Twin arginine translocation system in secretory expression of recombinant human growth hormone

Mohammad Reza Bagherinejad, Hamid Mir-Mohammad Sadeghi, Daryoush Abedi, C. Perry Chou, Fatemeh Moazen, Mohammad Rabbani

Abstract


Recombinant protein production in E. coli has several advantages over other expression systems. Misfolding, inclusion body formation, and lack of eukaryotic post translational modification are the most disadvantages of this system. Exporting of correctly folded proteins to the outside of reductive cytoplasmic environment through twin-arginine system could help to pass these limiting steps. Two signal sequences, TorA and SufI are used at N-terminal of human growth hormone (hGH) bearing DsbA gene sequence at C-terminal to enhance folding. The synthetic cassettes including the signal sequence, hGH and DsbA were transformed into E. coli BL21 (DE3) to study the effect of signal sequence and DsbA chaperone on translocation and folding of the protein. The results confirmed using signal sequence at N-terminal of targeted protein and coexpression with DsbA could transport proteins to the periplasmic space and culture media compared to control groups. Although there is no protein band of somatropin in SDS-Page of culture media samples when using SufI as signaling sequence, the study demonstrated TorA signal sequence could transport the target protein to the culture media. However, there was a considerable amount of hGH in periplasmic space when using SufI compared to control.


Keywords


Signal sequence; TorA; SufI; DsbA; Growth hormone; Twin arginine translocation

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References


Rosano GL, Ceccarelli EA. Recombinant protein expression in Escherichia coli: advances and challenges. Front Microbiol. 2014;5:172.

Assenberg R, Wan PT, Geisse S, Mayr LM. Advances in recombinant protein expression for use in pharmaceutical research. Curr Opin Struct Biol. 2013;23(3):393–402.

Ami D, Natalello A, Lotti M, Doglia SM. Why and how protein aggregation has to be studied in vivo. Microb Cell Fact. 2013;12:17.

Goltermann L, Good L, Bentin T. Chaperonins fight aminoglycoside-induced protein misfolding and promote short-term tolerance in Escherichia coli. J Biol Chem. 2013;288(15):10483–10489.

Mergulhão FJ, Summers DK, Monteiro GA. Recombinant protein secretion in Escherichia coli. Biotechnol Adv. 2005;23(3):177–202.

Albiniak AM, Matos CF, Branston SD, Freedman RB, Keshavarz-Moore E, Robinson C. High-level secretion of a recombinant protein to the culture medium with a Bacillus subtilis twin-arginine translocation system in Escherichia coli. FEBS J. 2013;280(16):3810–3821.

Kim YE, Hipp MS, Bracher A, Hayer-Hartl M, Hartl FU. Molecular chaperone functions in protein folding and proteostasis. Annu Rev Biochem. 2013;82:323–355.

Saibil H. Chaperone machines for protein folding, unfolding and disaggregation. Nat Rev Mol Cell Biol. 2013;14(10):630–642.

Klatt S, Konthur Z. Secretory signal peptide modification for optimized antibody-fragment expression-secretion in Leishmania tarentolae. Microb Cell Fact. 2012;11(1):97.

Palmer T, C. Berks B. The twine-arginine translocation (Tat) protein export pathway. Nature Rev Microbiol. 2012;10:483–496.

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(3):e89038.

Sockolosky JT, Szoka FC. Periplasmic production via the pET expression system of soluble, bioactive human growth hormone. Protein Expres Purifi. 2013;87(2):129–135.

Sambrook J, Russell DW. Molecular cloning: A laboratory manual, Vol. 3. 3rd edition. Cold Spring Harbor, NewYork, Cold Spring Harbor Laboratory Press; 2001.

Lin YH, Hsiao HC, Chou CP. Strain improvement to enhance the production of recombinant penicillin acylase in high-cell-density Escherichia coli cultures. Biotechnol Prog. 2002;18(6):1458–1461.

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(4):349–361.

Daleke MH, Ummels R, Bawono P, Heringa J, Vandenbroucke-Grauls CM, Luirink J, et al. General secretion signal for the mycobacterial type VII secretion pathway. Proc Natl Acad Sci U S A. 2012;109(28):11342–11347.

Chou CP, Tseng JH, Kuo BY, Lai KM, Lin MI, Lin HK. Effect of SecB chaperone on production of periplasmic penicillin acylase in Escherichia coli. Biotechnol Prog. 1999;15(3):439–445.

Berks BC, Lea SM, Stansfeld PJ. Structural biology of Tat protein transport. Cur Opin Struct Biol. 2014;27:32–37.

Palmer T, Berks BC. The twin-arginine translocation (Tat) protein export pathway. Nat Rev Microbiol. 2012;10(7):483–496.

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(2): 281–290.

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(10):2533–2542.

Rodriguez F, Rouse SL, Tait CE, Harmer J, De Riso A, Timmel CR, et al. Structural model for the protein-translocating element of the twin-arginine transport system. Proc Natl Acad Sci U S A. 2013;110(12):E1092-E1101.

Alanen HI, Walker KL, Lourdes Velez Suberbie M, Matos CF, Bönisch S, Freedman RB, et al. Efficient export of human growth hormone, interferon α2b and antibody fragments to the periplasm by the Escherichia coli Tat pathway in the absence of prior disulfide bond formation. Biochim Biophys Acta. 2015;1853(3):756–763.

Kim MJ, Park HS, Seo KH, Yang HJ, Kim SK, Choi JH, et al. Complete solubilization and purification of recombinant human growth hormone produced in Escherichia coli. PLoS One. 2013;8(2):e56168.

Levarski Z, Šoltýsová A, Krahulec J, Stuchlík S, Turňa J. High-level expression and purification of recombinant human growth hormone produced in soluble form in Escherichia coli. Protein Expr Purif. 2014;100:40–47.

Rezaei M, Zarkesh-Esfahani SH. Optimization of production of recombinant human growth hormone in Escherichia coli. J Res Med Sci. 2012;17(7):681–685.

Choi JH, Lee SY. Secretory and extracellular production of recombinant proteins using Escherichia coli. Appl Microbiol Biotechnol. 2004;64(5):625–635.

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.

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(3):261–268.

Kiany J, Zomorodipour A, Ahmadzadeh Raji M, Sanati MH. Construction of recombinant plasmids for periplasmic expression of human growth hormone in Escherichia coli under T7 and LAC promoters. J Sci, I.R. Iran. 2003; 14(4):311-316.


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