Abstract
This study shows expression of recombinant ovine growth hormone (roGH) and targeting to the inner membrane using signal sequence, DsbA, in Escherichia coli (E. coli) cell. Factors such as temperature, IPTG induction, and expression conditions were studied and show diverse optical density with different media compositions. The optimum expression level of roGH in terrific broth medium was at 25 °C on induction with 20 μM IPTG in early logarithmic phase. SDS-PAGE analysis of expression and subcellular fractions of recombinant constructs revealed the translocation of roGH to the inner membrane of E. coli with DsbA signal sequence at the N terminus of roGH. The protein was easily solubilized by 40 % acetonitrile with ~90 % purity and was identified by Western blot, and analysis on MALDI-TOF/TOF confirmed a size of 21,059 Da. Relatively high soluble protein yield of 65.3 mg/L of roGH was obtained. The biological function of roGH was confirmed by HeLa cell line proliferation. This is the first study describing achievement of biologically active soluble roGH targeted to the inner membrane of E. coli and rapid purification with high yield.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Bauman DE (1999) Bovine somatotropin and lactation: from basic science to commercial application. Domest Anim Endocrinol 17:101–116. doi:10.1016/S0739-7240(99)00028-4
Becker GW, Hsiung HM (1986) Expression, secretion and folding of human growth hormone in E. coli. Purification and characterization. FEBS Lett 204:145–150
Becker PB, Gloss B, Schmid W, Strahle U, Schutz G (1986) In vivo protein–DNA interactions in a glucocorticoid response element require the presence of the hormone. Nature 324:686–688
Bonneau M, Laarveld B, Aumaitre AL (1999) Biotechnology in animal nutrition, physiology and health. Livest Prod Sci (Korea) 59:223–241
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254
de Oliveira JE, Soares CR, Peroni CN, Gimbo E, Camargo IM, Morganti L, Bellini MH, Affonso R, Arkaten RR, Bartolini P, Ribela MT (1999) High-yield purification of biosynthetic human growth hormone secreted in E. coli periplasmic space. J Chromatogr 852:441–450
Foster AR, Houlihan DF, Gray C, Medale F, Fauconneau B, Kaushiki SJ, Le Bail PY (1990) The effects of ovine growth hormone on protein turnover in rainbow trout. Gen Comp Endocrinol 81:111–120
Goder V, Spiess M (2003) Molecular mechanism of signal sequence orientation in the endoplasmic reticulum. EMBO J 22:3645–3653. doi:10.1093/emboj/cdg361
Huber D, Boyd D, Xia Y, Olma MH, Gerstein M, Beckwith J (2005) Use of thioredoxin as a reporter to identify a subset of E. coli signal sequences that promote signal recognition particle-dependent translocation. J Bacteriol 187:2983–2991
Kaderbhai NN, Harding V, Kaderbhai MA (2008) Signal peptidase I-mediated processing of an engineered mammalian cytochrome b5 precursor is an exocytoplasmic post-translocational event in E. coli. Mol Membr Biol 25:388–399. doi:10.1080/09687680802154799
Khan RH, Rao KB, Eshwari AN, Totey SM, Panda AK (1998) Solubilization of recombinant ovine growth hormone with retention of native-like secondary structure and its refolding from the inclusion bodies of E. coli. Biotechnol Prog 14:722–728
Klein BK, Hill SR, Devine CS, Rowold E, Smith CE, Galosy S, Olins PO (1991) Secretion of active bovine somatotropin in E. coli. Biotechnology 9:869–872
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685. doi:10.1038/227680a0
Paladini AC, Pena C, Poskus E (1983) Molecular biology of growth hormone. CRC Crit Rev Biochem 15:25–56
Patra AK, Mukhopadhyay R, Mukhija R, Krishnan A, Garg LC, Panda AK (2000) Optimization of inclusion body solubilization and renaturation of recombinant human growth hormone from E. coli. Protein Expr Purif 18:182–192
Porollo VCA, Adamczak R, Meller J (2004) POLYVIEW: a flexible visualization tool for structural and functional annotations of proteins. Bioinformatics 20:2460–2462. doi:10.1093/bioinformatics/bth248
Puertas JM, Betton JM (2009) Engineering an efficient secretion of leech carboxypeptidase inhibitor in E. coli. Microb Cell Factories 8:57. doi:10.1186/1475-2859-8-57
Rao BCA, Garg LC, Panda AK, Totey SM (1997) High level expression of ovine growth hormone in E. coli: single step purification and characterization. Protein Expr Purif 11:201–208
Sadaf S, Khan MA, Wilson DB, Akhtar MW (2007) Molecular cloning, characterization, and expression studies of water buffalo (Bubalus bubalis) somatotropin. Biochem Mosc 72:162–169. doi:10.1134/S0006297907020058
Schierle CF, Berkmen M, Huber D, Kumamoto C, Boyd D, Beckwith J (2003) The DsbA signal sequence directs efficient, co-translational export of passenger proteins to the E. coli periplasm via the signal recognition particle pathway. J Bacteriol 185:5706–5713
Seeburg PH, Sias S, Adelman J, de Boer HA, Hayflick J, Jhurani P, Goeddel DV, Heyneker HL (1983) Efficient bacterial expression of bovine and porcine growth hormones. DNA 2:37–45
Soares CRJ, Gomide FIC, Ueda EKM, Bartolini P (2003) Periplasmic expression of human growth hormone via plasmid vectors containing the kPL promoter: use of HPLC for product quantification. Protein Eng 16:1131–1138
Teresa M, Ribela CP, Camargo IM, Oliveira JE, Bartolini P (2000) Single-step purification of recombinant human growth hormone (hGH) directly from bacterial osmotic shock fluids, for the purpose of (125) I-hGH preparation. Protein Expr Purif 2:115–120
Waddell WJ (1956) A simple UV spectrophotometric method for the determination of protein. J Lab Clin Med 48:311–314
Walli TK, Samanta AK (2000) Response of dairy animals to bovine somatotropin administration—a review. Indian Dairyman 52:7–20
Wallis OC, Sami AJ, Wallis M (1995) The effect of changes in nucleotide sequence coding for the N-terminus on expression levels of ovine growth hormone variants in E. coli. Biochim Biophys Acta 26:360–368
Zamani M, Nezafat N, Negahdaripour M, Dabbagh F, Ghasemi Y (2015) In silico evaluation of different signal peptides for the secretory production of human growth hormone in E. coli. Int J Pept Prot Res 21:1–8. doi:10.1007/s10989-015-9454-z
Conflict of Interest
The authors declare that they have no competing interests.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Durrani, F.G., Gul, R., Sadaf, S. et al. Expression and rapid purification of recombinant biologically active ovine growth hormone with DsbA targeting to Escherichia coli inner membrane. Appl Microbiol Biotechnol 99, 6791–6801 (2015). https://doi.org/10.1007/s00253-015-6751-6
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00253-015-6751-6