Effect of Twine-arginine Translocation-signaling Fusion System and Chaperones Co-expression on Secretory Expression of Somatropin

Bagherinejad, Mohammad Reza; Sadeghi, Hamid Mir-Mohammad; Abedi, Daryoush; Moazen, Fateme; Rabbani, Mohammad

Effect of Twine-arginine Translocation-signaling Fusion System and Chaperones Co-expression on Secretory Expression of Somatropin

Document Type : Original Article

Authors

Department of Pharmaceutical Biotechnology, School of Pharmacy, Isfahan University of Medical Sciences, Isfahan, Iran

Abstract

Background: Twine-arginine translocation (TAT) system is one of the exporting systems in Escherichia coli which could transport fully/semi-correctly folded proteins outside the reductive cytoplasmic space. In combination with co-expression with a chaperone system, the correctly folded proteins could be transported to oxidative periplasmic space and culture media to pass the main limitations in E. coli expression system such as misfolding and inclusion body formation. Materials and Methods: To study the effectiveness of signaling sequences and chaperone co-expression on the translocation of expressed protein, somatropin was selected as the target. Two common signal sequences in TAT system (TorA and SufI) were added at the N-terminal of somatropin and the cassettes were co-expressed in E. coli BL21 (DE3) by a chaperone team including DnaK/J-GrpeE. Results: The expression pattern studies including Western blotting and sodium dodecyl sulfate polyacrylamide gel electrophoresis confirmed that somatropin is expressed in two cassettes. However, the pattern was different for two signaling sequences. Conclusion: The results confirmed that the approach of using TAT-signaling sequences and co-expression with the chaperone team could enhance translocation of protein to periplasmic space and culture media compared to control groups. Western blotting results showed that the signal sequence TorA could transport more expressed proteins to the periplasmic space and culture media in comparison with SufI. However, there was a considerable amount of human growth hormone in the cytoplasm which could not be transported outside the cytoplasmic space.

Keywords

References

1.	Assenberg R, Wan PT, Geisse S, Mayr LM. Advances in recombinant protein expression for use in pharmaceutical research. Curr Opin Struct Biol 2013;23:393-402. [PUBMED]
2.	Rosano GL, Ceccarelli EA. Recombinant protein expression in Escherichia coli: Advances and challenges. Front Microbiol 2014;5:172. [PUBMED]
3.	Pérez-Rodríguez R, Fisher AC, Perlmutter JD, Hicks MG, Chanal A, Santini CL, et al. An essential role for the DnaK molecular chaperone in stabilizing over-expressed substrate proteins of the bacterial twin-arginine translocation pathway. J Mol Biol 2007;367:715-30.
4.	Calloni G, Chen T, Schermann SM, Chang HC, Genevaux P, Agostini F, et al. DnaK functions as a central hub in the E. coli chaperone network. Cell Rep 2012;1:251-64. [PUBMED]
5.	Klatt S, Konthur Z. Secretory signal peptide modification for optimized antibody-fragment expression-secretion in Leishmania tarentolae. Microb Cell Fact 2012;11:97. [PUBMED]
6.	Silverman JM, Brunet YR, Cascales E, Mougous JD. Structure and regulation of the type VI secretion system. Annu Rev Microbiol 2012;66:453-72. [PUBMED]
7.	Berks BC, Lea SM, Stansfeld PJ. Structural biology of Tat protein transport. Curr Opin Struct Biol 2014;27:32-7. [PUBMED]
8.	Kim MJ, Park HS, Seo KH, Yang HJ, Kim SK, Choi JH. Complete solubilization and purification of recombinant human growth hormone produced in Escherichia coli. PLoS One 2013;8:e56168. [PUBMED]
9.	Levarski Z, Šoltýsová A, Krahulec J, Stuchlík S, Turna J. High-level expression and purification of recombinant human growth hormone produced in soluble form in Escherichia coli. Protein Expr Purif 2014;100:40-7.
10.	Nguyen MT, Koo BK, Thi Vu TT, Song JA, Chong SH, Jeong B, et al. Prokaryotic soluble overexpression and purification of bioactive human growth hormone by fusion to thioredoxin, maltose binding protein, and protein disulfide isomerase. PLoS One 2014;9:e89038. [PUBMED]
11.	Palmer T, Berks BC. The twin-arginine translocation (Tat) protein export pathway. Nat Rev Microbiol 2012;10:483-96. [PUBMED]
12.	Sambrook J, Russell DW. Molecular Cloning: A Laboratory Manual. 3^rd ed. Vol. 3. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press; 2001.
13.	Choi JH, Lee SY. Secretory and extracellular production of recombinant proteins using Escherichia coli. Appl Microbiol Biotechnol 2004;64:625-35. [PUBMED]
14.	Georgiou G, Segatori L. Preparative expression of secreted proteins in bacteria: Status report and future prospects. Curr Opin Biotechnol 2005;16:538-45. [PUBMED]
15.	Mergulhão FJ, Summers DK, Monteiro GA. Recombinant protein secretion in Escherichia coli. Biotechnol Adv 2005;23:177-202.
16.	Low KO, Muhammad Mahadi N, Md Illias R. Optimisation of signal peptide for recombinant protein secretion in bacterial hosts. Appl Microbiol Biotechnol 2013;97:3811-26. [PUBMED]
17.	Kudva R, Denks K, Kuhn P, Vogt A, Müller M, Koch HG. Protein translocation across the inner membrane of Gram-negative bacteria: The Sec and Tat dependent protein transport pathways. Res Microbiol 2013;164:505-34.
18.	Narayanan N, Khan M, Chou CP. Enhancing functional expression of heterologous lipase B in Escherichia coli by extracellular secretion. J Ind Microbiol Biotechnol 2010;37:349-61. [PUBMED]
19.	Matos CF, Branston SD, Albiniak A, Dhanoya A, Freedman RB, Keshavarz-Moore E, et al. High-yield export of a native heterologous protein to the periplasm by the tat translocation pathway in Escherichia coli. Biotechnol Bioeng 2012;109:2533-42. [PUBMED]
20.	Patel R, Smith SM, Robinson C. Protein transport by the bacterial Tat pathway. Biochim Biophys Acta 2014;1843:1620-8. [PUBMED]
21.	Matos CF, Robinson C, Alanen HI, Prus P, Uchida Y, Ruddock LW, et al. Efficient export of prefolded, disulfide-bonded recombinant proteins to the periplasm by the Tat pathway in Escherichia coli CyDisCo strains. Biotechnol Prog 2014;30:281-90. [PUBMED]
22.	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 Pept Res Ther 2015;21:261-8. Available from: https://link.springer.com/article/10.1007/s10989-015-9454-z. [Last cited on 2016 Mar 08].
23.	Bagherinejad MR, Sadeghi HM, Abedi D, Chou CP, Moazen F, Rabbani M. Twin arginine translocation system in secretory expression of recombinant human growth hormone. Res Pharm Sci 2016;11:461-9. [PUBMED]

Advanced Biomedical Research