30 °C [3]. This narrow range of enzymatic activity with temperature is diVerent from the cold-adapted group I trypsins. Therefore, trypsin Y may be ideal for commercial use, for example, in food processing where low environmental temperatures are required for a short period of time to ensure quality. The dual substrate speciWcity of trypsin Y may also be an advantage for speciWc industrial or medical applications [18]. It appears that trypsin Y is produced in very low amounts in the Atlantic cod relative to trypsin I (results submitted for publication) and its isolation from the native source may not be feasible. Therefore, the industrial application of trypsin Y will probably depend on using the recombinant form produced in an eYcient and low-cost large-scale expression system like the His-Patch ThioFusion E. coli under study here. Materials and methods Host strains and vectors The ampicillin resistant E. coli TOP10 strain (recA, endA) (Invitrogen, CA, USA) was used for propagation of the recombinant plasmids as well as for the expression of the recombinant trypsin Y fusion constructs. The E. coli His-Patch ThioFusion expression system kit (Invitrogen), containing the pThioHis A expression vector, was used for cloning, expression, and partial puriWcation of the recombinant trypsin Y protein. The pThioHis A expression vector contains a trc promoter in addition to the gene encoding the Lac repressor (lacIq) that regulates expression from the trc promoter. IPTG1 (isopropyl--D-thiogalactopyrano-
1
Abbreviation used: IPTG, isopropyl--D-thiogalactopyranoside.
side) was used to induce production of the recombinant protein, as IPTG binds to the Lac repressor protein and blocks its action. The pThioHis A vector contains a histidine patch thioredoxin (HP-thioredoxin) gene mutated to create a metal-binding domain in the HP-thioredoxin protein to ease puriWcation of the recombinant fusion protein on a metal-chelating resin. The fusion to the HP-thioredoxin protein also confers solubility to insoluble heterologous proteins [19]. An enterokinase cleavage site is engineered into the pThioHis A vector between the HP-thioredoxin gene and the multiple cloning site. This enables cleavage of the desired recombinant protein from the HP-thioredoxin fusion part by enterokinase. Construction of expression vectors Two diVerent constructs of the pThioHis A vector and the trypsinogen Y cDNA were designed. This was done in order to determine if the cDNA sequence encoding the prepro part of trypsinogen Y was required for proper folding of the recombinant enzyme. The trypsinogen Y was cloned downstream from the trc promoter and the thioredoxin gene in the pThioHis A vectors. The Wrst construct contained the entire prepro sequence of the cDNA encoding trypsinogen Y. This included the signal sequence and activation peptide of the putative polypeptide starting at residue Ile(-6) according to the chymotrypsinogen numbering system. This cDNA construct was termed HP-thioredoxin–trypsin Y IGLA, where IGLA represents the Wrst four N-terminal amino acid residues of the putative trypsin Y polypeptide. Using the same nomenclature, the second construct, HP-thioredoxin–trypsin Y IIGG, was designed to encode the putative mature form of the enzyme starting at residue Ile16 in the recombinant trypsin Y polypeptide. The two diVerent trypsin Y cDNA constructs (IGLA and IIGG) were ampliWed in a PCR reaction according to standard protocols using a pBluescript vector containing the entire trypsinogen Y cDNA sequence. The PCR ampliWcations were performed in a PCR thermocycler 9700 system (Applied Biosystems, CA, USA). The following oligonucleotide primers (TAGCopenhagen, Denmark) were used to create the trypsin Y constructs: IGLA forward primer: 5⬘-TATATGGTACCTATCGGCCTGGCTCTG CTA-3⬘; IIGG forward primer: 5⬘-TATATGGTACCTAT CATCGGCGGACAGGAC-3⬘; IGLA and IIGG reverse primer: 5⬘-TATATTCTAGATCAGGGGTTGGCATCC AGTGA-3⬘. A KpnI restriction site was incorporated at the 5⬘-end of the trypsinogen Y cDNA and an XbaI site at its 3⬘-end. A stop codon was designed at the 3⬘-end between the trypsin Y construct and the XbaI restriction site. Transformation into E. coli TOP10 strain The two diVerent recombinant HP-thioredoxin–trypsin Y plasmids (IGLA and IIGG) were transformed into competent E. coli TOP10 cells using standard protocols [20]. A pThioHis A vector, without a trypsinogen Y insert, was
H.M. Pálsdóttir, Á. Gudmundsdóttir / Protein Expression and PuriWcation 51 (2007) 243–252
also transformed into competent E. coli TOP10 cells to be used as a control in the expression and puriWcation experiments. The transformed cells were spread onto Luria-Bertani (LB) agar plates containing 100 g/mL ampicillin (LBamp) for selection of positive colonies. Isolation of plasmids from positive transformants was carried out using the QIAGEN miniprep kit (QIAGEN, Hilden, Germany). The presence of trypsinogen Y inserts in the plasmids was determined by agarose gel electrophoresis after digestion with KpnI (Fermentas, St. Leon-Rot, Germany) and XbaI (GE Healthcare, UK) restriction enzymes. The plasmid–insert boundaries were sequenced to conWrm a correct reading frame of the inserts. This was done in a Perkin Elmer ABIPrism 377 DNA sequencer (Applied Biosystems) using ABI-prism Big-Dye Terminator Cycle Sequencing Kit with AmpliTaq DNA Polymerase, FS (Applied Biosystems). Expression of the recombinant HP-thioredoxin–trypsin Y fusion protein The two diVerent HP-thioredoxin–trypsin Y fusion constructs (IGLA and IIGG) were expressed in E. coli Top 10 cells. To analyse the time and temperature conditions for optimal expression of each recombinant protein, several colonies were grown for pilot expression using small-scale volumes (10 mL) in an Innova 4000 incubator shaker (New Brunswick ScientiWc Co. Inc., NJ, USA). This was followed by growing the cells in large-scale volumes in an incubator shaker (1–2 L). For the pilot expression, a single colony of each construct, previously grown on an LBamp agar plate, was used to inoculate a 1 mL LBamp medium. The culture was incubated overnight at 37 °C with shaking at 200– 225 rpm. Five hundred microlitres of the overnight culture were used to inoculate 10 mL of fresh LBamp medium in a 50 mL culture Xask and grown under same condition until the OD550 reached 0.5. Then IPTG was added to a Wnal concentration of 1 mM to induce expression of the recombinant HP-thioredoxin–trypsin Y fusion protein. Prior to the addition of IPTG, the zero time sample was prepared by pelleting a 1 mL sample at a maximum speed for 3 min in a microcentrifuge. The pellet was stored at ¡20 °C until further processing. In order to determine the optimum time of expression, samples were collected after 1, 4, 7, 10, 12, 19, and 25 h of incubation and handled the same way as the zero point sample. The expression was performed at four diVerent temperatures, 18, 25, 30, and 37 °C in order to Wnd the optimal temperature for expression. For the large-scale expression experiments in an incubator shaker, a 5 mL seed culture was grown overnight at 37 °C with vigorous shaking (200–225 rpm) in an LBamp medium. The overnight culture was then used to inoculate 1–2 L of fresh LBamp medium and grown until the OD550 reached 0.6. Protein expression was then induced by adding IPTG to a Wnal concentration of 1 mM. The culture was transferred to an incubator at 25 °C, the optimum temperature for expression of the fusion protein. The culture was grown with shaking (200–225 rpm) for 12 h followed by
245
harvesting the cells by centrifugation at 3000g for 10 min at 4 °C. Preparation of cell-free extracts The E. coli cell pellets containing the two diVerent HPthioredoxin–trypsin Y fusion proteins were resuspended in an imidazole buVer (20 mM Tris–HCl, pH 8, 2.5 mM EDTA, 5 mM imidazole) according to manufacturer’s instructions (Invitrogen) for analysis of the expression. Samples that were loaded directly onto a ProBond chromatography column (Invitrogen) were resuspended in Binding buVer (20 mM sodium phosphate, 500 mM NaCl, pH 7.8) instead of imidazole buVer. The cell suspension was placed on ice and sonicated with three 10 s bursts using a handheld sonicator (Sonics, CT, USA) to release the HP-thioredoxin–trypsin Y fusion protein from the cells. The lysate was then Xash-frozen in liquid nitrogen followed by thawing at 37 °C. After completion of three sonication-freezethaw cycles, the lysate was centrifuged at 3000 rpm for 15 min at 4 °C to pellet the cell debris and insoluble matter. Recombinant trypsin Y identity conWrmation Expression of the HP-thioredoxin–trypsin Y fusion protein was conWrmed by SDS–PAGE gel electrophoresis (12% separating gel with 5% stacking gel) and Western blot analysis using polyclonal anti-thio antibodies (Santa Cruz Biotechnology, CA, USA) or monoclonal anti-thio antibodies (Invitrogen). The protein bands on the SDS–PAGE gels were also stained with a SYPRO Red protein dye according to manufacturer’s instructions (GE Healthcare). The SYPRO Red stained proteins were detected by Xuorometric scanning in a TYPHOON 8600 variable mode imager spectrophotometer (GE Healthcare). Prestained low range molecular weight standards (21.2–110 kDa) (Bio-Rad, CA, USA) and a prestained protein molecular weight marker (20–118 kDa) (Fermentas) were used as markers on the SDS–PAGE gels. PuriWcation of the HP-thioredoxin–trypsin Y fusion protein on a ProBond column The cell supernatants suspended in ProBond Binding buVer were applied to a ProBond resin equilibrated in the ProBond Binding buVer according to manufacturer’s protocol (Invitrogen). Supposedly, the HP-thioredoxin–trypsin Y fusion protein binds to the ProBond metal-chelating column, whereas the majority of the host cell proteins will not bind. After loading the sample, the column was washed twice with two column volumes (CV) of ProBond Binding buVer. This was done by resuspending the resin in a ProBond Binding buVer followed by rocking the column on a platform shaker for 2 min and removal of the supernatant. The column was then washed with two CV of ProBond Washing buVer at pH 6.0 (20 mM sodium phosphate, 500 mM NaCl, pH 6.0) using the same method
246
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as described for the ProBond Binding buVer. This was repeated until the absorbance of the supernatant (Xow through) measured at 280 nm was less than 0.01. The protein was then eluted by applying consecutively 2.5 CV of each of the four ProBond imidazole elution buVers with increasing imidazole concentrations (50, 200, 350, and 500 mM imidazole, each in 20 mM sodium phosphate, 500 mM NaCl, pH 6.0). All the Xow-through fractions were collected for analysis. The elution of the HP-thioredoxin–trypsin Y fusion protein was monitored by measuring the absorbance of the fractions at 280 nm and by Western blot analysis using anti-thio antibodies for detection. Fractions showing measurable absorbance and a band on the Western blot corresponding to the HP-thioredoxin–trypsin Y fusion protein were mixed together for further puriWcation. HPLC puriWcation of the HP-thioredoxin–trypsin Y fusion protein on a MonoQ anion exchange column The ProBond puriWed supernatant was run through a MonoQ 5/5 ion exchange column in an automated HPLC system (Äkta PuriWer, GE Healthcare). The ProBond puriWed sample was Wrst dialysed against 20 mM Tris, pH 9.3, 5 mM ethanolamine, 10 mM CaCl2 (buVer A), and 1 mL of the dialysed sample was applied onto the MonoQ column at a Xow rate of 1.0 mL/min. The protein was eluted with an increasing linear gradient of NaCl up to 1.0 M after washing with buVer A. The absorbance at 280 nm was analysed by the software equipped with the FPLC system during the entire run. Analysis of the HP-thioredoxin–trypsin Y fusion protein expression The sample containing the recombinant HP-thioredoxin–trypsin Y fusion protein was tested for purity on SDS–PAGE gels by Western blot analysis and by SYPRO Red staining. The samples were activated by cleaving the HP-thioredoxin part from the trypsin Y fusion protein by using native Atlantic cod trypsin I. Approximately 25 g of HPthioredoxin–trypsin Y samples, in a total volume of 200 L were treated with 0.01, 0.1, and 1.0 U native Atlantic cod trypsin at 4 °C for 6 h. The trypsin and chymotrypsin activities of the cell supernatants and the partially puriWed recombinant HPthioredoxin–trypsin Y were routinely assayed with synthetic substrates after activation with native cod trypsin I. The synthetic substrates used were N-CBZ-Gly-Pro-ArgpNA (Sigma, MO, USA) for trypsin activity and suc-AlaAla-Pro-Phe-pNA (Sigma) for chymotrypsin activity. The substrate solutions used were 25 mM dissolved in dimethylsulfoxide (Sigma). The activation assays contained 50 L of the sample previously activated by native cod trypsin I, 100 L of 0.1 M Tris buVer at pH 9.0 and 10 L 25 mM synthetic substrate. The reactions were monitored
at RT, at 410 nm in a Spectra Max 384 Microplate spectrophotometer (Molecular Devices, CA, USA). Results Vector construction and expression Two diVerent trypsinogen Y cDNA constructs were cloned into the pThioHis A expression vector. The constructs were termed HP-thioredoxin–trypsin Y IGLA and HP-thioredoxin–trypsin Y IIGG based on the Wrst four amino acid residues in their polypeptide sequences. The calculated molecular mass of the cod trypsin Y IGLA construct fused with the HP-thioredoxin protein is approximately 40 kDa where the HP-thioredoxin part adds approximately 13 to 27 kDa trypsin Y. The molecular mass of the HP-thioredoxin–trypsin Y IIGG construct is slightly smaller or about 38 kDa due to the lack of a presequence in the part of the construct encoding trypsin Y. Western blots using polyclonal anti-thio antibodies showed that the HP-thioredoxin–trypsin Y constructs (IGLA and IIGG) were produced in the E. coli cells. Two control samples were used, i.e. E. coli cells containing the pThioHis A expression vector without the trypsinogen Y insert and E. coli cells with no vector inserts. Fig. 1 shows the 40 kDa protein band of the HP-thioredoxin–trypsin Y IGLA construct (lane 1). This band was not seen in the control samples (lanes 2 and 3). The 16 kDa HP-thioredoxin protein band was only detected in the control sample containing the pThioHis A expression vector (Fig. 1, lane 2). The optimal temperature for induction of expression of the HP-thioredoxin–trypsin Y fusion protein, as determined in pilot expression experiments, was found to be 25 °C and the optimal time of induction was about 12 h. The Western blot seen in Fig. 2 demonstrates that the amount of the putative 40 kDa recombinant HP-thioredoxin–trypsin Y IGLA fusion protein increases during the induction period (t0 to t12). At the zero time point (t0) (lane 1), no protein is expressed but after only 1 h of induction (t1) (lane 2), a 40 kDa protein band is detected. This band increased during the induction period and the strongest band was detected after 12 h (t12) (lane 6). Other cellular protein bands were also shown to increase during the induction period from t0 to t12. When the cells were induced for a longer time period (19 and 25 h), the intensity of the 40 kDa HP-thioredoxin–trypsin Y IGLA protein band started to decrease (data not shown). A 16 kDa protein band corresponding to the HP-thioredoxin control was observed on the Western blot (Fig. 2, lane 7) after 12 h of expression. The 40 kDa HP-thioredoxin–trypsin Y IGLA protein band was not seen in this sample. Pilot expression experiments demonstrated that only the full length trypsin Y IGLA construct could be activated by proteolytic cleavage with native cod trypsin I (data not shown). Therefore, this construct was used in the experiments presented in this paper.
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247
Fig. 2. Western blot analysis showing the optimal time of expression for HP-thioredoxin–trypsin Y IGLA production. Anti-thio antibodies were used for detection on the blot. The induction time of expression was from t0 to t10 (0–10 h). Lane 1 shows a sample taken prior to induction (t0), whereas lanes 2–6 show samples after 1, 4, 7, 10, and 12 h of expression (t1 to t12), respectively. The intensity of the 40 kDa HP-thioredoxin–trypsin Y IGLA fusion protein band increases during the induction period, where the strongest band is detected after 12 h (lane 6). The 16 kDa HPthioredoxin protein, expressed in E. coli for 12 h was used as a control (lane 7).
Fig. 1. Western blot analysis of the HP-thioredoxin–trypsin Y IGLA fusion protein and control samples from pilot expression experiments using anti-thio antibodies for detection. The 40 kDa HP-thioredoxin– trypsin Y protein band was clearly seen in the HP-thioredoxin–trypsin Y IGLA sample (lane 1) but it was absent in the HP-thioredoxin (lane 2) and E. coli (lane 3) control samples. The HP-thioredoxin control was seen as a band of »16 kDa on the blot (lane 2).
PuriWcation of the HP-thioredoxin–trypsin Y IGLA fusion protein The HP-thioredoxin–trypsin Y IGLA fusion protein was expressed on a large-scale and the cell lysates were puriWed on a ProBond column. The elution proWle of the fusion protein measured as absorbance at 280 nm is seen in Fig. 3A. Protein bands (40 kDa) corresponding to the peaks seen in part A (tubes 11–25) were observed in Western blot analysis using polyclonal (part B) and monoclonal (part C) anti-thio antibodies. Notably, the putative HP-thioredoxin–trypsin Y IGLA protein is not present in tube 10, the last wash fraction prior the HP-thioredoxin–trypsin Y IGLA elution (Fig. 3B). A great diVerence was observed on the Western blots with polyclonal (Fig. 3B) versus monoclonal (Fig. 3C) anti-thio antibodies. The polyclonal antibody cross-reacts with numerous cellular proteins in the cell lysates.
The HP-thioredoxin control protein was also puriWed on a ProBond column in the same manner as the HP-thioredoxin–trypsin Y IGLA sample and used as a positive control in this experiment. A strong 16 kDa HP-thioredoxin protein band was detected in Western blot analysis using polyclonal anti-thio antibodies (data not shown). The ProBond puriWcation method does not achieve puriWcation of the HP-thioredoxin–trypsin Y fusion protein to homogeneity. Therefore, further puriWcation of the ProBond puriWed sample was performed on a MonoQ anion exchange column in an automatic HPLC system. Fig. 4A shows the elution proWles of all fractions from the MonoQ column measured at 280 nm. An absorbance peak was seen after approximately 13 mL of elution, corresponding to a salt concentration of 0.18 M. The samples giving the absorbance peaks (13–37 mL) were run on SDS–PAGE gels and protein bands were visualized on Western blots using polyclonal anti-thio antibodies (Fig. 4B). The 40 kDa HP-thioredoxin–trypsin Y IGLA protein band was Wrst detected after elution with 25 mL of buVer corresponding to a salt concentration of 0.4 M. The ProBond puriWed HP-thioredoxin control sample was also run through the MonoQ-HPLC column. The MonoQ elution proWle measured at 280 nm is seen in Fig. 5A. A very sharp absorbance peak at 280 nm is seen when eluted with 31% NaCl. The samples giving the absorbance peak also gave a strong 16 kDa protein band on a Western blot when using polyclonal anti-thio antibodies (Fig. 5B, volumes 18–28). It can be noted that the sample is puriWed to homogeneity when compared with the non-puriWed sample (Fig. 5B, control).
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Fig. 3. Elution proWle and Western blots of the HP-thioredoxin–trypsin Y IGLA fusion protein puriWed on a ProBond metal-chelating resin. (A) Elution proWle measured at 280 nm. Tubes 1–10 show fractions collected after washing with wash buVer. Tubes 11–15 show the fractions collected after elution with 50 mM imidazole buVer, tubes 16–20 show the fractions collected after elution with 200 mM imidazole buVer, tubes 21–25 show the fractions collected after elution with 350 mM imidazole buVer and tubes 26–32 show the fractions collected after elution with 500 mM imidazole buVer. (B) Western blot analysis of the puriWed fractions (tubes 10–25) using polyclonal anti-thio antibodies. The numbers below the Wgure indicate the collection tube numbers according to the elution proWle in part A. (C) Western blot analysis of the puriWed fractions (tubes 12–20) using monoclonal anti-thio antibodies. The numbers below the Wgure indicate the collection tube numbers according to the elution proWle in part A.
Cleavage of the HP-thioredoxin part and activity measurements During the entire puriWcation process, the enzymatic activity of the HP-thioredoxin–trypsin Y IGLA protein was routinely assayed. The samples were activated by cleaving the HP-thioredoxin part from the recombinant trypsin Y IGLA polypeptide (r-trypsin Y) using native cod trypsin I. Our experiments showed that 0.01 U of native cod trypsin I per 25 g sample was the optimum amount for the activation process. Fig. 6 shows the activity measurements of activated MonoQ puriWed samples when measured towards the synthetic substrates N-CBZGly-Pro-Arg-pNA for trypsin activity (part A) and sucAla-Ala-Pro-Phe-pNA for chymotrypsin activity (part B). Notably, the HP-thioredoxin–trypsin Y IGLA showed higher enzymatic activity towards the trypsin and chymo-
trypsin synthetic substrates than the HP-thioredoxin control sample treated in the same manner. The activated r-trypsin Y was run on a SYPRO Red stained SDS– PAGE gel (Fig. 6C) and also analysed on a Western blot using polyclonal anti-thio antibodies for detection (Fig. 6D). The SYPRO Red stained gel shows the activated r-trypsin Y (Fig. 6C, lane 2) in comparison to the non-activated r-trypsin Y containing the HP-thioredoxin part (lane 3). The activated r-trypsin Y protein band is approximately 25 kDa and the HP-thioredoxin–trypsin Y band is about 40 kDa. The gel also shows a native cod trypsin I sample (23.5 kDa) used to cleave the HP-thioredoxin part from the r-trypsin Y polypeptide (lane 1). As expected, only the non-activated HP-thioredoxin–trypsin Y protein band is detected in Western blot analysis using anti-thio antibodies (Fig. 6D, lane 3). Cleavage of the HPthioredoxin part from the r-trypsin Y polypeptide should
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249
Fig. 4. Elution proWle and Western blots of the HP-thioredoxin–trypsin Y IGLA fusion protein puriWed on a MonoQ anion exchange column. (A) Elution proWle measured at 280 nm using a linear NaCl gradient from 0 to 1 M. (B) Western blot analysis of the puriWed fractions using polyclonal anti-thio antibodies. The numbers below the Wgure indicate the collection volume (mL) according to the elution proWle in part A.
result in a polypeptide free of the anti-thio part of the fusion protein (Fig. 6D, lane 2). The native cod trypsin I was not detected on the Western blot (Fig. 6D, lane 1). The puriWcation steps provided a sevenfold and an 87-fold increase in speciWc activity of the r-trypsin Y polypeptide as measured towards the synthetic substrate N-CBZ-Gly-Pro-Arg-pNA (Table 1). The Wnal preparation of r-trypsin Y was puriWed 631-fold with an overall yield of 43%. Starting with a 221 mg of non-puriWed proteins, this puriWcation process yielded around 150 g of puriWed r-trypsin Y polypeptide (Table 1). Discussion
Fig. 5. Elution proWle and Western blots of the HP-thioredoxin control protein puriWed on a MonoQ anion exchange column. (A) Elution proWle measured at 280 nm using a linear NaCl gradient from 0 to 1 M. (B) Western blot analysis of the puriWed fractions using polyclonal anti-thio antibodies. The numbers below the Wgure indicate the collection volume (mL) according to the elution proWle in part A.
The precursor form of Atlantic cod trypsin Y was readily produced in a soluble form as a 40 kDa fusion protein with HP-thioredoxin in the His-Patch ThioFusion E. coli expression system. Cleavage of the HP-thioredoxin–trypsin Y IGLA fusion protein with a minute amount of native trypsin I generated an active r-trypsin Y. Seemingly, using the native cod trypsin I to activate the recombinant HP-thioredoxin–trypsin Y IGLA fusion protein is a disadvantage due to its contribution to the activity of the Wnal preparation. However, this was shown to be the best enzyme for the activation process. Its use did not cause any confusion of false positive results of activity as a HP-thioredoxin control sample was prepared and treated with native cod trypsin I in the same manner as the activated HP-thioredoxin–trypsin Y IGLA fusion protein in all the activation experiments performed. Higher-vertebrate trypsins usually have a cluster of four consecutive negatively charged residues that serve as the
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Fig. 6. Enzymatic activity, SDS–PAGE, and Western blot analysis of the activated r-trypsin Y from the MonoQ column. (A) Trypsin activity of the r-trypsin Y sample was measured towards the synthetic substrate N-CBZGly-Pro-Arg-pNA at 410 nm in 0.1 M Tris buVer at pH 9.0. (B) The chymotrypsin activity of the r-trypsin Y sample was measured towards the synthetic substrate suc-Ala-Ala-Pro-Phe-pNA at 410 nm in 0.1 M Tris buVer at pH 9.0. (C) SYPRO red stained SDS–PAGE gel of the activated r-trypsin Y sample (lane 2) in comparison to the non-activated r-trypsin Y containing the HP-thioredoxin part (lane 3). The native cod trypsin I sample used to cleave the HP-thioredoxin part from the trypsin Y polypeptide is shown in lane 1. (D) Western blot analysis using polyclonal anti-thio antibodies. The non-activated r-trypsin Y containing the HP-thioredoxin part was detected (lane 3). As expected, the activated r-trypsin Y (lane 2) and the native cod trypsin I (lane 1) were not detected with the anti-thio antibodies.
recognition site for enterokinase, the physiologic activator of trypsinogen [21]. Also, an enterokinase cleavage site is engineered into the pThioHis A plasmid between the HPthioredoxin protein and the multiple cloning site. However, enterokinase cleavage did not activate the HP-thioredoxin– trypsin Y fusion protein. This may be due to the fact that the trypsin Y activation peptide contains only two negatively charged residues (Glu-Asp) instead of four found in most group I trypsins and therefore, lacks an enterokinase recognition site (Asp-Asp-Asp-Asp) [1,3].
As expected, the activated r-trypsin Y showed enzymatic activity towards the synthetic substrates speciWc for trypsin (N-CBZ-Gly-Pro-Arg-pNA) and chymotrypsin (suc-AlaAla-Pro-Phe-pNA). No activity was detected in this fraction without proteolytic cleavage by the native cod trypsin I. The E. coli cell lysates were used as the starting point to create Table 1. The puriWcation steps provided a sevenfold (ProBond) and an 87-fold (MonoQ) increase in speciWc activity as measured towards the synthetic substrate N-CBZ-Gly-Pro-Arg-pNA (Table 1). The Wnal preparation of the r-trypsin Y was puriWed 631-fold with an overall yield of 43% as compared to the non-puriWed recombinant HP-thioredoxin–trypsin Y fusion protein. Starting with a 221 mg of non-puriWed HP-thioredoxin–trypsin Y fusion protein, this puriWcation process yielded around 150 g of homogenous r-trypsin Y. The activation step of the HP-thioredoxin–trypsin Y fusion protein is the most problematic one in the process. Thus, to improve the yields of the active r-trypsin Y, this step needs to be further studied with respect to stability of the active recombinant enzyme generated as well as other factors. The His-Patch ThioFusion E. coli expression system has many advantages when compared with the P. pastoris expression system. However, the P. pastoris system has been used with good results for the expression of r-trypsin Y [3,18] as well as for the expression of Antarctic krill euphauserase [22]. The E. coli expression system oVers milder environmental conditions and a shorter time of cell growth with an induction time of only 12 h at 25 °C for trypsin Y expression, compared to 3–5 days at 20 °C in P. pastoris [3]. Also, due to the high amount of HP-thioredoxin–trypsin Y fusion protein produced in E. coli, the protein is easily detected during the expression process using either antibodies raised to the thioredoxin part of the fusion protein or by SDS–PAGE gel staining. Recombinant proteins tend to be poorly soluble and prone to molecular aggregation [13]. The presence of the highly soluble HP-thioredoxin part of the fusion protein contained at the N-terminal part of the cod trypsin Y sequence increases its solubility [19]. It also seems to protect the N-terminal end of the r-cod trypsin Y from proteolytic cleavage during expression and handling. In addition, it enables puriWcation of the fusion protein on a metal chelating ProBond aYnity column. Cold-adapted proteolytic enzymes from bacteria [14,23– 25] and marine invertebrates [22] have been successfully expressed in microorganisms. However, the recombinant forms of cold-adapted proteolytic enzymes from Wsh are diYcult to produce in an active form in mesophilic expression systems. This conclusion is based on literature search [26] as well as on our own experience with the expression of Wsh peptidases. The main reason seems to be their high thermal sensitivity and susceptibility to autolysis. Also, as is the case for many mesophilic enzymes, cold-adapted enzymes are prone to aggregation during their expression [13]. Combined, these problems lead to degradation and inactivation of the recombinant enzymes. Atlantic cod
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251
Table 1 PuriWcation of the r-trypsin Y PuriWcation step
Volume (mL)
Total activity (U)
Total protein (mg)
SpeciWc activity (U/mg)
Yield (%)
PuriWcation
Cell lysates ProBond MonoQ
100 35 10
0.42 0.32 0.18
221 23.5 0.15
0.0019 0.014 1.2
100 77 43
1.00 7.2 631
The HP-thioredoxin–trypsin Y IGLA fusion protein was isolated from a 1 L culture of E. coli containing approximately 9 g wet cell weights. The cells were resuspended in 100 mL of ProBond binding buVer. The enzymatic activity was measured at RT towards the synthetic substrate N-CBZ-Gly-Pro-ArgpNA. The activation assays contained 50 L of the sample activated with cod trypsin I, 100 L of 0.1 M Tris buVer, pH 9.0 and 10 L of 25 mM synthetic substrate. The measured values for HP-thioredoxin control samples treated in the same manner as the HP-thioredoxin–trypsin Y IGLA sample have been subtracted from the values presented.
trypsin I is exceptionally sensitive to autolysis, even at 18– 25 °C [27]. Isolation and puriWcation of cold-adapted proteases is also challenging for the same reasons as their expression [8,27–29]. Future experiments will focus on improvements of the expression, puriWcation, and activation of the recombinant cod trypsin Y. Site-speciWc mutagenesis of the cDNA encoding the enzyme may also be used to improve the stability of r-trypsin Y without sacriWcing the catalytic eYciency of the enzyme [30]. Such derivatives may broaden the commercial applicability of the r-trypsin Y. In conclusion, the recombinant form of Atlantic cod trypsin Y has been successfully expressed in the E. coli His-Patch ThioFusion expression system. Thus, an alternative method has been developed for cod trypsin Y production facilitating further studies and commercial use of this enzyme. Acknowledgments This work was supported by grants from the University of Iceland Research Fund, the Icelandic Research Fund, and the Icelandic Graduate Research Fund. References [1] R. Spilliaert, A. Gudmundsdottir, Atlantic cod trypsin Y-member of a novel trypsin group, Marine Biotechnol. 1 (1999) 598–607. [2] J.C. Roach, A clade of trypsins found in cold-adapted Wsh, Proteins 47 (2002) 31–44. [3] H.M. Palsdottir, Á. Gudmundsdottir, Recombinant trypsin Y from Atlantic cod—properties for commercial use, J. Aqua. Food Prod. Technol. 13 (2004) 85–100. [4] G. Feller, Molecular adaptations to cold in psychrophilic enzymes, Cell. Mol. Life Sci. 60 (2003) 648–662. [5] A.O. Smalås, H.K. Schrøder Leiros, V. Os, N.P. Willassen, Cold adapted enzymes, Biotechnol. Annu. Rev. 6 (2000) 1–57. [6] P. Zavodszky, J. Kardos, A. Svingor, G.A. Petsko, Adjustment of conformational Xexibility is a key event in the thermal adaptation of proteins, Proc. Natl Acad. Sci. USA 95 (1998) 7406–7411. [7] N. Aghajari, F. Van Petegem, V. Villeret, J.P. Chessa, C. Gerday, R. Haser, J. Van Beeumen, Crystal structures of a psychrophilic metalloprotease reveal new insights into catalysis by cold-adapted proteases, Proteins 50 (2003) 636–647. [8] B. Asgeirsson, J.W. Fox, J.B. Bjarnason, PuriWcation and characterization of trypsin from the poikilotherm Gadus morhua, Eur. J. Biochem. 180 (1989) 85–94. [9] S. D’Amico, J.C. Marx, C. Gerday, G. Feller, Activity-stability relationships in extremophilic enzymes, J. Biol. Chem. 278 (2003) 7891–7896.
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