Improved partitioning in aqueous two-phase system of tyrosine-tagged recombinant lactate dehydrogenase

Protein Expression and Purification 25 (2002) 263–269 www.academicpress.com

Improved partitioning in aqueous two-phase system of tyrosine-tagged recombinant lactate dehydrogenase Sara Fexby and Leif B€ ulow* Department of Pure and Applied Biochemistry, Center for Chemistry and Chemical Engineering, Lund University, P.O. Box 124, 221 00 Lund, Sweden Received 7 December 2001; and in revised form 25 January 2002

Abstract The partitioning of Bacillus stearothermophilus lactate dehydrogenase (LDH) in an aqueous two-phase system was studied. Particularly, the influence of tyrosine tags on the partitioning was evaluated. The hydrophobic effect, caused by the addition of tyrosine residues, was determined in a system based on dextran and the thermoseparating ethylene oxide–propylene oxide random copolymer (EO30PO70). Five different LDH variants were constructed with N-terminal tags containing tyrosines (Y3 and Y6), tyrosines and prolines (Y3P2 and Y6P2), and only prolines (P2). LDH fused with tags containing tyrosines increased the partitioning coefficient, and the more tyrosines added to the protein, the larger improvement in partitioning. When prolines were added between the tyrosine-rich tag and the protein, a further increased partitioning was obtained. The enhanced partitioning was attributed to the rigid structure of the proline, which in turn led to an increase in the exposure of the tag to the surroundings. The best tyrosine tag, Y6P2, increased the partition coefficient four times and additionally, a higher thermostability was observed. Ó 2002 Elsevier Science (USA). All rights reserved.

Partitioning in aqueous two-phase systems is a wellestablished method for separation and purification of proteins and cellular organelles [1,2]. These systems are formed because an aqueous solution of two water-soluble polymers separates into two phases when the concentrations of the polymers are larger than a critical value. After separation, both phases generally contain 70–95% water [3], which makes the two-phase systems favorable for biological materials. The partitioning of the sample is dependent upon its molecular size, hydrophobicity, charge, and biospecific recognition [4]. Aqueous two-phase systems are mostly used for primary purification of proteins, but the technique has also been applied to study interactions between different biomolecules [5], conformational changes [6], and oligomerization of proteins [7]. The partitioning behavior of a protein can be changed by altering its surface properties, which in turn can be achieved by chemical modifications [8,9] or genetic engineering techniques [10–12]. Alternatively, a ligand

*

Corresponding author. Fax: +46-46-222-4611. E-mail address: [email protected] (L. B€ ulow).

can be attached to one of the polymers. Proteins with high affinity for the ligand will be enriched in the aqueous phase containing the ligand-bound polymer [13]. In another approach, peptides that are directly specific to one of the two phases in the two-phase system are fused to the protein. Short peptides containing tryptophans have been added to the enzyme cutinase [14] and to the Z domain of protein A [15] to investigate the influence of the residues on the partitioning in a twophase system. Disadvantages of adding hydrophobic residues to the protein include a decrease in protein expression, perturbation of protein folding, and a tendency for the protein to adhere to cellular membranes that may in turn increase the risk of proteolysis [12]. While less hydrophobic than tryptophan, the polar amino acid tyrosine partitions in an EO30PO70–dextran system to the less hydrophilic EO30PO70-phase [16]. This partitioning effect of tyrosine is presumably due to the hydrophobicity of its aromatic group. Furthermore, tyrosine residues are more frequently occurring in proteins compared to tryptophans [17]. This motivated us to investigate the effects of tyrosine residues fused to a protein on the partitioning in a two-phase system as well as on the expression and stability.

1046-5928/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII: S 1 0 4 6 - 5 9 2 8 ( 0 2 ) 0 0 0 0 8 - 6

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The thermostable enzyme lactate dehydrogenase (LDH)1 from Bacillus stearothermophilus [18] was chosen as the model protein to evaluate how the hydrophobic effect of tyrosines influences the partitioning. Such information could be utilized to develop affinity tags for purification of other proteins. Various tags containing tyrosines were genetically linked to the Nterminal of the tetrameric enzyme, to optimize and evaluate the fused peptide. Proline residues have a very rigid ring structure and are known to act as a conformational ‘switch’ [19]. Prolines were added to some LDH constructs between the tyrosines and the protein to minimize conformational changes of the protein and also to disrupt constrained conformational structure of the tag caused by the protein. A random copolymer consisting of 30% ethylene oxide and 70% propylene oxide (EO30PO70) was used in this study. The copolymer is thermoseparating, and above a critical temperature (the cloud point) the polymer will precipitate in a water solution [20]. Potassium sulfate ðK2 SO4 Þ was the predominant salt in the system composed of EO30PO70–dextran and sodium phosphate buffer. K2 SO4 generates a chemical potential difference close to zero in an EO30PO70–dextran system [21] thereby minimizing potential salt effects. This is necessary for the study of potential hydrophobic effects caused by tyrosine residues.

into pTrc99A and each nucleotide carried a unique NheI restriction site. The ldh gene was subsequently ligated to the construct using the restriction enzyme BamHI and PstI.

Materials and methods

Aqueous two-phase system

Bacterial strain and plasmid

A two-phase system with a total weight of 2 g, containing 7.1% dextran and 6.8% EO30PO70, was prepared by weighing appropriate amounts of 24% polymer-stock solution of dextran (determined by polarimetry) and a 100% EO30PO70 into a 5 ml tube (calibrated by addition of known volumes of water). The dextran T500 (molecular weight 500,000) was purchased from Pharmacia Biotech, Sweden and the EO30PO70 polymer (molecular weight 3300) was obtained from Shearwater Polymers, USA. Five mM NaP buffer (both phosphate ions), pH 7.0, and 50 mM K2 SO4 were used in the systems. Water was added to give a final weight of 2 g. The systems contained 100–300 lg protein extract/g system. Partitioning was achieved by thoroughly mixing at room temperature, followed by centrifugation for 10 min at 1600g. Two well-separated phases were observed. The top and bottom phases were each diluted to appropriate concentrations before activity measurements. All polymer concentrations are given in % w/w. The phase diagram of this system has been published earlier [15]. The protein partitioning was described by the partition coefficient K, which is defined as LDH enzyme activity in the top phase divided by the activity in the bottom phase. The mass balance of LDH was

Escherichia coli TG1 ½F ; traD36; lacIq ; f ðlacZÞM15;  proAþ Bþ =supE; f ðhsdM-mcrBÞ5; ðr k ; mk –mrcB–Þ; thi; f ðlac-proABÞ was used as the host in all cloning procedures. pTrc99A was used as cloning vector [22]. The native ldh gene from Barstow et al. [18] was inserted in pUC18 by Carlsson et al. [23]. All cloning procedures were performed according to Sambrook et al. [24]. Ldh constructions Ten different oligonucleotides were designed (Table 1) for 50 -end ligation to the ldh gene. All nucleotides were synthesized at the Biomolecular Unit, Lund University, Sweden. Complementary forward and reverse oligonucleotides were hybridized at 98 °C for 5 min, followed by slow cooling to room temperature. The nucleotides were designed with an NcoI site at the 50 -end and a BamHI at the 30 -end used for insertion 1 Abbreviations used: LDH, lactate dehydrogenase; IPTG, isopropylb-D -thiogalactoside; SDS–PAGE, SDS–polyacrylamide gel electrophoresis; MES, 2-[N-morpholino]ethanesulfonic acid; EO30PO70, 30% ethylene oxide and 70% propylene oxide.

Protein expression All cells were grown in LB medium (10 g/L tryptone, 5 g/L yeast extract, and 10 g/L NaCl) containing 100 lg/ml ampicillin. A shake flask containing 200 ml LB medium and 100 lg/ml ampicillin was inoculated with 0.5 ml of an overnight culture and gene expression was induced directly with 0.1 mM isopropyl-b-D thiogalactoside (IPTG). The cells were harvested at late logarithmic phase (3000g, 10 min) and resuspended in 5 ml of 50 mM sodium phosphate (NaP) buffer, pH 7.0. The cell slurry was sonicated and centrifugated (20,000g, 15 min). The supernatants were subsequently heated at 65 °C for 10 min and centrifugated (20,000g, 15 min). The purity of the proteins was routinely checked by SDS–polyacrylamide gel electrophoresis (SDS–PAGE) performed according to Sambrook et al. [24]. A commercially available molecular mass marker (Novex Experimental Technology, Mark 12 Wide Range Protein) was used and the proteins were detected by staining with Coomassie brilliant blue.

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Table 1 Sequences of oligonucleotides used in the cloning of ldh constructs Name

Oligonucleotides

P2-forward P2-reverse Y3-forward Y3-reverse Y3P2-forward Y3P2-reverse Y6-forward Y6-reverse Y6P2-forward Y6P2-reverse

50 -CATGGCTCCGCCGGCTAGCG-30 50 -GATCCGCTAGCCGGCGGAGC-30 50 -CATGGCTTACTATTACGCTAGCG-30 50 -GATCCGCTAGCGTAATAGTAAGC-30 50 -CATGGCTTACTATTACCCGCCGGCTAGCG-30 50 -GATCCGCTAGCCGGCGGGTAATAGTAAGC-30 50 -CATGGCTTACTATTACTATTACTATGCTAGCG-30 50 -GATCCGCTAGCATAGTAATAGTAATAGTAAGC-30 50 -CATGGCTTACTATTACTATTACTATCCGCCGGCTAGCG-30 50 -GATCCGCTAGCCGGCGGATAGTAATAGTAATAGTAAGC-30

The sequences in bold code for the specific residues in the tags.

determined by calculating the total activity added to the system and the activities were found in the different phases. A mass balance between 90% and 110% was accepted as satisfactory. The yields in the top and bottom phases were calculated as the ratio between total LDH activity in the phase and the total LDH activity added to the system. Assays of protein concentration and enzyme activity Sonicated and heat-treated protein extracts were assayed for total protein concentrations using a protein assay (Bio-Rad Laboratories) based on the Bradford Coomassie blue dye-binding procedure [25] and using bovine serum albumin as the standard. Assays of LDH activity were performed in 0.1 M 2-[N-morpholino]ethanesulfonic acid (MES) buffer, pH 6.5, containing 30 mM pyruvate and 0.2 mM NADH. The decrease in absorbance at 340 nm was recorded spectrophotometrically. One unit of enzyme reduces 1 lmol pyruvate per minute at room temperature. To minimize effects from the phase forming components on LDH, all measurements were performed with a constant background. Therefore, an equal volume of an opposite phase containing no protein was added to each sample collected phase. Extra water was also added to create a single, homogenous phase. Both phases from the system were measured at least twice and each system was performed four times. Design of protein models The tertiary structure data of LDH (PDB, 1LDN [26]) were used to create protein models of Y6-LDH and Y6P2-LDH. The 3-D structure was visualized with the software Swiss-PdbViewer v3.7b1 [27]. The peptide tags were added to the N-terminal, one amino acid at a time, using the function ‘‘Add Residue.’’ By maximizing potential H-bonds and minimizing poor contacts, the staggered conformations (rotamers) that best fitted the local environments were chosen.

Results Construction and expression of LDH variants To investigate the influence of tyrosine residues on the partitioning in an EO30PO70–dextran system, different tags were fused to the N-terminal of LDH. Five different LDH constructs were prepared (Table 2). The proteins were expressed intracellularly in E. coli under IPTG induction. Protein extracts were obtained from cells that were grown to late exponential phase followed by sonication and heat treatment to denature most of the native bacterial proteins. No inclusion body formation was observed. However, addition of a tag to a protein may affect its expression and stability. Analysis by SDS–PAGE of total protein contents and heattreated protein extracts revealed that all tagged proteins, with the exception of P2-LDH and Y6-LDH, were well expressed (Fig. 1). The relative levels of LDH expression were approximately 100, 20, 120, 110, 40, and 90 for native, P2-, Y3-, Y3P2-, Y6-, and Y6P2-LDH, respectively estimated with AlphaImager 2200 Documentation and Analysis system using a horizontal baseline. Before partitioning, we included a heat treatment step to denature a substantial fraction of the native E. coli proteins. After heating at 65 °C for 10 min, the specific enzymatic activity of LDH was lowered by 76% when six tyrosines were added to the protein (Fig. 2), but if the tag also contained two prolines the specific enzymatic activity only decreased by 36% compared to native LDH. Similarly, Y3-LDH showed the same specific

Table 2 Amino acid sequences of LDH constructs Name

Protein constructs

P2-LDH Y3-LDH Y3P2-LDH Y6-LDH Y6P2-LDH

MAPPASGANA-LDH MAYYYASGANA-LDH MAYYYPPASGANA-LDH MAYYYYYYASGANA-LDH MAYYYYYYPPASGANA-LDH

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activity as native LDH, but the specific activity was increased by 25% when prolines were added between the tag and the protein. A heat stability test was also performed on sonicated bacterial extracts of the native and tagged LDH proteins. The protein extracts were heated to 80 °C for 60 min and the enzymatic activity was measured every 10 min for all proteins (Fig. 3). The tyrosine-tagged proteins generally showed an improved stability. Only P2-LDH exhibited a significantly lower thermostability compared with the native LDH protein. Partition experiments The heat-treated proteins were partitioned in an aqueous two-phase system composed of 7.1% dextran, 6.8% EO30PO70, 5 mM NaP, pH 7.0, and 50 mM K2 SO4 . The native LDH protein exhibited a partition coefficient close to one and a yield of 52% in the EO30PO70-phase. The K value increased as tyrosine residues were added to the protein (Fig. 4), followed by an increase in the top phase yield to 72–82%. There was

Fig. 1. Twelve percent SDS–PAGE gel analysis of native and engineered LDH proteins. (A) Total protein patterns from E. coli TG1 carrying the indicated LDH constructs and (B) heat treatment at 65 °C for 10 min (cf. Materials and methods). The molecular mass marker (MW) to the left followed by LDH (I), P2-LDH (II), Y3-LDH (III), Y3P2-LDH (IV), Y6-LDH (V), and Y6P2-LDH (VI).

Fig. 2. Specific enzymatic activity in U/mg total protein of all LDH constructs. All protein extracts were heat-treated at 65 °C for 10 min. The presented values represent means of three independent measurements.



Fig. 3. Heat stability test of LDH (N), P2-LDH (), Y3-LDH ( ), Y3P2-LDH (d), Y6-LDH (}), and Y6P2-LDH (r). The protein extracts were exposed to 80 °C and the enzymatic activities were measured every 10 min during an hour. All activity results were normalized to the activity of non-heated proteins.

Fig. 4. The partitioning coefficient, K, of native LDH and engineered LDH proteins in the EO30PO70–dextran two-phase system. The system contained 50 mM K2 SO4 as predominant salt and 5 mM NaP buffer, pH 7.0.

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Table 3 Specific activities and yields of native LDH and Y6P2-LDH after purification in an E030P079–dextran two-phase system LDH Protein (U/mg) After sonication After heat treatment at 65 °C for 10 min Top phase (E030P070)

25.3 29.0 148

Y6P2-LDH Yield (%) 100 73.8 17.5

Protein (U/mg) 16.9 28.0 841

Yield (%) 100 84.0 40.5

to 0.15 when prolines are introduced in the peptide tag. The partition improvement (PI) is defined as [14] Fig. 5. The partition coefficient, K, as a function of the amount of added tyrosines. LDH is a tetramer and to each monomer a tag was fused containing three or six tyrosines, which resulted in a total increase of 12 or 24 tyrosine residues. LDH proteins with tyrosine tags ( ) and tyrosine and proline tags (r). The equations for the linear regression were for tyrosine peptides K ¼ 0:08½Tyrn þ 0:99 and for tyrosine and proline peptides K ¼ 0:15½Tyrn þ 0:96 and the correlation coefficient was 0.99 for both.

a distinct difference between the constructs when three and six tyrosines were added. When two prolines were linked to the tag the K value was increased further. This may be attributed to a higher degree of exposure of the tag to the surrounding medium. The K value seems to be proportional to the number of added tyrosines (Fig. 5). The slope in the linear regression corresponds to the tyrosine contribution and the slope increases from 0.08

PI ¼ Ktagged

protein =Kprotein

ð1Þ

and PI values were obtained in the region of 1.7–4.3. A complete purification of Y6P2-LDH from sonicated bacterial extracts using the E030P070–dextran two-phase system resulted in a purification factor of 50. When compared with native LDH the purification factor was increased at least eightfold. Specific activities and yields are shown in Table 3. Protein models Protein models of Y6-LDH and Y6P2-LDH were created with the program Swiss-PdbViewer. Since the Nterminal parts of the LDH subunits are pairwise close in space, two tags are closely associated in the protein. In Fig. 6, the extra tyrosines (pink) in Y6P2-LDH, the right

Fig. 6. Protein models of Y6-LDH (left) and Y6P2-LDH (right). The N-terminal parts of the LDH subunits are pairwise close in space and the tags, fused to each monomer, are closely associated in the protein. The four monomers are shown in different colors and the tags are colored in mostly yellow or green. In the tags tyrosines are shown in pink and prolines in blue.

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figure, are more exposed compared to Y6-LDH, the left figure. The models thereby support the experimental results, indicating that the tags are more exposed to the surroundings when two prolines are added.

Discussion A number of different tags have been explored to facilitate protein purification. Some examples include FLAG [28], glutathinone S-transferase [29], polyhistidines [30], polycysteines and polyphenylalanine [31], and Z domains [32]. Several requirements should be put on an affinity tag. The yield of protein, protein stability, and for some applications, ease of removal are essential features. This often implies that the tail should be short and exposed. In this study, we have evaluated tyrosine tags for the first time. The protein chosen, B. stearothermophilus lactate dehydrogenase, is a tetrameric protein and large in size for partitioning in a two-phase system, 140 kDa. In our study we could demonstrate that the more tyrosines added, the higher partition coefficient was obtained. The increase in partitioning appeared to be proportional to the number of added tyrosines. Our experimental results showed a higher contribution to the K value when the tagged LDH contained both tyrosine and proline residues compared with tyrosines alone. However, the observed effects were still not as high as the contribution of free tyrosines partitioned in the two-phase system by Berggren et al. [16]. When fusing amino acid residues to a protein, it is reasonable to assume that the residues may be restricted in their exposure to the surroundings. Thus, the lower tyrosine contribution is probably due to limited exposure of the tag to the surroundings and to the protein size. According to Sasakawa et al. [33] smaller molecules have higher K values and partitioning of larger biomolecules has a lower partition coefficient. This was observed in two-phase systems where partitionings were performed close to the isoelectric point of each respective protein. The addition of tyrosine-containing tags in the exposed N-terminal also resulted in an increased thermostability of the protein. By addition of randomized peptides to a protein instead of performing random mutagenesis, Matsuura et al. [34] could easily increase further diversification of valuable properties of a protein. For instance, the thermostability of catalase could be enhanced by adding randomized peptide tags to the C-terminal of the enzyme. Similarly, an increased stability was also observed when proteins were chemically modified with polyethylene glycol [8,9]. To improve our understanding for designing suitable affinity tags, it would be interesting to study the tag effect on proteins with different sizes, since the tyrosine contribution is dependent on the protein size and the

degree of exposure of the tag to the surroundings. To further optimize the tyrosine tags for both protein purification and gene expression, an approach based on random peptide libraries may be particularly helpful. The randomized tyrosine containing peptides can be attached directly to the target protein or, alternatively, to phages [35] or cells [36]. Peptide libraries displayed on phages have previously been extensively used for selection of ligand specifically binding to target proteins [37]. To conclude, the best tyrosine tag, Y6P2, increased the partition coefficient four times and a higher thermostability was observed. This is particularly valuable since heating of the EO30PO70-phase above its cloud point would result in precipitation of the polymer. The thermostable LDH protein can then be recovered from a nearly polymer-free water solution and the polymer can easily be recycled and reused in other two-phase experiments [38,39]. This approach could thus be especially useful for primary recovery of thermotolerant proteins in large-scale applications of two-phase systems.

Acknowledgments This project was supported by the Swedish Center for Bioseparation. We thank Folke Tjerneld and his group, especially the former member Kristina Berggren, for fruitful discussions.

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