title: Effects of Trx2p and Sec23p expression on stable production of hepatitis B surface antigen S domain in recombinant Saccharomyces cerevisiae.
abstract: The S domain of hepatitis B virus surface antigen (sHBsAg) is the primary component for vaccine development against virus infection. For stable expression of sHBsAg in recombinant Saccharomyces cerevisiae, new accessory genes necessary for foreign protein expression were screened by DNA microarray. Among 600 genes of interest, genes coding for an activating protein of ATPase in Hsp90 (Aha1p), S. cerevisiae DnaJ (Scj1p), thioredoxin 2 (Trx2p) and a GTPase-activator specific for Sar1 (Sec23p) as well as Pdi1p were selected in transcriptome analysis, which are known to facilitate disulfide bond formation or induce protein transport in the secretion pathway. Individual and combinatorial expression of SEC23, TRX2 and PDI1 increased total sHBsAg concentration by 1.9-6.5-fold, relative to the control strain expressing sHBsAg only. Additionally, moderate expression of Kex2p protease able to cut off the signal peptide enhanced the portion of the authentic sHBsAg to total sHBsAg. Fed-batch fermentation of the S. cerevisiae 2805 strain coexpressing the sHBsAg, SEC23, PDI1 and KEX2 genes resulted in 70.6mg/L final sHBsAg concentration which was 5.6 times higher than that of the control. Transmission electron microscopic analysis of the yeast cells elucidated the effects of the accessory gene coexpression on the intracellular localization of sHBsAg. Like PDI1, coexpression of both SEC23 and/or TRX2 newly isolated in this study is expected to improve the target protein expression in S. cerevisiae.
authors.affiliation: Department of Agricultural Biotechnology, Seoul National University, Seoul 151-921, Republic of Korea.
authors.forename: Young-Kyoung
authors.initials: YK
authors.lastname: Park
authors.forename: Sang-Min
authors.initials: SM
authors.lastname: Jung
authors.forename: Hyung-Kwon
authors.initials: HK
authors.lastname: Lim
authors.forename: Young-Jin
authors.initials: YJ
authors.lastname: Son
authors.forename: Yong-Cheol
authors.initials: YC
authors.lastname: Park
authors.forename: Jin-Ho
authors.initials: JH
authors.lastname: Seo
mesh_terms: D022181:COP-Coated Vesicles / Q000235:genetics / Q000378:metabolism*; D020690:GTPase-Activating Proteins / Q000235:genetics / Q000378:metabolism*; D006514:Hepatitis B Surface Antigens / Q000096:biosynthesis* / Q000235:genetics; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000378:metabolism; D011995:Recombination, Genetic / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000502:physiology*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D013879:Thioredoxins / Q000235:genetics / Q000378:metabolism*
chemical_list: D020690:GTPase-Activating Proteins; D006514:Hepatitis B Surface Antigens; D011994:Recombinant Proteins; C079037:SEC23 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C541128:Trx3 protein, S cerevisiae; D013879:Thioredoxins