Optimization of the expression of hepatitis B virus e gene in Pichia pastoris and immunological characterization of the product

Department of Laboratory Medicine, The Third Affiliated Hospital of Sun Yat-sen University, Guangzhou, Guangdong Province 510630, PR China.
Journal of Biotechnology (Impact Factor: 2.87). 08/2008; 138(1-2):1-8. DOI: 10.1016/j.jbiotec.2008.07.1989
Source: PubMed


Escherichia coli-derived hepatitis B e antigen (HBeAg) is widely used for serological tests of hepatitis B virus (HBV). Because it exhibits cross-reactivity with HBcAb in human sera, current antibody to HBeAg (HBeAb) immunoassays are based on competitive inhibition enzyme-linked immunosorbent assay (ELISA) rather than sandwich ELISA, which interfere with the specificity and sensitivity of HBeAb detection. Pichia pastoris has advantages of eukaryotic cells, while having the capacity of high-level secretion of foreign proteins. To explore the diagnostic suitability of recombinant HBeAg (rHBeAg), we expressed the wild type HBV e gene (wt-e-gene) and the synthetic HBV e gene (syn-e-gene; native HBV e gene modified based on synonymous codon usage bias) in P. pastoris. The recombinant antigen was secreted into the medium. The expression level of rHBeAg was enhanced by optimizing HBV e gene. The yield of syn-e-gene product was approximately five-fold greater than wt-e-gene. The protein represented 66% of the total supernatant protein, and was simply purified to 90%. P. pastoris-derived HBeAg showed high HBe antigenicity, while lacking any HBc antigenicity and cross-reactivity between all proteins derived from the culture of P. pastoris and normal human sera. P. pastoris-derived HBeAg has higher specificity and sensitivity for detection HBeAb in the diagnostic assay than the commercial HBeAb ELISA kits.

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    • "To increase the secretion expression level, we optimized the gene of interest by means of substituting rare codons with P. pichia preferable triplets. At the same time, we balanced the G+C content, removed the A/T or G/C repeats, and avoided the formation of hairpin or special secondary structure of hIL-25 mRNA(Li et al. 2008). The productivity of rhIL-25 was much higher using codon optimized rhIL-25. "
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    ABSTRACT: Interleukin (IL)-25 (also known as IL-17E) is a distinct member of the IL-17 cytokine family which induces IL-4, IL-5, and IL-13 expression and promotes pathogenic T helper (Th)-2 cell responses in various organs. IL-25 has been shown to have crucial role between innate and adaptive immunity and also a key component of the protection of gastrointestinal helminthes. In this study, to produce bioactive recombinant human IL-25 (rhIL-25), the cDNA of mature IL-25 was performed codon optimization based on methylotropic yeast Pichia pastoris codon bias and cloned into the expression vector pPICZαA. The recombinant vector was transformed into P. pichia strain X-33 and selected by zeocin resistance. Benchtop fermentation and simple purification strategy were established to purify the rhIL-25 with about 17 kDa molecular mass. Functional analysis showed that purified rhIL-25 specifically bond to receptor IL-17BR and induce G-CSF production in vitro. Further annexin V-FITC/PI staining assay indicated that rhIL-25 induced apoptosis in two breast cancer cells, MDA-MB-231 and HBL-100. This study provides a new strategy for the large-scale production of bioactive IL-25 for biological and therapeutic applications.
    Full-text · Article · Oct 2013 · Applied Microbiology and Biotechnology
    • "An example of this is the production of secreted human albumin in P. pastoris[23]. However, some other reports have shown that the optimization of codon usage would, in some cases, lead to better results242526. As an example, production of a secreted hepatitis B gene e would in- Biotechnol. "
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    ABSTRACT: Membrane proteins play key roles in diverse cellular functions and have become the target for a large number of pharmacological drugs. Despite representing about 20-30% of cellular proteins, their characterization is long overdue since they are difficult to handle, to purify from their natural source or to obtain as recombinant proteins. Pichia pastoris is a methylotrophic yeast species increasingly used as a host for heterologous protein expression for both research and industrial purposes. Over the past few years many efforts have allowed important advances in the development of this expression system for the expression and production of membrane proteins. The most recent achievements in improving yield and proper folding of integral membrane proteins are summarized in this review.
    No preview · Article · Jun 2011 · Biotechnology Journal
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    ABSTRACT: To obtain the protein expression of a hybrid xylanase in yeast, the gene encoding it was modified according to the codon bias of Pichia pastoris and expressed extracellularly in this yeast as an active xylanase, MBtx, exhibited a molecular mass of approximately 35kDa on SDS–PAGE. The pH behavior of MBtx in terms of both activity and stability was similar to that of Btx, original gene product in Escherichia coli, while a certain difference was observed in optimal temperature for activity and in thermal stability. HPLC analysis revealed the xylan in wheat could be hydrolyzed by MBtx and the major hydrolysis product was xylotriose. These results showed codon usage played a key role in regulating the expression of the hybrid xylanase in P. pastoris and the recombinant hybrid xylanase, MBtx, produced by P. pastoris could be potentially useful in feed industry.
    Preview · Article · Aug 2009 · World Journal of Microbiology and Biotechnology
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