AJMB

Evaluation of Recombinant Human Growth Hormone Secretion in E. coli using the L-asparaginase II signal Peptide

PDF - Export to EndNote - PubMed Central XML format - PubMed Central XML format - PubMed Central XML format
PMID: 27920886 (PubMed) - PMCID: PMC5124255 - View online: PubReader
Volume 8, Issue 4, October-December , Page 182 to 187
Tuesday, December 29, 2015 :Received , Saturday, April 9, 2016 :Accepted


  • - Pharmaceutical Sciences Research Center, Shiraz University of Medical Sciences, Shiraz, Iran
    - Department of Pharmaceutical Biotechnology, Faculty of Pharmacy, Shiraz University of Medical Sciences, Shiraz, Iran

  • - Pharmaceutical Sciences Research Center, Shiraz University of Medical Sciences, Shiraz, Iran
  • Corresponding author Shiraz University of Medical Sciences, Shiraz, Iran, Tel: +98 917 7125495, E-mail: ghasemiy@sums.ac.ir
    - Pharmaceutical Sciences Research Center, Shiraz University of Medical Sciences, Shiraz, Iran
    - Department of Pharmaceutical Biotechnology, Faculty of Pharmacy, Shiraz University of Medical Sciences, Shiraz, Iran

Abstract:

Background: In the recent years, there has been an increasing interest in secretory production of recombinant proteins, due to its various advantages compared with intracellular expression. Signal peptides play a critical role in prosperous secretion of recombinant proteins. Accordingly, different signal peptides have been assessed for their ability to produce secretory proteins by trial-and-error experiments. The aim of this study was to evaluate the effect of L-asparaginase II signal peptide on the recombinant human Growth Hormone (hGH) protein secretion in the Escherichia coli (E. coli) host.
Methods: Cloning and expression of a synthetic hGH gene, containing L-asparaginase II signal sequence was performed in E. coli BL21 (DE3) using 0.1 mM IPTG as an inducer at 23oC overnight. Periplasmic protein extraction was performed using three methods, including osmotic shock, osmotic shock in the presence of glycine and combined Lysozyme/EDTA osmotic shock. Afterwards, the hGH expression was determined by SDS-PAGE.
Results: Based on experimentally obtained results, hGH protein is expressed as inclusion body even in the presence of L-asparaginase II signal peptide.
Conclusion: Therefore, this signal peptide is not effective for secretory production of the recombinant hGH.


 

 

Introduction :

Human Growth Hormone (hGH) or somatotropin, a single chain polypeptide contains 191 amino acid residues with a molecular mass of 22 kDa, is synthetized and secreted by the anterior pituitary gland 1,2. The mature form of the hGH is created from its 217 amino acid precursor after removal of the signal peptide (26 amino terminal residues). Owing to a substantial role of the hGH in various biological functions, it has a wide range of therapeutic applications like hormone therapy of hypopituitary dwarfism, skin burns, bone fractures, bleeding ulcers, HIV wasting syndrome and genetic disorders such as Turner’s and Down’s syndromes 3-5. Since this protein does not need post-translational modifications like glycosylation, Escherichia coli (E. coli) host is considered a prokaryotic powerhouse for industrial production of recombinant hGH 6-8. Notwithstanding different advantages of E. coli, including appropriate growth on low-cost media, fast biomass accumulation 6,9 and extensive knowledge of its genetics and physiology 10, there are some obstacles in acquiring considerable yields of correctly folded recombinant proteins. Failure of protein to rapidly reach a correct folding and disulfide bond formation leads to protein degradation by proteases and accumulation as inactive aggregates in the cytoplasm called inclusion bodies 6,11,12. Transferring the protein into the bacterial periplasmic region of E. coli or directly into the extracellular medium, using an appropriate signal peptide at the N-terminal of the protein, is an applicable approach to solve these problems 11. Efficient secretion of the recombinant protein is achieved using an optimal signal sequence, which is compatible with the secretory protein. Unfortunately, there is no general rule for selecting a suitable signal peptide to guarantee successful protein secretion. Therefore, trial-and-error experimental approach is universally applied to evaluate different signal peptides 11. Uptill now, various signal peptides have been evaluated for secretory production of hGH in E. coli, like OmpA 13,14, PhoA 15, pelB 14,16, LTB 17, npr 18, StII 19,20, DsbA 8, penicillinase 21 and natural hGH signal peptide 22.
In a continuing effort, the purpose of the current study was to investigate the influence of a bacterial signal peptide, L-asparaginase II, on the recombinant hGH protein secretion for the first time.

 

Materials and Methods :

Bacterial strain, plasmid, and culture growth conditions: The BL21 (DE3) E. coli host strain (Novagen, Germany) carrying T7 RNA polymerase gene and pET15b plasmid (Pasteur Institute, Iran) carrying T7 promoter were applied for cloning and expression steps. Cells were grown at 37oC in Luria-Bertani (LB) broth medium consisting of 10 g/L tryptone, 5 g/L yeast extract, and 5 g/L NaCl with shaking at 200 rpm. LB medium was supplemented with 100 μg/ml ampicillin for the growth of recombinant cells 23.

Design and construction of hGH secretory expression plasmid: Codon optimization of the hGH gene (Accession no. NM_000515) was performed by Codon Usage Wrangler server at http://www.mrc-lmb.cam.ac.uk/ms/methods/codon.html to apply the E. coli preferred codons and consequently, achieve high-level expression in this prokaryotic expression host.
In order to evaluate the effect of L-asparaginase II signal peptide on secretion of hGH, its nucleotide sequence from E. coli K-12 (Accession no. NC_ 000913.3) was obtained from database resources of the National Center for Biotechnology Information (NCBI) at www.ncbi.nlm.nih.gov and inserted in the N-terminal of the optimized hGH gene. The signal peptide cleavage site was predicted using SignalP server (version 4.0), which is based on neural network method 24. Codon-optimized synthetic hGH containing L-asparaginase II signal sequence was prepared from Generay Biotech (Shanghai, China), after embedding the NdeI and BamHI restriction endonuclease cut sites in the N- and C-terminal of the gene construct, respectively. Synthetic gene and the bacterial expression vector (pET-15b) were digested with NdeI and BamHI enzymes (Fermentas, EU), and the ligation step was performed using Rapid DNA Ligation Kit (Fermentas, EU). Competent E. coil BL21 (DE3) cells were transformed with ligation mixture using TransformAid TM Bacterial Transformation Kit (Fermentas, EU). After extraction of recombinant plasmids from transformed E. coli, PCR analysis was applied to check the proper insertion of the gene in the recombinant plasmid using T7 promoter-specific primers. The nucleotide sequence of recombinant insert was verified by automated DNA sequencing (SinaClon BioScience Co., Iran).

Induction of protein expression and periplasmic protein extraction: Transformed cells were cultivated in LB medium containing 100 μg/ml ampicillin at 37oC until a cell density of OD600=0.5-0.7 was achieved before induction. 0.1 mM IPTG (isopropyl- β -d-thiogalactopyrano-side), purchased from Fermentas, EU, was applied to induce protein expression and cultivation was continued at 23oC with shaking (200 rpm) overnight. The cell pellets were then harvested for periplasmic protein extraction by centrifugation at 4,000xg for 10 min and the supernatant was saved for analysis by SDS-PAGE. Periplasmic extraction of the protein was performed using three methods. According to the first method, osmotic shock, cell pellets were resuspended in sucrose/EDTA solution (20%). After incubation for 15 min at room temperature, the osmotically fragile cells were harvested by centrifugation at 4000xg for 10 min. The supernatant was removed and the cell pellet was resuspended in 1 ml cold distilled water (4°C) and incubated with gentle agitation at 4°C for 15 min. The resulting spheroplasts were harvested by centrifugation (13000xg at 4°C) and the supernatant, referred to as the periplasmic fraction, was recovered. The pellet was saved for analysis by SDS-PAGE. The second method was the osmotic shock in the presence of glycine, in which 1% glycine was added together with IPTG in the induction time. In the third procedure, combined Lysozyme/EDTA osmotic shock, the cell pellets were resuspended in sucrose/lysozyme solution containing 20% sucrose, 0.2 M Tris-HCl, 0.05% lysozyme, and 1 mM EDTA. The cell suspension was then incubated statically for 15 min at room temperature. Thereafter, equal volume of cold water was added and the suspension was incubated on ice for a further 15 min. Finally, the periplasmic fraction was recovered by centrifugation (13000xg at 4°C). The supernatant was removed and labeled as periplasmic fraction 25.

Expression analysis using SDS-PAGE: SDS-PAGE was performed in accordance with Laemmli 26 under reducing conditions, and protein bands were stained by Coomassie Brilliant Blue. Resolving and stacking gels were prepared in final concentrations of the 15 and 5%, respectively.

 

Results :

High-level cytoplasmic expression of recombinant hGH in E. coli frequently leads to aggregation of misfolded protein 8,27. To overcome this obstacle, an alternative expression approach can be applied to secrete the protein into the periplasmic space of the E. coli by inserting a signal sequence to the N-terminal of the hGH gene. It has been demonstrated that naturally secreted proteins like hGH, are universally considered to be appropriate candidates for the secretory production in heterologous systems 8. An optimal balance between all stages of the secretory cascade is required for a prosperous secretion process. One of the most important factors that has a considerable effect on all steps of secretion pathway and finally, on the yield of the recombinant secreted protein is the signal peptide 28. This short amino-terminal extension is then proteolytically removed by specific enzymes called signal peptidases during translocation to yield the correctly folded proteins with the formation of proper disulfide bonds 11,29. Due to the lack of universal rule for selecting an appropriate signal peptide, extensive trial-and-error research has been undertaken to express periplasmic recombinant proteins using different signal peptides 11. E. coli L-asparaginase II, a periplasmic enzyme with high affinity for L-asparagine, has been applied clinically for treatment of acute lymphoblastic leukemia 30. The ansB gene encoding this enzyme naturally comprises a secretory signal peptide of 22 amino terminal residues 31,32. A previous report indicated efficient secretory expression of recombinant hirudin III in E. coli exploiting L-asparaginase II signal sequence 33. Another study represents the improvement of recombinant cyclodextrin glucanotransferase secretion in the presence of mutant L-asparaginase II signal peptide 34. However, to our knowledge, there is no experimental study to evaluate this signal peptide in connection with hGH and its probable effect on appropriate secretion of this protein. Therefore, this objective was pursued in the present study.

Codon optimization and cloning: The nucleotide sequence encoding hGH was optimized by Codon Usage Wrangler supplier to be expressed with E. coli preferred codons. It has been demonstrated that substantial difference of eukaryotic and prokaryotic codon usages is one of the principal reasons for non-efficient expression of the foreign genes in E. coli 35,36. Previous reports also indicated that codon optimization for E. coli considerably enhances the mammalian protein expressions in this host 37,38.
The native sequence of L-asparaginase II signal peptide from E. coli K-12 was fused to the N terminal of the optimized hGH gene to assist in protein secretion across the cytoplasmic membrane. The cleavage site of this signal peptide was evaluated using SignalP server (version 4.0). Although several computational tools are available for prediction of the signal sequences and location of their cleavage site, SignalP ranks among the most precise and reliable tools, which provides high-throughput processing of protein sequences with accuracy of 87% 24. Figure 1 schematically demonstrates SignalP outputs, including various scores (C, S, Y, S-mean and D), and the cleavage site. Predicted D-score for L-asparaginase II signal peptide in connection with hGH gene was 0.61 (>0.5), which indicated an acceptable probability of acting as a signal peptide. The sequence was flanked by NdeI/BamHI restriction sites and successfully inserted into the corresponding sites on the expression vector pET15b to transform E. coli BL21 (DE3) cells. The structure of the synthetic gene is illustrated in figure 2. As shown in figure 3, PCR analysis using T7 promoter-specific primers verified the exact insertion of the gene in the recombinant plasmid. Automated DNA sequencing (SinaClon BioScience Co., Iran) also confirmed the correct insertion of the synthetic nucleotide sequence (data not shown).

Induction of protein expression and periplasmic protein extraction: It has been demonstrated that, reduction of culture temperature and IPTG concentration can be helpful to decrease the probability of protein aggregation in the form of inclusion bodies 11. Therefore, protein expression was induced by 0.1 mM IPTG at 23oC overnight. Different strategies have been employed to enhance the permeability of the outer membrane and promote extracellular secretion of recombinant proteins from E. coli. These procedures include mechanical methods (ultrasound), physical methods (osmotic shock, freezing and thawing), chemicals (addition of magnesium, calcium, EDTA, glycine, and Triton X-100), and enzymatic (lysozyme) treatments 11,39-41. In our study, three methods were applied to extract the periplasmic proteins, after protein expression. The first applied method for this purpose was the osmotic shock, as one of the most widely used methods of periplasmic release, which leads to the outer membrane of the cells to become permeable using hypertonic solutions 25,42,43. Previous studies reported that osmotic shock alone did not efficiently release the Streptomyces thermoviola-ceus (S. thermoviolaceus) α-amylase from the periplasm of E. coli 42. It has been also demonstrated that glycine supplementation may slightly disrupt peptidoglycan cross-linkages and cell membrane integrity 44. On the other hand, glycine supplemented to the medium retarded formation of inclusion bodies and enhanced the efficiency of extracellular production of recombinant proteins 44,45. Hence, as the second method in this study, osmotic shock was performed on cells, which were cultured in the glycine-supplemented medium. In 1996, French et al demonstrated that in comparison with other periplasmic extraction methods, combined lysozyme/osmotic shock was the most appropriate method for recovery of a recombinant protein from the periplasm of E. coli cells 25. Therefore, this method was also performed for periplasmic protein extraction in our study. The SDS-PAGE analysis of protein expression and extraction using these three methods are described in figure 4.

 

Discussion :

Based on the obtained results, no hGH protein band was observed in the supernatant and periplasmic fractions. In contrast, a major band of hGH protein was seen in the cytoplasmic fraction. Consequently, hGH protein is expressed as inclusion body.
Based on the obtained results, almost all of the recombinant hGH was expressed in the cytoplasm as inclusion body even in the presence of L-asparaginase II signal sequence, which indicates no efficient role for this signal peptide in the hGH protein secretion in E. coli BL21 (DE3). There are several interpretations that could explain why this problem arose. A prosperous secretion process generally needs an optimal balance between all stages of the secretory cascade. Inefficient secretion might be due to several factors like the structural characteristics of the signal peptide, ineffective cleavage of signal peptide from hGH protein, incompatibility between the mature protein and the signal peptide, inability to cross over the membrane and reach the periplasmic space or insufficient balance between the capacity of the export machinery and rate of protein synthesis 28. One of the most important concerns in secretory production is to understand the characteristics of the target protein that permits the secretion process to occur. This still remains unsolved and makes scientists apply the trial-and-error approach rather than rational evaluation 46. Taken together, although native and mutant L-asparaginase II signal peptide were efficient for recombinant hirudin III and cyclodextrin glucanotransferase secretion in E. coli, respectively 33,34, hGH secretion was not achieved in the presence of this signal peptide. The experiences gained from this study and other previous investigations, which reported low secretion yields, prompted us to apply bioinformatics tools as an alternative approach instead of time consuming, costly and labor-intensive trial-and-error strategy. Although our in silico study was conducted after the present study, its results were published prior to the current experimental report 47. Results of both studies were in agreement and indicated that L-asparaginase II signal peptide is not suitable for hGH secretion.

 

Conclusion :

Considering the significant advantages of secretory production of recombinant proteins, this study was designed to evaluate the possible role of a bacterial signal peptide, L-asparaginase II, in the hGH protein secretion in the E. coli host. The results of the current experimental study and our earlier in silico published study have demonstrated that successful secretion of hGH is not achieved using L-asparaginase II signal sequence. This study provides confirmatory evidence that although one signal peptide can be suitable for secretion of one protein, it is not necessarily appropriate for secretion of other proteins.

 

Acknowledgement :

This study was supported by a grant from the Research Council of Shiraz University of Medical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran.

 

Conflict of Interest :

The authors declare that they have no conflict of interest.

 

Ethical Approval :

This article does not contain any studies with human participants or animals performed by any of the authors.

 



<p>Figure 1. Sequence analysis of the L-asparaginase II signal peptide by SignalP 4.0 server.</p> <p>Signalp 4.0 determines five different scores including C, S, Y, S-mean and D scores. Cleavage sites and signal peptide positions are recognized by C and S-scores, respectively. A derivative of the C and S-scores called Y-score eventuates in more precise prediction of the cleavage sites than the raw C-score. S-mean is the average of S scores. D-score is also the average of the S-mean and Y-max which represents premiere discrimination between secretory and non-secretory proteins. There is a high possibility of being signal peptides for sequences with D-score &gt;0.5. The results for L-asparaginase II signal sequence in connection with hGH sequence are as follows: C-score: 0.838, S-score: 0.707, Y-score: 0.644, S-mean: 0.555, D-score: 0.61.</p>

Figure 1. Sequence analysis of the L-asparaginase II signal peptide by SignalP 4.0 server.

Signalp 4.0 determines five different scores including C, S, Y, S-mean and D scores. Cleavage sites and signal peptide positions are recognized by C and S-scores, respectively. A derivative of the C and S-scores called Y-score eventuates in more precise prediction of the cleavage sites than the raw C-score. S-mean is the average of S scores. D-score is also the average of the S-mean and Y-max which represents premiere discrimination between secretory and non-secretory proteins. There is a high possibility of being signal peptides for sequences with D-score >0.5. The results for L-asparaginase II signal sequence in connection with hGH sequence are as follows: C-score: 0.838, S-score: 0.707, Y-score: 0.644, S-mean: 0.555, D-score: 0.61.


<p>Figure 2. DNA sequence of the synthetic gene coding for hGH (Sense strand).</p> <p>Cut sites for <em>Nde</em>I (CATATG) and <em>Bam</em>HI (GGATCC) enzymes are shown in boldface on the N and C-terminal of the sense strand, respectively. The underlined residues also represent the L-asparaginase II signal sequence.</p>

Figure 2. DNA sequence of the synthetic gene coding for hGH (Sense strand).

Cut sites for NdeI (CATATG) and BamHI (GGATCC) enzymes are shown in boldface on the N and C-terminal of the sense strand, respectively. The underlined residues also represent the L-asparaginase II signal sequence.


<p>Figure 3. PCR confirmation of the recombinant plasmids using T7 specific primers. Lane M: DNA size marker, lane 1: Intact pET15b plasmid as a negative control, lanes 2, 3, 4: Recombinant plasmids.</p>

Figure 3. PCR confirmation of the recombinant plasmids using T7 specific primers. Lane M: DNA size marker, lane 1: Intact pET15b plasmid as a negative control, lanes 2, 3, 4: Recombinant plasmids.


<p>Figure 4. SDS-PAGE analysis of protein expression using three methods for periplasmic protein extraction consisting of A) osmotic shock, B) osmotic shock in the presence of glycine and C) combined Lysozyme/EDTA osmotic shock. Lane M: protein size marker, lanes 1, 2, 3<em>: </em>supernatant, periplasmic and cytoplasmic fractions of recombinant <em>E. coli</em> respectively. Lanes 4, 5, 6: supernatant, periplasmic and cytoplasmic fractions of <em>E. coli</em> containing intact pET15b as a negative control, respectively</p>

Figure 4. SDS-PAGE analysis of protein expression using three methods for periplasmic protein extraction consisting of A) osmotic shock, B) osmotic shock in the presence of glycine and C) combined Lysozyme/EDTA osmotic shock. Lane M: protein size marker, lanes 1, 2, 3: supernatant, periplasmic and cytoplasmic fractions of recombinant E. coli respectively. Lanes 4, 5, 6: supernatant, periplasmic and cytoplasmic fractions of E. coli containing intact pET15b as a negative control, respectively


References :
  1. Tritos NA, Mantzoros CS. Recombinant human growth hormone: old and novel uses. Am J Med 1998;105(1):44-57.   [PubMed]
  2. Isaksson OG, Edén S, Jansson JO. Mode of action of pituitary growth hormone on target cells. Annu Rev Physiol 1985;47:483-499.   [PubMed]
  3. Hahm MS, Chung BH. Secretory expression of human growth hormone inSaccharomyces cerevisiae using three different leader sequences. Biotechnol Bioprocess Eng 2001;6(4):306-309.
  4. Ozdamar TH, Sentürk B, Yilmaz OD, Calik G, Celik E, Calik P. Expression system for recombinant human growth hormone production from Bacillus subtilis. Biotechnol Prog 2009;25(1):75-84.   [PubMed]
  5. Roehr B. The many faces of human growth hormone. BETA 2003;15(4):12-16.   [PubMed]
  6. Baneyx F, Mujacic M. Recombinant protein folding and misfolding in Escherichia coli. Nat Biotechnol 2004;22(11):1399-1408.   [PubMed]
  7. Ramanan RN, Tik WB, Memari HR, Azaman SNA, Ling TC, Tey BT, et al. Effect of promoter strength and signal sequence on the periplasmic expression of human interferon-α2b in Escherichia coli. African J Biotechnol 2010;9(3):285-292.   [PubMed]
  8. Soares CR, Gomide FI, Ueda EK, Bartolini P. Periplasmic expression of human growth hormone via plasmid vectors containing the lambdaPL promoter: use of HPLC for product quantification. Protein Eng 2003;16(12):1131-1138.   [PubMed]
  9. Arora D, Khanna N. Method for increasing the yield of properly folded recombinant human gamma interferon from inclusion bodies. J Biotechnol 1996;52(2):127-133.   [PubMed]
  10. Marr AG. Growth rate of Escherichia coli. Microbiol Rev 1991;55(2):316-333.   [PubMed]
  11. Choi JH, Lee SY. Secretory and extracellular production of recombinant proteins using Escherichia coli. Appl Microbiol Biotechnol 2004;64(5):625-635.   [PubMed]
  12. Ventura S, Villaverde A. Protein quality in bacterial inclusion bodies. Trends Biotechnol 2006;24(4):179-185.   [PubMed]
  13. Becker GW, Hsiung HM. Expression, secretion and folding of human growth hormone in Escherichia coli. Purification and characterization. FEBS lett 1986;204(1):145-150.   [PubMed]
  14. Sockolosky JT, Szoka FC. Periplasmic production via the pET expression system of soluble, bioactive human growth hormone. Protein Expr Purif 2013;87(2):129-135.   [PubMed]
  15. Gray GL, Baldridge JS, McKeown KS, Heyneker HL, Chang CN. Periplasmic production of correctly processed human growth hormone in Escherichia coli: natural and bacterial signal sequences are interchangeable. Gene 1985;39(2-3):247-254.   [PubMed]
  16. Zomorodipour A. Construction of recombinant plasmids for periplasmic expression of human growth hormone in Escherichia coli under T7 and lac promoters. J Sci Islam Repub Iran 2003;14(4):311-316.   [Abstract]
  17. Ghorpade A, Garg LC. Efficient processing and export of human growth hormone by heat labile enterotoxin chain B signal sequence. FEBS lett 1993;330(1):61-65.   [PubMed]
  18. Uchida H, Naito N, Asada N, Wada M, Ikeda M, Kobayashi H, et al. Secretion of authentic 20-kDa human growth hormone (20K hGH) in Escherichia coli and properties of the purified product. J Biotechnol 1997;55(2):101-112.   [PubMed]
  19. Chang CN, Rey M, Bochner B, Heyneker H, Gray G. High-level secretion of human growth hormone by Escherichia coli. Gene 1987;55(2-3):189-196.   [PubMed]
  20. Chang JY, Pai RC, Bennett WF, Bochner BR. Periplasmic secretion of human growth hormone by Escherichia coli. Biochem Soc Trans 1989;17(2):335-337.   [PubMed]
  21. Kato C, Kobayashi T, Kudo T, Furusato T, Murakami Y, Tanaka T, et al. Construction of an excretion vector and extracellular production of human growth hormone from Escherichia coli. Gene 1987;54(2-3):197-202.   [PubMed]
  22. Dalmora S, de Oliveira JE, Affonso R, Gimbo E, Ribela MT, Bartolini P. Analysis of recombinant human growth hormone directly in osmotic shock fluids. J Chromatogr A 1997;782(2):199-210.   [PubMed]
  23. Ghasemi Y, Dabbagh F, Ghasemian A. Cloning of a fi-brinolytic enzyme (subtilisin) gene from Bacillus subtilis in Escherichia coli. Mol Biotechnol 2012;52(1):1-7.   [PubMed]
  24. Petersen TN, Brunak S, von Heijne G, Nielsen H. SignalP 4.0: discriminating signal peptides from transmembrane regions. Nat Methods 2011;8(10):785-786.   [PubMed]
  25. French C, Keshavarz-Moore E, Ward JM. Development of a simple method for the recovery of recombinant proteins from the Escherichia coli periplasm. Enzyme Microb Technol 1996;19(5):332-338.
  26. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970;227(5259):680-685.   [PubMed]
  27. Zamani M, Berenjian A, Hemmati S, Nezafat N, Ghoshoon MB, Dabbagh F, et al. Cloning, Expression, and Purification of a synthetic human growth hormone in Escherichia coli using response surface methodology. Mol Biotechnol 2015;57(3):241-250.   [PubMed]
  28. Baradaran A, Sieo CC, Foo HL, Illias RM, Yusoff K, Rahim RA. Cloning and in silico characterization of two signal peptides from Pediococcus pentosaceus and their function for the secretion of heterologous protein in Lactococcus lactis. Biotechnol Lett 2013;35(2):233-238.   [PubMed]
  29. Hiller K, Grote A, Scheer M, Münch R, Jahn D. PrediSi: prediction of signal peptides and their cleavage positions. Nucleic Acids Res 2004;32(Web Server issue):W375-379.   [PubMed]
  30. Ghasemi Y, Ebrahimnezhad A, Rasoul-Amini S, Zarrini Gh, Ghoshoon MB, Raee MJ. An optimized medium for screening of L-asparaginase production by Escherichia coli. Am J Biochem Biotechnol 2008;4(4):422-424.
  31. Jennings MP, Beacham IR. Analysis of the Escherichia coli gene encoding L-asparaginase II, ansB, and its regulation by cyclic AMP receptor and FNR proteins. J Bacteriol 1990;172(3):1491-1498.   [PubMed]
  32. Ghoshoon MB, Berenjian A, Hemmati Sh, Dabbagh F, Karimi Z, Negahdaripour M, et al. Extracellular pro-duction of recombinant L-Asparaginase II in Escherichia coli: Medium optimization using response surface methodology. Int J Pept Res Ther 2015;21(4):487-495.
  33. Tan S, Wu W, Liu J, Kong Y, Pu Y, Yuan R. Efficient expression and secretion of recombinant hirudin III in E. coli using the L-asparaginase II signal sequence. Protein Expr Purif 2002;25(3):430-436.   [PubMed]
  34. Ismail NF, Hamdan S, Mahadi NM, Murad AM, Rabu A, Bakar FD, et al. A mutant L-asparaginase II signal peptide improves the secretion of recombinant cyclodextrin glucanotransferase and the viability of Escherichia coli. Biotechnol Lett 2011;33(5):999-1005.   [PubMed]
  35. Makrides SC. Strategies for achieving high-level expression of genes in Escherichia coli. Microbiol Rev 1996;60(3):512-538.   [PubMed]
  36. Gurvich OL, Baranov PV, Gesteland RF, Atkins JF. Expression levels influence ribosomal frameshifting at the tandem rare arginine codons AGG_AGG and AGA_AGA in Escherichia coli. J Bacteriol 2005;187(12):4023-4032.   [PubMed]
  37. Li A, Kato Z, Ohnishi H, Hashimoto K, Matsukuma E, Omoya K, et al. Optimized gene synthesis and high expression of human interleukin-18. Protein Expr Purif 2003;32(1):110-118.   [PubMed]
  38. Li Y, Chen CX, von Specht BU, Hahn HP. Cloning and hemolysin-mediated secretory expression of a codon-optimized synthetic human interleukin-6 gene in Escherichia coli. Protein Expr Purif 2002;25(3):437-447.   [PubMed]
  39. Mergulhão FJ, Summers DK, Monteiro GA. Recom-binant protein secretion in Escherichia coli. Biotechnol Adv 2005;23(3):177-202.   [PubMed]
  40. Shokri A, Sandén AM, Larsson G. Cell and process design for targeting of recombinant protein into the culture medium of Escherichia coli. Appl Microbiol Biotechnol 2003;60(6):654-664.   [PubMed]
  41. Fu XY. Extracellular accumulation of recombinant protein by Escherichia coli in a defined medium. Appl Microbiol Biotechnol 2010;88(1):75-86.   [PubMed]
  42. Nossal NG, Heppel LA. The release of enzymes by osmotic shock from Escherichia coli in exponential phase. J Biol Chem 1966;241(13):3055-3062.   [PubMed]
  43. Neu HC, Heppel LA. The release of enzymes from Escherichia coli by osmotic shock and during the formation of spheroplasts. J Biol Chem 1965;240(9):3685-3692.   [PubMed]
  44. Yang J, Moyana T, MacKenzie S, Xia Q, Xiang J. One hundred seventy-fold increase in excretion of an FV fragment-tumor necrosis factor alpha fusion protein (sFV/TNF-alpha) from Escherichia coli caused by the synergistic effects of glycine and triton X-100. Appl Environ Microbiol 1998;64(8):2869-2874.   [PubMed]
  45. Jang Kh, Seo JW, Song KB, Kim Ch, Rhee SK. Extracellular secretion of levansucrase from Zymomonas mobilis in Escherichia coli. Bioprocess Eng 1999;21(5):453-458.
  46. Low KO, Muhammad Mahadi N, Md Illias R. Optimisation of signal peptide for recombinant protein secretion in bacterial hosts. Appl Microbiol Biotechnol 2013;97(9):3811-3826.   [PubMed]
  47. Zamani M, Nezafat N, Negahdaripour M, Dabbagh F, Ghasemi Y. In silico evaluation of different signal peptides for the secretory production of human growth hormone in E. coli. Int J Peptide Res Ther 2015;21(3):261-268.