Evaluation of bottlenecks in proinsulin secretion by Escherichia coli

Journal of Biotechnology 109 (2004) 31–43

Evaluation of bottlenecks in proinsulin secretion by Escherichia coli F.J.M. Mergulhão, M.A. Taipa, J.M.S. Cabral, G.A. Monteiro∗ Centro de Engenharia Biológica e Qu´ımica, Instituto Superior Técnico, Av. Rovisco Pais, 1049-001 Lisbon, Portugal Received 16 November 2002; received in revised form 8 September 2003; accepted 14 October 2003

Abstract This work evaluates three potential bottlenecks in recombinant human proinsulin secretion by Escherichia coli: protein stability, secretion capacity and the effect of molecular size on secretion efficiency. A maximum secretion level of 7.2 mg g−1 dry cell weight was obtained in the periplasm of E. coli JM109(DE3) host cells. This value probably represents an upper limit in the transport capacity of E. coli cells secreting ZZ-proinsulin and similar proteins with the protein A signal peptide. A selective deletion study was performed in the fusion partner and no effect of the molecular size (17–24 kDa) was detected on secretion efficiency. The protective effect against proteolysis provided by the ZZ domain was thoroughly demonstrated in the periplasm of E. coli and it was also shown that a single Z domain is able to provide the same protection level without compromising the downstream processing. The use of this shorter fusion partner enables a 1.6-fold increase in the recovery of the target protein after cleavage of the affinity handle. © 2004 Elsevier B.V. All rights reserved. Keywords: Recombinant; Human; Proinsulin; Secretion; ZZ domain; Purification

1. Introduction Escherichia coli was the first host to produce a recombinant DNA pharmaceutical enabling the approval of Eli Lilly’s recombinant human insulin in 1982 (Swartz, 2001). Although in the last 20 years we have witnessed rapid advances in the development of expression systems, E. coli still remains one of the most attractive hosts for heterologous protein production (Pines and Inouye, 1999). This is due to its ability to grow rapidly and at high density on inexpensive substrates, its well-characterised genetics, and the ∗ Corresponding author. Tel.: +351-218-419-065; fax: +351-218-419-062. E-mail address: [email protected] (G.A. Monteiro).

availability of an increasingly large number of cloning vectors and mutant host strains (Baneyx, 1999). Despite these advantages, the production of some proteins has often proved to be difficult in E. coli due to the degradation of cloned gene products by hostspecific proteases (Belagaje et al., 1997; Meerman and sGeorgiou, 1994). Several proteases and peptidases have been identified in the cytoplasm and periplasm of E. coli (Gottesman, 1996) and data has accumulated suggesting that low-molecular weight proteins or peptides are particularly prone to proteolytic degradation (Tang and Hu, 1993). To circumvent the problem of protein instability and also to facilitate protein purification, fusion techniques have been widely used (Hellebust et al., 1989; Murby et al., 1991a,b; Nilsson et al., 1992, 1997; Nygren et al., 1994).

0168-1656/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.jbiotec.2003.10.024

32

F.J.M. Mergulhão et al. / Journal of Biotechnology 109 (2004) 31–43

One common strategy to stabilise a heterologous protein is to express it as a chimera with a homologous protein. Human proinsulin has been traditionally expressed in a fusion form. Earlier studies on proinsulin expression report the use of ␤-galactosidase as a fusion partner (Belagaje et al., 1997; Goeddel et al., 1979; Guo et al., 1984). In this particular case, the target protein (96 amino acids) fused to a partner with 1007 amino acids accounts for only 9% of the total recombinant protein. Such long fusion partners are obviously undesirable (Flores et al., 1986) and usually it is not necessary to fuse the target protein with the entire host protein as only part of the homologous protein may suffice to efficiently protect against proteolytic activity (Lukacsovich et al., 1990). Another strategy to increase the stability of foreign proteins is by targeting their production to the periplasmic space of E. coli since it is known that most of the proteases are located in the cytoplasm (Fricke et al., 1995; Makrides, 1996; Swamy and Goldberg, 1982). It was shown that the half-life of recombinant proinsulin is increased 10-fold (from 2 to 20 min) when the protein is translocated to the periplasmic space (Talmadge and Gilbert, 1982). However, E. coli naturally does not secrete high amounts of proteins (Francetic et al., 2000) and their transport to the periplasm or to the culture medium is a particularly complex process (Economou, 1999; Pugsley, 1993). The most important mechanism for protein secretion in E. coli is the type II secretion pathway (Zhou et al., 1999). This is a Sec-dependent process in which unfolded proteins carrying an N-terminal sequence are transported across the inner membrane to the periplasm where they are processed and folded prior to their translocation across the outer membrane (Palacios et al., 2001). It has been reported that the size of the passenger polypeptide may influence the secretion efficiency in E. coli (Koster et al., 2000; Palacios et al., 2001; Sauvonnet et al., 1995) and that large cytoplasmic proteins may be physically impossible to translocate (Baneyx, 1999). Efforts to secrete smaller eukaryotic molecules have met with greater success than those of large and complex peptides (Stader and Silhavy, 1990) suggesting that, all other factors being equal, smaller proteins or peptides may be easier to be secreted than larger proteins. The ZZ-affinity tag was created by engineering an IgG binding domain based on the B domain of

staphylococcal protein A (SpA) (Moks et al., 1987). Besides the affinity purification of recombinant proteins, the properties of this tag include a stabilisation because of protection against proteolysis, facilitated folding and increase of the in vivo half-lifes of therapeutical gene products (Nygren et al., 1994). The promoter and secretion signals of SpA have shown to be functional in E. coli and therefore protein A fusions (or fusions to its engineered domain) can be translocated to the periplasm of the bacteria and in some cases to the culture medium (Stahl et al., 1999; Stahl and Nygren, 1997). Human proinsulin is composed of two peptide chains (A and B) connected by a spacer peptide (C peptide) (Mackin, 1998). The fusion proteins produced using the expression vectors developed in the present work have the Z domain(s) fused to the proinsulin B chain (e.g. ZZ-BCA). The aim of the work hereby reported was to evaluate three potential bottlenecks in recombinant human proinsulin secretion in E. coli: protein stability, secretion capacity and the effect of molecular size on secretion efficiency. To accomplish this task two expression strains were used to attain different expression levels and a selective deletion study was performed in the affinity tail to assess the effect of the multiplicity of Z domains on protein stability and secretion efficiency. The implications of these deletions in downstream processing were also evaluated.

2. Materials and methods 2.1. Expression vector construction The two exons of the human proinsulin gene were assembled as previously reported (Mergulhão et al., 1999). The fragment was cloned in the pEZZ18 vector (Amersham Pharmacia) and this construction was named pFM7 (Mergulhão et al., 2000). A cloning fragment (containing the Shine-Dalgarno sequence, SpA leader peptide, ZZ domain and the proinsulin gene) was obtained from plasmid pFM7 enabling the construction of the pFM15 vector, expressing ZZ-proinsulin under the control of the lacUV5 promoter (Mergulhão et al., 2003b). Another vector, named pFM17 (encoding for Z-proinsulin), was obtained by incomplete digestion of pFM7 with AccI

F.J.M. Mergulhão et al. / Journal of Biotechnology 109 (2004) 31–43

33

^

pFM7 : LKKKNIYSIRKLGVGIASVTLGTLLISGGVTPAANA AQHDEAVDNKFNKEQQNAFYEI ^ pFM17: LKKKNIYSIRKLGVGIASVTLGTLLISGGVTPAANA AQHDEAVDNKFNKEQQNAFYEI ^ pFM18: LKKKNIYSIRKLGVGIASVTLGTLLISGGVTPAANS …..…………………………………. pFM7 : LHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDNKFNKEQQNAFY pFM17: LHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKV..……………………. pFM18: …………………………………………………………………………………………………. pFM7 : EILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDANSHM-proinsulin pFM17: ……………………………………………………………………………. DANSHM-proinsulin pFM18: …………………………………………………………………………………… HM-proinsulin

Fig. 1. Amino acid alignment of the three expressed proteins. Predicted cleavage site (by the program PSORT, http://psort.nibb.ac.jp/) is indicated by ∧. Amino acids from the SpA signal peptide are italicised. First Z domain is underlined and second Z domain is double-underlined. The amino acids from the human proinsulin moiety which are fused to the last methionine of the alignment in all three constructs were omitted for clarity.

(Promega), thus removing one Z domain. After gel purification of the relevant fragment, self-ligation was performed with T4 DNA ligase (Promega). Site-directed mutagenesis was performed in the pFM7 vector to introduce an extra EcoRI site, by using the QuickChangeTM Site-Directed Mutagenesis Kit (Stratagene) with the following primers (the two substitutions are indicated in bold and underlined): • 5 -GGCGTAACACCTGCTGCGAATTCTGCG¯ CAACACGATGAAGCCG-3 ¯  • 5 -CGGCTTCATCGTGTTGCGCAGAATTCGCAGCAGGTGTTACGCC-3 ¯ ¯ The plasmid was digested with EcoRI (Promega), removing both Z domains, and after gel electrophoresis and purification, self-ligation was performed with T4 DNA ligase (Promega) yielding vector pFM18 (encoding for proinsulin without Z domains). Automated DNA sequencing was performed on both strands of all three vectors. The amino acid alignments of the proteins produced by these expression vectors are represented in Fig. 1. 2.2. Cultivation conditions and growth medium E. coli JM109(DE3) (Promega) or AF1000 (Sandén et al., 2003) (kindly provided by Prof. Gen Larsson, Swedish Centre for Bioprocess Technology, Sweden) was grown in 500 ml shake-flasks (37 ◦ C, 220 rpm) with 100 ml of LB or M9 medium containing 4 g l−1 of glucose (Sambrook et al., 1989) supplemented with 100 ␮g ml−1 of ampicillin (Sigma). For the

JM109(DE3) cultures, 5 mM of thiamine (Sigma) was added to the M9 medium. 2.3. Protein analysis 2.3.1. Periplasmic fraction analysis A periplasmic extract was prepared by an osmotic shock procedure from 1 ml of cell culture as previously described (Mergulhão et al., 2001) and analysed by an indirect ELISA method. Samples of 20 ␮l of periplasmic extract were dissolved in 100 ␮l of coating buffer and adsorbed onto the wells of Maxisorp® microplates (Nalge Nunc). Samples for the construction of a calibration curve were also prepared by adding up to 540 ng of ZZ-proinsulin standard in 80 ␮l of water spiked with 20 ␮l of periplasmic extract of AF1000 cells without plasmid. After incubation (2 h at room temperature), the wells were washed and non-specific binding sites were blocked with 350 ␮l of phosphate-buffered-saline (PBS)–0.05% Tween® 20 (PBST). The first antibody, a mouse anti-human proinsulin monoclonal antibody (Advanced Immunochemical) was then added (170 ng in 100 ␮l of PBST) and after a 2 h incubation at room temperature the wells were washed as before. Anti-mouse Ig, horseradish peroxidase-linked from sheep (Amersham) (100 ␮l in a 1:1000 dilution in PBST) was used as detection antibody. The plate was incubated with the conjugate for 2 h at room temperature and washed as before. A volume of 200 ␮l of substrate solution (3.6 mM o-phenylenediamine, 0.036% H2 O2 in 5 mM Na2 HPO4 and 2 mM citric acid) was added and the colorimetric reaction developed for 1 h in the

34

F.J.M. Mergulhão et al. / Journal of Biotechnology 109 (2004) 31–43

dark, at room temperature. The reaction was stopped by adding 50 ␮l of 2 M H2 SO4 to each well and absorbance at 490 nm was read with a multiplate reader (Bio-Rad). Total protein in periplasmic fractions and purified recombinant protein was also analysed by the bicinchoninic acid (BCA) method (Smith et al., 1985). Periplasmic secretion results are presented in milligrams of protein per gram of dry cell weight (mg g−1 ). 2.3.2. Supernatant analysis The culture supernatant, obtained by harvesting the cells from 1 ml of cell culture (10 000 g, 5 min) was analysed by an ELISA method similar to the one used for the periplasmic fractions. Sample volumes of 50 ␮l were used and a calibration curve was constructed by adding up to 120 ng of ZZ-proinsulin standard in 50 ␮l of water spiked with 50 ␮l of culture supernatant of AF1000 cells without plasmid. The substrate incubation in the dark lasted for 15 min before the acid was added. Culture medium secretion results are presented in milligrams of protein per gram of dry cell weight (mg g−1 ). 2.3.3. Total expression analysis Culture volumes of 7.5–15 ␮l (cells and medium) were analysed by Western blotting assay (Mergulhão et al., 2000) to monitor the total protein expression. After colorimetric staining, the intensity of the bands was analysed by densitometry using the Total Lab® software (Phoretix, Nonlinear Dynamics). Affinity purified samples of ZZ-proinsulin (up to 200 ng) from periplasmic fractions, prepared as previously described (Mergulhão et al., 2001), were loaded on the SDS-PAGE gels in parallel with the samples, and used as standards for the construction of a calibration curve. Due to some type of interference (Mergulhão et al., 2003a), this method underestimated the total expression levels (total expressed ZZ-proinsulin was apparently less than secreted fraction assayed by ELISA). Hence, total expression results are presented in arbitrary units per gram of dry cell weight (AU g−1 ). 2.4. Protein purification Proinsulin fusions were affinity-purified using an IgG-coupled Sepharose column (Pharmacia) as de-

scribed previously (Mergulhão et al., 2001). The fusion proteins were eluted with 0.5 mM acetic acid, pH 2.8, and 10 fractions of 1 ml were collected for analysis.

3. Results and discussion Three potential bottlenecks on the secretory production of human proinsulin were analysed by using two different expression strains and three proinsulin derivatives. The protein stability, secretion capacity and the effect of molecular size on secretion efficiency were evaluated on expression systems using the SpA promoter and leader peptide in which the proinsulin moiety was fused to none, one or two Z domains derived from the B domain of staphylococcal protein A. The implications of the number of Z domains to be used as fusion partners were also evaluated regarding protein purification by affinity chromatography. 3.1. Effects of the host strain and medium composition on expression A comparative study was performed with the pFM7 vector on JM109(DE3) and AF1000 cells grown in rich LB medium and minimal M9 medium (Fig. 2). The maximum expression levels obtained with the JM109(DE3) strain were higher (5.2-fold in M9 and 1.6-fold in LB) than those obtained with AF1000 cells in both cultivation media (Fig. 2A). The two strains have similar genotypes, the most significant difference being that JM109 is relA− , spoT+ whereas AF1000 is relA+ , spoT+ . During starvation, a series of modifications, designated as the stringent response, have been reported in E. coli (Jishage et al., 2002; Joseleau-Petit et al., 1999). The first event in this response is the production of a specific nucleotide, guanosine-5 -diphosphate-3 -diphosphate (ppGpp) which interacts with the RNA polymerase. Two enzymes can catalyse ppGpp synthesis: ppGpp synthetase I, the relA gene product, is activated upon amino acid starvation and ppGpp synthetase II, the spoT gene product, is responsible for ppGpp synthesis during carbon source limitation (Barker et al., 2001a,b). Since RNA polymerase is a target for ppGpp, the stringent response is extremely important in plasmid replication as RNA polymerase plays a key

F.J.M. Mergulhão et al. / Journal of Biotechnology 109 (2004) 31–43 8

-1

ZZ-Proinsulin (mg g )

20

-1

ZZ-Proinsulin (AU g )

25

15

10

5

6

4

2

0

0 0

(A)

35

10

20 Time (h)

0

30

(B)

10

20 Time (h)

30

Fig. 2. Effect of medium composition and expression host in ZZ-proinsulin expression using the pFM7 vector. (䊊) JM109 in M9, (䊉) JM109 in LB, () AF1000 in M9, (䉱) AF1000 in LB. (A) Total expression, (B) periplasmic extract. Total expression values result from a single determination of a representative cultivation and are presented in arbitrary units per gram of dry cell weight (AU g−1 ). Periplasmic results are an average of two independent cultivations. Values are presented in milligrams of ZZ-proinsulin per gram of dry cell weight (mg g−1 ), standard deviation was lower than 25%.

role in the initiation of replication of several replicons (Wegrzyn et al., 1991). It was demonstrated that most of the ColE1-like plasmids, like pFM7, are unable to replicate efficiently in a relA+ host during starvation for different amino acids, although several of these plasmids were capable of replicating in a relA− mutant under the same starvation conditions (Wrobel and Wegrzyn, 1998). Furthermore, it was reported (Kvint et al., 2000) that ppGpp causes a rapid reduction in rRNA transcription, probably by reducing the stability of the open promoter–RNA polymerase complexes at rRNA promoters. With strain JM109, the production is maximal in M9 medium (Fig. 2A). It has been reported (Wrobel and Wegrzyn, 1998) that relA− mutants do not contain ppGpp synthetase I and since the minimal medium contains glucose, ppGpp synthetase II is inactive therefore preventing ppGpp production under these conditions. In LB medium, carbon source availability is lower than in M9 (because there are no carbohydrates) and therefore some ppGpp can be synthesised by ppGpp synthetase II, resulting in lower expression levels with the JM109 strain. With the AF1000 strain, the highest expression levels were found in LB medium (Fig. 2A). Since AF1000 is relA+ , the amino acid starvation in minimal medium can cause a ppGpp build-up by ppGpp synthetase I, which lowers

the expression levels. This effect is apparently more important than the basal production of ppGpp that can occur in LB medium due to the ppGpp synthetase II. The influence of protein molecular size on the expression level was also evaluated. The maximum expression level of Z-proinsulin (pFM17) was similar to the one obtained with ZZ-proinsulin (pFM7), whereas expression without a Z domain fusion (pFM18) could not be detected by Western blot analysis (Fig. 3A). The promoter and Shine-Dalgarno sequences were the same in the three constructs and mRNA analysis in silico did not show significant differences between them (not shown). If the transcriptional and translational levels attained with the pFM18 construct are indeed similar to those obtained with the pFM7 and pFM17 vectors, then the absence of signal in non-fused proinsulin samples analysed by Western blotting can be explained by protein degradation (Belagaje et al., 1997; Cowley and Mackin, 1997; Guo et al., 1984; Jonasson et al., 1996; Kang and Yoon, 1994; Tang and Hu, 1993) combined with a relatively low sensitivity of the analytical method. 3.2. Protection from proteolytic activity From what was previously discussed, the fusion peptide comprising the SpA signal peptide and proin-

36

F.J.M. Mergulhão et al. / Journal of Biotechnology 109 (2004) 31–43

Recombinant protein (AU g-1)

30

20

10

0 0

10

(A)

20

30

Time (h)

Recombinant protein (mg g-1)

8

6

4

2

0 0

10

(B)

20

30

Time (h)

Recombinant protein (mg g-1)

8

6

4

2

0 0

(C)

10

20

30

Time (h)

Fig. 3. Effect of multiplicity of Z domains in expression and secretion of proinsulin and derivatives. (䊊) pFM7 (ZZ-proinsulin), (䊉) pFM17 (Z-proinsulin), (䊏) pFM18 (proinsulin). (A) Total expression, (B) periplasmic extract, (C) culture medium. Total expression values result from a single determination of a representative cultivation and the results are presented in arbitrary units per gram of dry cell weight (AU g−1 ). Periplasmic and supernatant results are an average of two independent cultivations and the values are presented in milligrams of ZZ-proinsulin per gram of dry cell weight (mg g−1 ). Standard deviations for the periplasmic extract and culture medium results were lower than 22 and 13%, respectively.

sulin might be expressed at the same rate as the remaining ZZ- and Z-fusion peptides. The reasons for its apparent instability may be related to the existence of several proteases capable of proinsulin degradation (Fricke et al., 1995). At least two metalloproteases (named Pi and Ci) degrade Insulin and similar sized peptides (Gottesman, 1996). Protease Ci is a cytoplasmic enzyme whereas protease Pi (also referred to as protease III, pitrilysin) is mainly active in the periplasm (Meerman and Georgiou, 1994; Swamy and Goldberg, 1982), although it can also be active in the cytoplasm (Kowit et al., 1976). Furthermore, protease Pi can be efficiently secreted through the outer cell membrane to the culture medium (Diaz-Torres et al., 1991). To assess the extension of periplasmic degradation, the amount of ZZ-proinsulin in the periplasmic extract was determined by ELISA prior and after loading on the IgG coupled Sepharose column. The protein eluted from the column was further quantified by the BCA method (Smith et al., 1985) and the three assays yielded similar results. This means that the fusion protein (ZZ-BCA, where B, C and A are the three proinsulin chains) that was secreted to the periplasmic space had to contain the ZZ domain (in order to bind the column) and also the site of connection of the C-chain and the carboxyl-end of the B chain of human proinsulin (where the epitope for the monoclonal antibody is located) to be recognised in the ELISA assay. Protease Pi cleaves the oxidised insulin B chain at two sites with an initial rapid cleavage at Tyr16 –Leu17 and a second slower cut at Phe25 –Tyr26 (Cheng and Zipser, 1979). Our results show that the secreted peptide contains the fusion tag, the B chain and has the correct size (Fig. 4, lanes 1–3) and therefore it is likely that ZZ-proinsulin is indeed protected from proteolysis at least when it reaches the periplasmic space. It has been suggested (Kang and Yoon, 1994) that the ZZ-tag protects human proinsulin from proteolytic activity in the cytoplasm of E. coli cells. The results obtained in this work indicate that the ZZ domain efficiently protects recombinant human proinsulin from proteolytic attack in the periplasmic space, where one of the most important degrading enzymes (Pi) is located. It has been suggested that this protection might be due to electrostatic interactions between the basic residues of the product and acidic regions of the ZZ domain which may induce the formation of protected

F.J.M. Mergulhão et al. / Journal of Biotechnology 109 (2004) 31–43

Fig. 4. SDS-PAGE of ZZ-proinsulin purification. Lanes 1–3: affinity purified ZZ-proinsulin (0.1, 0.07 and 0.05 ␮g, respectively), lane 4: molecular weight markers, lane 5: periplasmic extract (5 ␮g), lane 6: cell culture (≈2 ␮g), lanes 7 and 8: column flow-through (≈5 ␮g). Numbers on the left represent the molecular weight markers (kDa) of lane 4. The localisation and sharpness of each marker band are also depicted for clarity.

soluble forms (Nilsson and Abrahmsen, 1990). Our results show that for recombinant human proinsulin, the protective effect associated with a fusion with two Z domains can be attained by fusing the peptide with a single Z domain without loss in performance (Fig. 3). The absolute requirement of a fusion partner for a production scheme aimed at proinsulin secretion was also demonstrated. Further deletions within the Z domain were not performed as this synthetic domain had been previously optimised for optimum binding properties, proteolytic resistance and cleavage from the target protein (Abrahmsen et al., 1986; Moks et al., 1987; Nilsson and Abrahmsen, 1990; Stahl and Nygren, 1997). 3.3. Periplasmic secretion The highest value of periplasmic ZZ-proinsulin secretion was obtained with the JM109(DE3) strain in M9 minimal medium. With this strain, a concentration of 7.2 ± 0.5 mg g−1 was reached (Fig. 2B) whereas only 1.4 ± 0.3 mg g−1 was obtained with AF1000. A comparable ZZ-proinsulin secretion level (6.2 mg g−1 ) was previously obtained with a secretion vector with the malK promoter and the SpA leader peptide (Mergulhão et al., 2003b). Although in that case the total expression level was five-fold lower

37

than the one here reported with the pFM7 vector in JM109(DE3) cells, the periplasmic secretion was only slightly decreased (1.2-fold). These results evidence the existence of a secretion bottleneck in terms of the export capacity of E. coli. The fact that the export capacity of the E. coli secretion machinery is limited has already been recognised by several authors (Rosenberg, 1998; Simmons and Yansura, 1996). The existence of a secretion bottleneck in the production of recombinant proteins was reported in the secretion of leptin using the outer membrane protein A signal sequence (Guisez et al., 1998) and in the secretory production of the Pediococcus antimicrobial peptide, pediocin AcH in E. coli (Miller et al., 1998). Interestingly, even when the expressed protein is slightly modified, as in the case of Z-proinsulin (Fig. 3B), the bottleneck subsists as long as the leader peptide is maintained. This result suggests that although the size of the secreted peptide decreased (27% as compared to ZZ-proinsulin), there is no size limitation on the secretion efficiency within the size range analysed (17–24 kDa). In silico analysis has predicted that the probability of existence of a signal sequence is the same in the pFM7 (ZZ-proinsulin) and pFM17 (Z-proinsulin) constructions and that the probability of cleavage of that sequence is equal in both systems. These probabilities are lower in the case of pFM18 (proinsulin), especially with regard to the cleavage of the signal sequence (Table 1) which can be explained by the amino acid substitution in position −1 relative to the cleavage site (alanine to serine, Fig. 1). A lower predicted secretion efficiency can also be explained by the +1 substitution (alanine to histidine, Fig. 1) since it is known that positively charged amino acid residues directly following the cleavage site have a negative effect on secretion by the Sec pathway (MacIntyre et al., 1990). These results, together with the amino acid composition analysis, indicate that ZZ-proinsulin and Z-proinsulin should be secreted with the same efficiency and non-fused proinsulin should also be introduced into the general secretory pathway albeit with a lower efficiency (Table 1). Our experimental results are in good accordance with these predictions for ZZ-proinsulin and Z-proinsulin (Fig. 3B). In terms of target protein production, best results were obtained with the pFM17 vector (Zproinsulin). With this system, periplasmic secretion

38

F.J.M. Mergulhão et al. / Journal of Biotechnology 109 (2004) 31–43

Table 1 In silico analysis results ZZ-proinsulin

Z-proinsulin

Proinsulin

Prevision of secretion probability Existence of SP 9.36 Cleavage of SP 6.32 Cleavage site 36 Secretion 0.94

9.36 6.32 36 0.94

8.75 3.13 36 0.73

Properties of the secreted peptide Amino acids 215 MW (kDa) 24.05 Theoretical pI 5.07 % Positive 9.3 % Negative 14 % Hydrophobic 23.7 Aliphatic index 79.95 Hydropathicity −0.65

157 17.42 5.04 8.3 13.4 24.2 80.83 −0.52

88 9.66 5.48 6.8 10.2 27.3 86.36 −0.11

Prevision of secretion probability was made with the program PSORT (http://psort.nibb.ac.jp/). These predictions include the probability of existence of a signal peptide (SP), whether it is cleaved or not, and the probability of cleavage within that sequence. Higher scores represent higher probabilities. The cleavage site and the probability of periplasmic secretion are also indicated (Nakai and Kanehisa, 1991; Nielsen et al., 1997). The number of amino acids, molecular weight (MW), theoretical isoelectric point (pI), aliphatic index, and the grand average of hydropathicity, were predicted by the program PROTPARAM (http://www.expasy.ch/tools/protparam.html). Amino acid analysis was performed by the program SAPS (http://www.isrec.isbsib.ch/software/SAPS form.html).

levels of 3.5 mg g−1 of proinsulin (corresponding to 6.4 ± 0.7 mg g−1 of Z-proinsulin) were obtained. 3.4. Extracellular secretion Extracellular secretion was achieved with all the expression systems (Fig. 3C). Maximum secretion to the culture medium was obtained with the pFM7 vector (5.6 ± 0.1 mg g−1 of ZZ-proinsulin, corresponding to 2.2 mg g−1 of proinsulin). However, in terms of target protein, the best result was obtained with the pFM17 vector, enabling the recovery of 3.0 mg g−1 of proinsulin (corresponding to 5.4 ± 0.1 mg g−1 of Z-proinsulin). No effect of the molecular size could be detected when comparing the extracellular secretion of ZZ-proinsulin and Z-proinsulin (Fig. 3C). Analysis of the amino acid composition of the secreted peptides (Table 1) shows some differences between the prod-

ucts of the three secretion systems (pFM7, pFM17 and pFM18). When the Z domains are deleted, the percentage of charged amino acids decreases and the percentage of hydrophobic residues increases. The theoretical isoelectric point increases as well as the aliphatic index and average hydropathicity (Table 1). However, these increases are more pronounced when comparing the products of the pFM17 vector (Z-proinsulin) with the ones from pFM18 (proinsulin). The deletion of a single Z domain does not produce dramatic changes in the properties of the secreted peptide as compared to the ZZ-proinsulin fusion (Table 1). Despite the differences found when comparing the properties of secreted peptides, the amount of protein secreted to the culture medium is proportional to that found in the periplasm (Fig. 3B and C). Protein release into the culture medium can fall into three main categories, which include the utilisation of existing pathways for secreted proteins, like the type I secretion system (Bingle et al., 2000; Blight and Holland, 1994; Fernandez et al., 2000), or membrane transport through a type II mechanism (Pugsley, 1993), leakage-associated translocation (Blight and Holland, 1994; del Castillo et al., 2001; Shokri et al., 2002) and cell lysis (Lee et al., 2001). We have previously demonstrated with the pFM7 vector that ZZ-proinsulin transport to the periplasmic space occurs with cleavage of a signal peptide (Mergulhão et al., 2000) suggesting a type II secretion mechanism. However, it is unlikely that the fusion proteins are exported to the culture medium by a dedicated export system as these are usually highly specific for their cognate substrate proteins (Pugsley et al., 1997). Additionally we have also demonstrated previously that cell lysis is not a major contributor to recombinant protein release to the culture medium (Mergulhão et al., 2000). Periplasmic leakage can be of some importance on the culture medium release of proinsulin derivatives and there are at least three mechanisms by which it can occur. During cell division, leakage of periplasmic contents can happen prior to the formation of individual outer membranes. On the other hand, the accumulation of recombinant protein in the periplasm may cause an osmotic-pressure build-up which acts as the driving force for transport across the outer membrane (Hasenwinkle et al., 1997). Additionally, the produc-

F.J.M. Mergulhão et al. / Journal of Biotechnology 109 (2004) 31–43

tion of a recombinant protein can result in severe perturbations in the membrane (Pugsley et al., 1997), thus increasing its selective permeability (Slos et al., 1994) and facilitating the leakage process. If leakage is responsible for the release of the fusion proteins to the culture medium, then a proportional ratio between extracellular secretion and periplasmic contents should exist, as it was observed in this study (Fig. 3B and C).

39

is indicated and a recovery yield was calculated. Full recovery of a ZZ-fusion was obtained with the pFM15 expression vector and the purity of the purified recombinant product was very high as it can be observed on a silver-stained SDS-PAGE (Fig. 4). When comparing similar amounts of target protein loaded onto the column (pFM7 and pFM17 in Table 2), it is evident that the deletion of a single Z domain does not affect the performance of the affinity chromatography step for protein recovery. It has been suggested (Moks et al., 1987) that the number of Z domains to be used as fusion partners depends on the steric interference with the target protein, the efficiency of secretion, and the desired strength of the IgG interaction. The use of two Z domains was found to be optimal for the expression of human insulin-like growth factor I (IGF-I) (Moks et al., 1987), although expression and purification of Z-IGF-I has also been reported (Samuelsson et al., 1996). On the other hand, using two Z domains as fusion partners was insufficient to protect ZZ-IGF-II from proteolysis, although the growth factors are structurally similar and despite the fact that the two arginines recognised by a protease in E. coli are present in both molecules (Hammarberg et al., 1989). Analysis of different repeats of Z domains has shown that the binding strength of a single Z domain is lower than the one attained with two Z domains and that there is no increase in the binding capacity when using more than two Z domains (Ljungquist et al., 1989). However, in the case of proinsulin, similar yields were obtained when using one or two Z domains as fusion partners (Table 2). The similarities in performance of

3.5. Affinity purification The impact of consecutive deletion of Z domains on the affinity purification of ZZ-proinsulin was evaluated. Secretion of proinsulin in a non-fusion form has several advantages like the guarantee of N-terminal authenticity along with the absence of an additional cleavage step to obtain the target product. Our results show that there is no sufficient accumulation of the non-fused product to establish a purification process. These results are in accordance with those obtained by other authors (Kang and Yoon, 1994). Expressing a recombinant protein as a fusion with a Z domain allows its specific purification by a single chromatography step using an IgG-coupled matrix (Nilsson et al., 1997). Table 2 shows the results of several experiments using the two protein fusions (with one or two Z domains) for affinity purification. As the amount of fusion protein loaded onto the column decreases, more diluted fractions are obtained in the eluate thus preventing an accurate protein quantitation in some fractions. The amount of protein that was recovered and quantified in each of these experiments

Table 2 Effect of the number of Z domains on recombinant protein purification System

Protein load

Expression vector

Fusion tag

pFM15 pFM15 pFM7 pFM7 pFM17 pFM17

ZZ ZZ ZZ ZZ Z Z

Recovery

Fusion protein

Total protein

mg l−1

␮g

mg ml−1

mg

21.2 20.2 13.3 8.2 13.9 9.9

635 584 153 45 160 54

1.7 1.1 0.3 0.3 0.4 0.3

50.9 30.6 3.2 1.6 4.6 1.6

Fusion (%)

1.2 1.9 4.7 2.8 3.5 3.4

Fusion protein mg l−1

␮g

92.7 65.5 11.8 2.0 17.9 2.2

649 524 107 16 125 17

Recovery (%)

100 90 70 36 78 32

Proinsulin (␮g)a

261 210 43 6 69 10

Fusion protein results were obtained by indirect ELISA assay and total protein results were obtained by the BCA method. The maximum amount of proinsulin that can be obtained after cleavage of the fusion partner is indicated in the last column. a Estimation of the amount of proinsulin obtained after tag cleavage.

40

F.J.M. Mergulhão et al. / Journal of Biotechnology 109 (2004) 31–43

the chromatographic step may be explained by several factors. The small size of human proinsulin may avoid significant steric interference in the column binding step, the efficiency of secretion is not affected in this size range as previously demonstrated and the binding strength of a single domain is sufficient for the purification of this protein. Furthermore, except for the size, the properties of the secreted peptides are not very different when comparing ZZ-proinsulin with Z-proinsulin (Table 1). Using a single Z domain instead of the ZZ domain as a fusion partner for human proinsulin can lead to the recovery of a 1.6-fold higher amount of proinsulin after cleavage of the fusion tag (Table 2). With the pFM17 expression vector a maximum yield of 3.5 mg g−1 of “native proinsulin” (corresponding to 6.4 ± 0.7 mg g−1 of Z-proinsulin) was achieved. This value is 14-fold higher than the one obtained by other groups (0.25 mg g−1 ) expressing proinsulin as a DsbA fusion in the periplasm of E. coli JM109 cells (Winter et al., 2001). The same authors demonstrated that by careful choice of the expression host and through the optimisation of cultivation conditions and medium composition this yield could be increased to 9.2 mg g−1 (approximately 2.6 times higher than in our case). The use of low-molecular weight additives and the co-secretion of chaperones have been used to increase the amount of secreted proinsulin to the periplasm of E. coli (Schaffner et al., 2001). These approaches may also be effective for Z-proinsulin secretion.

4. Conclusions The results hereby reported show that there is a bottleneck in proinsulin secretion due to the limited capacity of the E. coli transport machinery. The leader peptide used for protein introduction in the general secretory pathway probably plays a key role in the definition of this upper limit for secretion. The data obtained along with previous results suggest that this limit should be around 7 mg g−1 for secretion of ZZ-proinsulin and a similar protein (Z-proinsulin) in two different E. coli strains using the same signal sequence (SpA leader peptide). In the search for shorter fusion partners for recombinant protein expression we have found that specific deletion of a single Z domain in a ZZ-proinsulin fusion is not deleterious

for downstream processing. Furthermore, fusion with a single Z domain also guarantees protection against proteolytic activity, a problem that makes secretion of non-fused proinsulin unattractive for large-scale process development. Using this shorter tag in proinsulin expression resulted in a 1.6-fold increase in the amount of proinsulin that can be obtained after cleavage of the fusion tag, although no effect of the molecular size was seen on the secretion efficiency of the system. The implications of a single Z deletion will vary with the size and amino acid composition of each target protein and therefore the multiplicity of Z domains to be used as fusion partners should be optimised in each particular case.

Acknowledgements F.J.M. Mergulhão acknowledges a Ph.D. fellowship from the PRAXIS XXI Programme, Ministério da Ciˆencia e Tecnologia, Portugal. Prof. Gen Larsson (Swedish Centre for Bioprocess Technology, Stockholm, Sweden) and Prof. Anne Farewell (Department of General and Marine Microbiology, University of Gothenburg, Sweden) are acknowledged for their helpful contributions and discussions in some parts of the work related to proinsulin expression and secretion.

References Abrahmsen, L., Moks, T., Nilsson, B., Uhlen, M., 1986. Secretion of heterologous gene products to the culture medium of Escherichia coli. Nucl. Acids Res. 14, 7487–7500. Baneyx, F., 1999. Recombinant protein expression in Escherichia coli. Curr. Opin. Biotechnol. 10, 411–421. Barker, M.M., Gaal, T., Gourse, R.L., 2001a. Mechanism of regulation of transcription initiation by ppGpp. II. Models for positive control based on properties of RNAP mutants and competition for RNAP. J. Mol. Biol. 305, 689–702. Barker, M.M., Gaal, T., Josaitis, C.A., Gourse, R.L., 2001b. Mechanism of regulation of transcription initiation by ppGpp. I. Effects of ppGpp on transcription initiation in vivo and in vitro. J. Mol. Biol. 305, 673–688. Belagaje, R.M., Reams, S.G., Ly, S.C., Prouty, W.F., 1997. Increased production of low molecular weight recombinant proteins in Escherichia coli. Protein Sci. 6, 1953–1962. Bingle, W.H., Nomellini, J.F., Smit, J., 2000. Secretion of the Caulobacter crescentus S-layer protein: further localization of the C-terminal secretion signal and its use for secretion of recombinant proteins. J. Bacteriol. 182, 3298–3301.

F.J.M. Mergulhão et al. / Journal of Biotechnology 109 (2004) 31–43 Blight, M.A., Holland, I.B., 1994. Heterologous protein secretion and the versatile Escherichia coli haemolysin translocator. Trends Biotechnol. 12, 450–455. Cheng, Y.S., Zipser, D., 1979. Purification and characterization of protease III from Escherichia coli. J. Biol. Chem. 254, 4698– 4706. Cowley, D.J., Mackin, R.B., 1997. Expression, purification and characterization of recombinant human proinsulin. FEBS Lett. 402, 124–130. del Castillo, F.J., Moreno, F., del Castillo, I., 2001. Secretion of the Escherichia coli K-12 SheA hemolysin is independent of its cytolytic activity. FEMS Microbiol Lett. 204, 281–285. Diaz-Torres, M.R., Dykstra, C.C., Claverie-Martin, F., Kushner, S.R., 1991. Extracellular release of protease III (ptr) by Escherichia coli K12. Can. J. Microbiol. 37, 718–721. Economou, A., 1999. Following the leader: bacterial protein export through the Sec pathway. Trends Microbiol. 7, 315–320. Fernandez, L.A., Sola, I., Enjuanes, L., de Lorenzo, V., 2000. Specific secretion of active single-chain Fv antibodies into the supernatants of Escherichia coli cultures by use of the hemolysin system. Appl. Environ. Microbiol. 66, 5024–5029. Flores, N., de Anda, R., Guereca, L., Cruz, N., Antonio, S., Balbas, P., Bolivar, F., Valle, F., 1986. A new expression vector for the production of fused proteins in Escherichia coli. Appl. Microbiol. Biotechnol. 25, 267–271. Francetic, O., Belin, D., Badaut, C., Pugsley, A.P., 2000. Expression of the endogenous type II secretion pathway in Escherichia coli leads to chitinase secretion. EMBO J. 19, 6697–6703. Fricke, B., Betz, R., Friebe, S., 1995. A periplasmic insulin-cleaving proteinase (ICP) from Acinetobacter calcoaceticus sharing properties with protease III from Escherichia coli and IDE from eucaryotes. J. Basic Microbiol. 35, 21–31. Goeddel, D.V., Kleid, D.G., Bolivar, F., Heyneker, H.L., Yansura, D.G., Crea, R., Hirose, T., Kraszewski, A., Itakura, K., Riggs, A.D., 1979. Expression in Escherichia coli of chemically synthesized genes for human insulin. Proc. Natl. Acad. Sci. U.S.A. 76, 106–110. Gottesman, S., 1996. Proteases and their targets in Escherichia coli. Annu. Rev. Genet. 30, 465–506. Guisez, Y., Fache, I., Campfield, L.A., Smith, F.J., Farid, A., Plaetinck, G., Van der Heyden, J., Tavernier, J., Fiers, W., Burn, P., Devos, R., 1998. Efficient secretion of biologically active recombinant OB protein (leptin) in Escherichia coli, purification from the periplasm and characterization. Protein Expr. Purif. 12, 249–258. Guo, L.H., Stepien, P.P., Tso, J.Y., Brousseau, R., Narang, S., Thomas, D.Y., Wu, R., 1984. Synthesis of human insulin gene. VIII. Construction of expression vectors for fused proinsulin production in Escherichia coli. Gene 29, 251–254. Hammarberg, B., Nygren, P.A., Holmgren, E., Elmblad, A., Tally, M., Hellman, U., Moks, T., Uhlen, M., 1989. Dual affinity fusion approach and its use to express recombinant human insulin-like growth factor II. Proc. Natl. Acad. Sci. U.S.A. 86, 4367–4371.

41

Hasenwinkle, D., Jervis, E., Kops, O., Liu, C., Lesnicki, G., Haynes, C., Kilburn, D., 1997. Very high-level production and export in Escherichia coli of a cellulose binding domain for use in a generic secretion-affinity fusion system. Biotechnol. Bioeng. 55, 854–863. Hellebust, H., Murby, M., Abrahmsen, L., Uhlen, M., Enfors, S., 1989. Different approaches to stabilize a recombinant fusion protein. BioTechnology 7, 165–168. Jishage, M., Kvint, K., Shingler, V., Nystrom, T., 2002. Regulation of sigma factor competition by the alarmone ppGpp. Genes Dev. 16, 1260–1270. Jonasson, P., Nilsson, J., Samuelsson, E., Moks, T., Stahl, S., Uhlen, M., 1996. Single-step trypsin cleavage of a fusion protein to obtain human insulin and its C peptide. Eur. J. Biochem. 236, 656–661. Joseleau-Petit, D., Vinella, D., D’Ari, R., 1999. Metabolic alarms and cell division in Escherichia coli. J. Bacteriol. 181, 9–14. Kang, Y., Yoon, J.W., 1994. Effect of modification of connecting peptide of proinsulin on its export. J. Biotechnol. 36, 45–54. Koster, M., Bitter, W., Tommassen, J., 2000. Protein secretion mechanisms in Gram-negative bacteria. Int. J. Med. Microbiol. 290, 325–331. Kowit, J.D., Choy, W.N., Champe, S.P., Goldberg, A.L., 1976. Role and location of “protease I” from Escherichia coli. J. Bacteriol. 128, 776–784. Kvint, K., Farewell, A., Nystrom, T., 2000. RpoS-dependent promoters require guanosine tetraphosphate for induction even in the presence of high levels of sigmas. J. Biol. Chem. 275, 14795–14798. Lee, J., Saraswat, V., Koh, I., Song, K.B., Park, Y.H., Rhee, S.K., 2001. Secretory production of Arthrobacter levan fructotransferase from recombinant Escherichia coli. FEMS Microbiol. Lett. 195, 127–132. Ljungquist, C., Jansson, B., Moks, T., Uhlen, M., 1989. Thiol-directed immobilization of recombinant IgG-binding receptors. Eur. J. Biochem. 186, 557–561. Lukacsovich, T., Baliko, G., Orosz, A., Balla, E., Venetianer, P., 1990. New approaches to increase the expression and stability of cloned foreign genes in Escherichia coli. J. Biotechnol. 13, 243–250. MacIntyre, S., Eschbach, M.L., Mutschler, B., 1990. Export incompability of N-terminal basic residues in a mature polypeptide of Escherichia coli can be alleviated by optimising the signal peptide. Mol. Gen. Genet. 221, 466–474. Mackin, R.B., 1998. Proinsulin: recent observations and controversies. Cell Mol. Life Sci. 54, 696–702. Makrides, S.C., 1996. Strategies for achieving high-level expression of genes in Escherichia coli. Microbiol. Rev. 60, 512–538. Meerman, H.J., Georgiou, G., 1994. Construction and characterization of a set of E. coli strains deficient in all known loci affecting the proteolytic stability of secreted recombinant proteins. Biotechnology (NY) 12, 1107–1110. Mergulhão, F., Monteiro, G., Kelly, A., Taipa, M., Cabral, J., 2000. Recombinant human proinsulin: a new approach in gene assembly and protein expression. J. Microbiol. Biotechnol. 10, 690–693.

42

F.J.M. Mergulhão et al. / Journal of Biotechnology 109 (2004) 31–43

Mergulhão, F., Monteiro, G., Larsson, G., Sandem, A., Farewell, A., Nystrom, T., Cabral, J., Taipa, M., 2003a. Medium and copy number effects on the secretion of human proinsulin in Escherichia coli using the universal stress promoters uspA and uspB. Appl. Microbiol. Biotechnol. 61, 495–501. Mergulhão, F.J., Kelly, A.G., Monteiro, G.A., Taipa, M.A., Cabral, J.M., 1999. Troubleshooting in gene splicing by overlap extension: a step-wise method. Mol. Biotechnol. 12, 285–287. Mergulhão, F.J., Monteiro, G.A., Cabral, J.M., Taipa, M.A., 2001. A quantitative ELISA for monitoring the secretion of ZZ-fusion proteins using SpA domain as immunodetection reporter system. Mol. Biotechnol. 19, 239–244. Mergulhão, F.J.M., Monteiro, G.A., Larsson, G., Bostrom, M., Farewell, A., Nystrom, T., Cabral, J.M.S., Taipa, M.A., 2003b. Evaluation of inducible promoters on the secretion of a ZZ-proinsulin fusion protein. Biotechnol. Appl. Biochem. 38, 87–93. Miller, K.W., Schamber, R., Chen, Y., Ray, B., 1998. Production of active chimeric pediocin AcH in Escherichia coli in the absence of processing and secretion genes from the Pediococcus pap operon. Appl. Environ. Microbiol. 64, 14–20. Moks, T., Abrahmsen, L., Holmgren, E., Bilich, M., Olsson, A., Uhlen, M., Pohl, G., Sterky, C., Hultberg, H., Josephson, S., et al., 1987. Expression of human insulin-like growth factor I in bacteria: use of optimized gene fusion vectors to facilitate protein purification. Biochemistry 26, 5239–5244. Murby, M., Cedergren, L., Nilsson, J., Nygren, P.A., Hammarberg, B., Nilsson, B., Enfors, S.O., Uhlen, M., 1991a. Stabilization of recombinant proteins from proteolytic degradation in Escherichia coli using a dual affinity fusion strategy. Biotechnol. Appl. Biochem. 14, 336–346. Murby, M., Nygren, P.A., Rondahl, H., Hellman, U., Enfors, S.O., Uhlen, M., 1991b. Differential degradation of a recombinant albumin-binding receptor in Escherichia coli. Eur. J. Biochem. 199, 41–46. Nakai, K., Kanehisa, M., 1991. Expert system for predicting protein localization sites in gram-negative bacteria. Proteins 11, 95– 110. Nielsen, H., Engelbrecht, J., Brunak, S., von Heijne, G., 1997. Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Eng. 10, 1–6. Nilsson, B., Abrahmsen, L., 1990. Fusions to staphylococcal protein A. Meth. Enzymol. 185, 144–161. Nilsson, B., Forsberg, G., Moks, T., Hartmanis, M., Uhlen, M., 1992. Fusion proteins in biotechnology. Curr. Opin. Biotechnol. 3, 363–369. Nilsson, J., Stahl, S., Lundeberg, J., Uhlen, M., Nygren, P.A., 1997. Affinity fusion strategies for detection, purification, and immobilization of recombinant proteins. Protein Expr. Purif. 11, 1–16. Nygren, P.A., Stahl, S., Uhlen, M., 1994. Engineering proteins to facilitate bioprocessing. Trends Biotechnol. 12, 184–188. Palacios, J.L., Zaror, I., Martinez, P., Uribe, F., Opazo, P., Socias, T., Gidekel, M., Venegas, A., 2001. Subset of hybrid eukaryotic proteins is exported by the type I secretion system of Erwinia chrysanthemi. J. Bacteriol. 183, 1346–1358.

Pines, O., Inouye, M., 1999. Expression and secretion of proteins in E. coli. Mol. Biotechnol. 12, 25–34. Pugsley, A.P., 1993. The complete general secretory pathway in Gram-negative bacteria. Microbiol. Rev. 57, 50–108. Pugsley, A.P., Francetic, O., Possot, O.M., Sauvonnet, N., Hardie, K., 1997. Recent progress and future directions in studies of the main terminal branch of the general secretory pathway in Gram-negative bacteria—a review. Gene 192, 13–19. Rosenberg, H.F., 1998. Isolation of recombinant secretory proteins by limited induction and quantitative harvest. Biotechniques 24, 188–191. Sambrook, J., Fritsch, E.F., Maniatis, T., 1989. Molecular cloning: a laboratory manual. CSH Press, Cold Spring Harbour. Samuelsson, E., Jonasson, P., Viklund, F., Nilsson, B., Uhlen, M., 1996. Affinity-assisted in vivo folding of a secreted human peptide hormone in Escherichia coli. Nat. Biotechnol. 14, 751– 755. Sandén, A.M., Prytz, I., Tubulekas, I., Forberg, C., Le, H., Hektor, A., Neubauer, P., Pragai, Z., Harwood, C., Picon, A., Teixeira de Mattos, J., Postma, P., Farewell, A., Nystrom, T., Reeh, S., Pedersen, S., Larsson, G., 2003. Limiting factors in Escherichia coli fed-batch production of recombinant protein. Biotechnol. Bioeng. 81, 158–166. Sauvonnet, N., Poquet, I., Pugsley, A.P., 1995. Extracellular secretion of pullulanase is unaffected by minor sequence changes but is usually prevented by adding reporter proteins to its N- or C-terminal end. J. Bacteriol. 177, 5238–5246. Schaffner, J., Winter, J., Rudolph, R., Schwarz, E., 2001. Co-secretion of chaperones and low-molecular-size medium additives increases the yield of recombinant disulfide-bridged proteins. Appl. Environ. Microbiol. 67, 3994–4000. Shokri, A., Sanden, A.M., Larsson, G., 2002. Growth rate-dependent changes in Escherichia coli membrane structure and protein leakage. Appl. Microbiol. Biotechnol. 58, 386–392. Simmons, L.C., Yansura, D.G., 1996. Translational level is a critical factor for the secretion of heterologous proteins in Escherichia coli. Nat. Biotechnol. 14, 629–634. Slos, P., Speck, D., Accart, N., Kolbe, H.V., Schubnel, D., Bouchon, B., Bischoff, R., Kieny, M.P., 1994. Recombinant cholera toxin B subunit in Escherichia coli: high-level secretion, purification, and characterization. Protein Expr. Purif. 5, 518–526. Smith, P.K., Krohn, R.I., Hermanson, G.T., Mallia, A.K., Gartner, F.H., Provenzano, M.D., Fujimoto, E.K., Goeke, N.M., Olson, B.J., Klenk, D.C., 1985. Measurement of protein using bicinchoninic acid. Anal. Biochem. 150, 76–85. Stader, J.A., Silhavy, T.J., 1990. Engineering Escherichia coli to secrete heterologous gene products. Meth. Enzymol. 185, 166– 187. Stahl, S., Nilsson, J., Hober, S., Uhlen, M., Nygren, P., 1999. Affinity fusions, gene expression. In: Flickinger, M., Drew, S. (Eds.), Encyclopedia of Bioprocess Technology: Fermentation, Biocatalysis and Bioseparation. Wiley, New York, pp. 49–63. Stahl, S., Nygren, P.A., 1997. The use of gene fusions to protein A and protein G in immunology and biotechnology. Pathol. Biol. (Paris) 45, 66–76.

F.J.M. Mergulhão et al. / Journal of Biotechnology 109 (2004) 31–43 Swamy, K.H., Goldberg, A.L., 1982. Subcellular distribution of various proteases in Escherichia coli. J. Bacteriol. 149, 1027– 1033. Swartz, J.R., 2001. Advances in Escherichia coli production of therapeutic proteins. Curr. Opin. Biotechnol. 12, 195–201. Talmadge, K., Gilbert, W., 1982. Cellular location affects protein stability in Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 79, 1830–1833. Tang, J., Hu, M., 1993. Production of human proinsulin in E. coli in a non-fusion form. Biotechnol. Lett. 15, 661–666. Wegrzyn, G., Neubauer, P., Krueger, S., Hecker, M., Taylor, K., 1991. Stringent control of replication of plasmids derived from coliphage lambda. Mol. Gen. Genet. 225, 94–98.

43

Winter, J., Neubauer, P., Glockshuber, R., Rudolph, R., 2001. Increased production of human proinsulin in the periplasmic space of Escherichia coli by fusion to DsbA. J. Biotechnol. 84, 175–185. Wrobel, B., Wegrzyn, G., 1998. Replication regulation of ColE1-like plasmids in amino acid-starved Escherichia coli. Plasmid 39, 48–62. Zhou, S., Yomano, L.P., Saleh, A.Z., Davis, F.C., Aldrich, H.C., Ingram, L.O., 1999. Enhancement of expression and apparent secretion of Erwinia chrysanthemi endoglucanase (encoded by celZ) in Escherichia coli B. Appl. Environ. Microbiol. 65, 2439–2445.