Abstract
A study of the Mut+ phenotype for the expression of recombinant hepatitis B surface antigen (HBsAg) in Pichia pastoris strain GS115 using the pPIC3.5K vector with a two-phase fed-batch protocol in a shake flask system is described. Expression levels of HBsAg protein of 6.74 g L−1 Dry Cell Weight (DCW) and 26.07 mg L−1 of HBsAg concentration were achieved 48 h from the induction point which added to a 120 h reduction in production period in comparison with MutS expression (168 h). The use of the pPIC3.5K-HBsAg plasmid in the Mut+ phenotype enhanced the expression of HBsAg by a nearly 13 times higher volumetric productivity in the first 24 h and 35 times higher at peak production concentration. Comparison of AOX expression cassettes relative to the HBsAg gene in the role of mRNA secondary structure during translation initiation revealed that HBsAg possesses lower folding stability with AOX1 Mut+ phenotype. The results from this study demonstrated that expression of HBsAg with Mut+ AOX1 promoter is adequate as an alternative for the production of HBsAg. In addition, the AOX promoter of the Mut+ phenotype was observed to be better suited for HBsAg expression, which correlates with the ease of translation initiation under shake flask conditions.
[1] Liaw Y.F., Antiviral therapy of chronic hepatitis B: opportunities and challenges in Asia, J. Hepatol., 2009, 51(2), 403–410. http://dx.doi.org/10.1016/j.jhep.2009.04.00310.1016/j.jhep.2009.04.003Search in Google Scholar
[2] Yap S.F., Chronic hepatitis B infection in Malaysians, Malay J. Pathol., 1994, 16(1), 3–4. Search in Google Scholar
[3] WHO. Hepatitis B. Fact sheet. 2013. http://www.who.int/mediacentre/factsheets/fs204/en/index.html. Accessed December 2013. Search in Google Scholar
[4] Yu A.S., Cheung R.C., Keeffe E.B., Hepatitis B vaccines, Clin. Liver Dis., 2004, 8(2), 283. http://dx.doi.org/10.1016/j.cld.2004.02.01010.1016/j.cld.2004.02.010Search in Google Scholar
[5] Bruss V., Ganem D., Mutational analysis of hepatitis B surface antigen particle assembly and secretion, J. Virol., 1991, 65(7), 3813–3820. 10.1128/jvi.65.7.3813-3820.1991Search in Google Scholar
[6] Kouichi S., Kenichi T., Shin-ichiro N., Hiroshi M., Kenichi M., Production of hepatitis B virion-like particles in yeast, Gene, 1991, 106(2), 143–149. http://dx.doi.org/10.1016/0378-1119(91)90193-F10.1016/0378-1119(91)90193-FSearch in Google Scholar
[7] Zhang W.U.Y., Ying W., Dao Guo L., Yan T., Wei Z., A mimotope of pre-S2 region of surface antigen of viral hepatitis B screened by phage display, Cell Res., 2001, 11(3), 203–208. http://dx.doi.org/10.1038/sj.cr.729008710.1038/sj.cr.7290087Search in Google Scholar
[8] Ottone S., Nguyen X., Bazin J., Bérard C., Jimenez S., Letourneur O., Expression of hepatitis B surface antigen major subtypes in Pichia pastoris and purification for in vitro diagnosis, Protein Expression Purif., 2007, 56(2), 177–188. http://dx.doi.org/10.1016/j.pep.2007.07.00810.1016/j.pep.2007.07.008Search in Google Scholar
[9] Tan W.S., Tan G.H., Yusoff K., Seow H.F., A phagedisplayed cyclic peptide that interacts tightly with the immunodominant region of hepatitis B surface antigen, J. Clin. Virol., 2005, 34(1), 35–41. http://dx.doi.org/10.1016/j.jcv.2005.01.00710.1016/j.jcv.2005.01.007Search in Google Scholar
[10] Cereghino J.L., Cregg J.M., Heterologous protein expression in the methylotrophic yeast Pichia pastoris, FEMS Microbiol. Rev., 2006, 24(1), 45–66. http://dx.doi.org/10.1111/j.1574-6976.2000.tb00532.x10.1111/j.1574-6976.2000.tb00532.xSearch in Google Scholar
[11] Sudbery P.E., The expression of recombinant proteins in yeasts, Curr. Opin. Biotechnol., 1996, 7(5), 517–524. http://dx.doi.org/10.1016/S0958-1669(96)80055-310.1016/S0958-1669(96)80055-3Search in Google Scholar
[12] Zhang W., Inan M., Meagher M.M., Fermentation strategies for recombinant protein expression in the methylotrophic yeast Pichia pastoris, Biotechnol. Bioprocess Eng., 2000, 5(4), 275–287. http://dx.doi.org/10.1007/BF0294218410.1007/BF02942184Search in Google Scholar
[13] Vassileva A., Chugh D.A., Swaminathan S., Khanna N., Expression of hepatitis B surface antigen in the methylotrophic yeast Pichia pastoris using the GAP promoter, J. Biotechnol., 2001, 88(1), 21–35. http://dx.doi.org/10.1016/S0168-1656(01)00254-110.1016/S0168-1656(01)00254-1Search in Google Scholar
[14] Krainer F.W., Dietzsch C., Hajek T., Herwig C., Spadiut O., Glieder A., Recombinant protein expression in Pichia pastoris strains with an engineered methanol utilization pathway, Microb. Cell Fact., 2012, 11(1), 22. http://dx.doi.org/10.1186/1475-2859-11-2210.1186/1475-2859-11-22Search in Google Scholar
[15] Macauley-Patrick S., Fazenda M.L., McNeil B., Harvey L.M., Heterologous protein production using the Pichia pastoris expression system, Yeast, 2005, 22(4), 249–270. http://dx.doi.org/10.1002/yea.120810.1002/yea.1208Search in Google Scholar
[16] Cregg J., Tschopp J., Stillman C., Siegel R., Akong M., Craig W., et al., High-Level expression and efficient assembly of hepatitis B surface antigen in the methylotrophic yeast, Pichia pastoris, Nat. Biotechnol., 1987, 5(5), 479–485. http://dx.doi.org/10.1038/nbt0587-47910.1038/nbt0587-479Search in Google Scholar
[17] Charoenrat T., Ketudat-Cairns M., Stendahl-Andersen H., Jahic M., Enfors S.O., Oxygenlimited fed-batch process: an alternative control for Pichia pastoris recombinant protein processes, Bioprocess Biosyst. Eng., 2005, 27(6), 399–406. http://dx.doi.org/10.1007/s00449-005-0005-410.1007/s00449-005-0005-4Search in Google Scholar
[18] Gu M.B., Park M.H., Kim D.I., Growth rate control in fed-batch cultures of recombinant Saccharomyces cerevisiae producing hepatitis B surface antigen (HBsAg), Appl. Microbiol. Biotechnol., 1991, 35(1), 46–50. http://dx.doi.org/10.1007/BF0018063410.1007/BF00180634Search in Google Scholar
[19] Morvarid A., Zeenathul N., Tam Y., Zuridah H., Mohd-azmi M., Azizon B., Effect of glycerol feed in methanol induction phase for hepatitis B surface antigen expression in Pichia pastoris strain KM71, Pertanika J. Sci. & Technol., 2012, 20(1), 31–42. Search in Google Scholar
[20] Gurramkonda C., Adnan A., Gäbel T., Lünsdorf H., Ross A., Nemani S.K., et al., Simple highcell density fed-batch technique for high-level recombinant protein production with Pichia pastoris: Application to intracellular production of hepatitis B surface antigen, Microb. Cell Fact., 2009, 8(1), 13. http://dx.doi.org/10.1186/1475-2859-8-1310.1186/1475-2859-8-13Search in Google Scholar
[21] Zhang W., Bevins M.A., Plantz B.A., Smith L.A., Meagher M.M., Modeling Pichia pastoris growth on methanol and optimizing the production of a recombinant protein, the heavy-chain fragment C of botulinum neurotoxin, serotype A, Pap. Biotechnol., 2000, 18. 10.1002/1097-0290(20001005)70:1<1::AID-BIT1>3.0.CO;2-YSearch in Google Scholar
[22] Cregg J.M., Barringer K., Hessler A., Madden K., Pichia pastoris as a host system for transformations, Mol. Cell. Biol., 1985, 5(12), 3376–3385. 10.1128/mcb.5.12.3376-3385.1985Search in Google Scholar
[23] Romanos M., Advances in the use of Pichia pastoris for high-level gene expression, Curr. Opin. Biotechnol., 1995, 6(5), 527–533. http://dx.doi.org/10.1016/0958-1669(95)80087-510.1016/0958-1669(95)80087-5Search in Google Scholar
[24] Linder S., Schliwa M., Kube-Granderath E., Direct PCR screening of Pichia pastoris clones, BioTechniques, 1996, 20(6), 980. 10.2144/96206bm08Search in Google Scholar
[25] Tam Y.J., Allaudin Z.N., Lila M.A., Bahaman A.R., Tan J.S., Rezaei M.A., Enhanced cell disruption strategy in the release of recombinant hepatitis B surface antigen from Pichia pastoris using response surface methodology, BMC Biotechnol., 2012, 12(1), 70. http://dx.doi.org/10.1186/1472-6750-12-7010.1186/1472-6750-12-70Search in Google Scholar
[26] Kruger N.J., The Bradford Method for Protein Quantitation, In: Walker JM, ed. The Protein Protocols Handbook, Vol: Humana Press, 2002, 15–21. http://dx.doi.org/10.1385/1-59259-169-8:1510.1385/1-59259-169-8:15Search in Google Scholar
[27] Trentmann O., Khatri N.K., Hoffmann F., Reduced oxygen supply increases process stability and product yield with recombinant Pichia pastoris, Biotechnol. Prog., 2008, 20(6), 1766–1775. http://dx.doi.org/10.1021/bp049711h10.1021/bp049711hSearch in Google Scholar
[28] Chauhan A., Arora D., Khanna N., A novel feeding strategy for enhanced protein production by fedbatch fermentation in recombinant Pichia pastoris, Process Biochem. (Amsterdam, Neth.), 1999, 34(2), 139–145. 10.1016/S0032-9592(98)00080-6Search in Google Scholar
[29] Sunil Kumar G., Ganapathi T., Revathi C., Prasad K., Bapat V., Expression of hepatitis B surface antigen in tobacco cell suspension cultures, Protein Expression Purif., 2003, 32(1), 10–17. http://dx.doi.org/10.1016/j.pep.2003.07.00410.1016/j.pep.2003.07.004Search in Google Scholar
[30] Smith M.L., Mason H.S., Shuler M.L., Hepatitis B surface antigen (HbsAg) expression in plant cell culture: kinetics of antigen accumulation in batch culture and its intracellular form, Biotechnol. Bioeng., 2002, 80(7), 812–822. http://dx.doi.org/10.1002/bit.1044410.1002/bit.10444Search in Google Scholar
[31] Cos O., Serrano A., Montesinos J.L., Ferrer P., Cregg J.M., Valero F., Combined effect of the methanol utilization (Mut) phenotype and gene dosage on recombinant protein production in Pichia pastoris fed-batch cultures, J. Biotechnol., 2005, 116(4), 321–335. http://dx.doi.org/10.1016/j.jbiotec.2004.12.01010.1016/j.jbiotec.2004.12.010Search in Google Scholar
[32] Kozak M., Initiation of translation in prokaryotes and eukaryotes, Gene, 1999, 234(2), 187–208. http://dx.doi.org/10.1016/S0378-1119(99)00210-310.1016/S0378-1119(99)00210-3Search in Google Scholar
[33] Kozak M., Downstream secondary structure facilitates recognition of initiator codons by eukaryotic ribosomes, Proc. Natl. Acad. Sci. U. S. A., 1990, 87(21), 8301–8305. http://dx.doi.org/10.1073/pnas.87.21.830110.1073/pnas.87.21.8301Search in Google Scholar PubMed PubMed Central
[34] Makrides S.C., Strategies for achieving high-level expression of genes in Escherichia coli, Microbiol. Rev., 1996, 60(3), 512–538. 10.1128/mr.60.3.512-538.1996Search in Google Scholar
[35] Kozak M., Influences of mRNA secondary structure on initiation by eukaryotic ribosomes, Proc. Natl. Acad. Sci. U. S. A., 1986, 83(9), 2850–2854. http://dx.doi.org/10.1073/pnas.83.9.285010.1073/pnas.83.9.2850Search in Google Scholar
[36] Chang J.T., Green C., Wolf R., Inhibition of translation initiation on Escherichia coli gnd mRNA by formation of a long-range secondary structure involving the ribosome binding site and the internal complementary sequence, J. Bacteriol., 1995, 177(22), 6560–6567. 10.1128/jb.177.22.6560-6567.1995Search in Google Scholar
[37] Le Calvez H., Green J.M., Baty D., Increased efficiency of alkaline phosphatase production levels in Escherichia coli using a degenerate Pe1B signal sequence, Gene, 1996, 170(1), 51–55. http://dx.doi.org/10.1016/0378-1119(95)00850-010.1016/0378-1119(95)00850-0Search in Google Scholar
[38] Satchidanandam V., Shivashankar Y., Availability of a second upstream AUG can completely overcome inhibition of protein synthesis initiation engendered by mRNA secondary structure encompassing the start codon, Gene, 1997, 196(1), 231–237. http://dx.doi.org/10.1016/S0378-1119(97)00232-110.1016/S0378-1119(97)00232-1Search in Google Scholar
[39] Kozak M., Circumstances and mechanisms of inhibition of translation by secondary structure in eucaryotic mRNAs, Mol. Cell. Biol., 1989, 9(11), 5134–5142. 10.1128/mcb.9.11.5134-5142.1989Search in Google Scholar
[40] Mathews D.H., Turner D.H., Prediction of RNA secondary structure by free energy minimization, Curr. Opin. Struct. Biol., 2006, 16(3), 270–278. http://dx.doi.org/10.1016/j.sbi.2006.05.01010.1016/j.sbi.2006.05.010Search in Google Scholar
[41] de Smit M.H., van Duin J., Secondary structure of the ribosome binding site determines translational efficiency: a quantitative analysis, Proc. Natl. Acad. Sci. U. S. A., 1990, 87(19), 7668–7672. http://dx.doi.org/10.1073/pnas.87.19.766810.1073/pnas.87.19.7668Search in Google Scholar
[42] Penton E., Muzio V., González M., The Hepatitis B virus (HBV) infection and its prevention by a recombinant-DNA viral surface antigen (rec HBsAG) vaccine, Biotecnología Aplicada, 1994, 11(1), 1–11. Search in Google Scholar
[43] Tleugabulova D., Falcón V., Sewer M., Pentón E., Aggregation of recombinant hepatitis B surface antigen in Pichia pastoris, Journal of Chromatography B: Biomedical Sciences and Applications, 1998, 716(1), 209–219. http://dx.doi.org/10.1016/S0378-4347(98)00297-710.1016/S0378-4347(98)00297-7Search in Google Scholar
[44] Wampler D., Lehman E., Boger J., McAleer W., Scolnick E., Multiple chemical forms of hepatitis B surface antigen produced in yeast, Proc. Natl. Acad. Sci. U. S. A., 1985, 82(20), 6830–6834. http://dx.doi.org/10.1073/pnas.82.20.683010.1073/pnas.82.20.6830Search in Google Scholar PubMed PubMed Central
[45] Hauser P., Voet P., Simoen E., Thomas H., Petre J., De Wilde M., et al., Immunological properties of recombinant HBsAg produced in yeast, Postgrad. Med. J., 1987, 63, 83. Search in Google Scholar
[46] Valenzuela P., Medina A., Rutter W.J., Ammerer G., Hall B.D., Synthesis and assembly of hepatitis B virus surface antigen particles in yeast, Nature, 1982, 298(5872), 347. http://dx.doi.org/10.1038/298347a010.1038/298347a0Search in Google Scholar PubMed
© 2014 Versita Warsaw
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.
