Recombinant hepatitis B Vaccines – Disease Characterization and Vaccine Production

Brocke, Pascale; Schaefer, Stephan; Melber, Karl; Jenzelewski, Volker; Müller, Florian; Dahlems, Ulrike; Bartelsen, Oliver; Park, Kyung‐Nam; Janowicz, Zbigniew A.; Gellissen, Gerd

doi:10.1002/3527602356.ch12

Cited by 24 publications

(24 citation statements)

References 65 publications

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“…First, procedure for HBsAg extraction was optimized. Since HBsAg lipophylic particles tend to associate with the membrane fraction (Schaefer et al, 2002), their release is strongly dependent on the composition of the extraction buffer used. We examined different compositions of the extractions buffers, including published elsewhere (Janowicz et al, 1991;Vassileva et al, 2001), and found buffer containing 10 mM phosphate buffer (pH 7.5), 5 mM EDTA, 0.5 M NaCl, 0.5% Triton X-100, and 1 mM PMSF to be the most efficient.…”

Section: Hbsag Assaysmentioning

confidence: 99%

“…The plasmid pHB320 (V. I. Kordium and O. F. Vozianov, unpublished work) was used as a source of the HBsAg gene (serotype ayw3, genotype D (Schaefer et al, 2002)). The HBsAg gene was isolated as a 0.68 kb long PCR fragment with pHB320 as a template and primers OL3 (5 0 -GGAAGCTTATGGAGAACATCACATC-3 0 ) and OL4…”

Section: Construction Of the Hbsag Expression Vector Pko6mentioning

confidence: 99%

“…Another reason was that different fermentation modes could be employed to express these proteins . In fact, HBsAg production is the only industrial manufacturing process that involves methanol feeding in H. polymorpha, as it apparently requires extensive microbody membrane proliferation (Schaefer et al, 2002;Brocke et al, 2005). In our mutant strains deficient in glucose repression, peroxisomes, and, correspondingly, peroxisome membranes are constitutively synthesized in glucose medium without methanol (Stasyk et al, 2002(Stasyk et al, , 2004.…”

Section: Introductionmentioning

confidence: 98%

“…Methanol level in the medium must be controlled within an optimum range, as the maximum promoter activity is achieved at low methanol levels, and its excess is toxic to the cells . Methanol feeding also demands extensive aeration because of a high oxygen requirement for the alcohol oxidase reaction (Jenzelewski, 2002;Schaefer et al, 2002).…”

Section: Introductionmentioning

confidence: 99%

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Glucose‐induced production of recombinant proteins in Hansenulapolymorpha mutants deficient in catabolite repression

Krasovska

Stasyk

Nahorny

et al. 2007

Biotech & Bioengineering

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The most commonly used expression platform for production of recombinant proteins in the methylotrophic yeast Hansenula polymorpha relies on the strong and strictly regulated promoter from the gene encoding peroxisomal enzyme alcohol (or methanol) oxidase (P(MOX)). Expression from P(MOX) is induced by methanol and is partially derepressed in glycerol or xylose medium, whereas in the presence of hexoses, disaccharides or ethanol, it is repressed. The need for methanol for maximal induction of gene expression in large-scale fermentation is a significant drawback, as this compound is toxic, flammable, supports a slow growth rate and requires extensive aeration. We isolated H. polymorpha mutants deficient in glucose repression of P(MOX) due to an impaired HpGCR1 gene, and other yet unidentified secondary mutations. The mutants exhibited pronounced defects in P(MOX) regulation only by hexoses and xylose, but not by disaccharides or ethanol. With one of these mutant strains as hosts, we developed a modified two-carbon source mode expression platform that utilizes convenient sugar substrates for growth (sucrose) and induction of recombinant protein expression (glucose or xylose). We demonstrate efficient regulatable by sugar carbon sources expression of three recombinant proteins: a secreted glucose oxidase from the fungus Aspergillus niger, a secreted mini pro-insulin, and an intracellular hepatitis B virus surface antigen in these mutant hosts. The modified expression platform preserves the favorable regulatable nature of P(MOX) without methanol, making a convenient alternative to the traditional system.

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Section: Hbsag Assaysmentioning

confidence: 99%

Section: Construction Of the Hbsag Expression Vector Pko6mentioning

confidence: 99%

Section: Introductionmentioning

confidence: 98%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Glucose‐induced production of recombinant proteins in Hansenulapolymorpha mutants deficient in catabolite repression

Krasovska

Stasyk

Nahorny

et al. 2007

Biotech & Bioengineering

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show abstract

“…HBV aşıları üretiminde S. cerevisiae'nın yanı sıra metilotrofik maya olan H. polymorpha'nın daha üstün ekspresyon özelliği olduğu belirlenmiş ve birçok HBV aşısında kullanılmaya başlanmıştır (22). Rekombinasyon teknolojisi ile üretilen aşılardan bazıları da maya hücresi yerine memeli hücresi (fare over hücresi) kullanılarak üretilmektedir (23).…”

Section: Hepatit B Aşılarıunclassified