high-level expression of hepatitis b surface antigen in pichia pastoris, its purification and immunological characterization

Selamoğlu, Hande
Hepatitis B virus (HBV), which belongs to the family Hepadnaviridae, is responsible for acute and chronic hepatitis. The vaccines presently used to immunize patients against HBV are recombinant subunit vaccines consisting of viral surface antigens (S protein). However, they are expensive and their use is limited in poor countries. For that reason, HBV remains an important worldwide health problem. Of the 2 billion people who have been infected with the HBV, more than 350 million have chronic (lifelong) infections, who face increased risk of developing cirrhosis and hepatocellular carcinoma. In this study, high-level expression of recombinant Hepatitis B surface Antigen (rHBsAg), PreS2-S was achieved in the methylotrophic yeast, Pichia pastoris. For this aim, a single copy of HBV M gene (PreS2-S) was inserted at the downstream of the alcohol oxidase (AOX1) promoter of the pPICZA vector. rHBsAg protein could then be expressed intracellularly by induction with methanol. High cell density fermentation was followed by chromatographic separation to obtain pure rHBsAg. Humoral response after immunization with the purified protein was observed in mice using commercial Hepatitis B surface antigen kits. It was verified by the atomic force microscopy that rHBsAg has been produced in the desired conformation.


Molecular evaluation and antimicrobial susceptibility testing of Escherichia coli isolates from food products in Turkey
Kyere, Emmanuel Owusu; Bulut, Ece; AVŞAROĞLU ERKAN, MÜRŞİDE DİLEK; Soyer, Yeşim (Springer Science and Business Media LLC, 2015-06-01)
Some strains of Escherichia coli can be important food borne pathogens. Characterization and antimicrobial resistance testing of 28 E. coli isolates from random food samples obtained in Van, Turkey were performed. Primers for 6 indicator genes (fliC, stx1, stx2, eae, hlyA, and rfbE) for shiga toxin-producing E. coli and 5 indicator genes for each pathogroup (bfpA, aggR, ipaH, daaD, st, and lt) were used. E. coli isolates were also typed using pulsed field gel electrophoresis with the XbaI restriction enzyme...
Influence of controlled-pH and uncontrolled-pH operations on recombinant benzaldehyde lyase production by Escherichia coli
Çalık, Pınar; Demir, AS (Elsevier BV, 2006-03-02)
To select the host microorganism having the highest benzaldehyde lyase (BAL) production capacity, pUC 18::bal gene was transferred into four Escherichia coli strains. As the highest enzyme activity was obtained with E. coli K12 (ATCC 10798) carrying pUC18::bal gene, BAL production medium was designed for K 12. Using the designed medium containing 8.0 kg m(-3) glucose, 5.0 kg m(-3) (NH4)(2)HPO4 and the salt solution, the effects of uncontrolled-pH and controlled-pH operations were investigated at uncontrolle...
Comparison of benzaldehyde lyase production capacity in recombinant Escherichia coli and recombinant Bacillus species
Kaya, Hande; Çalık, Pınar; Department of Chemical Engineering (2006)
In this study, the benzaldehyde lyase (BAL, EC production in E. coli BL21 (DE3) pLySs as intracellular and in Bacillus species as extracellular were investigated, and comparison of the production capacity of the enzyme in the developed recombinant microorganisms were compared. For this purpose, firstly, PCR amplified bal gene was cloned into pRSETA vector which is under the control of strong T7 promoter and expressed in E. coli BL21 (DE3) pLysS strain. With developed recombinant E. coli BL21 (DE3)...
Bioprocess parameters and oxygen transfer characteristics in beta-lactamase production by Bacillus species
Celik, E; Çalık, Pınar (Wiley, 2004-03-01)
After screening potential beta-lactamase producers in a medium containing penicillin G, an inducible (Bacillus subtilis NRS 1125) and a constitutive (Bacillus licheniformis 749/C ATCC 25972) P-lactamase producer were selected. As the highest enzyme activity was obtained with B. licheniformis 749/C, the effects of the concentration of carbon sources, i.e., glucose, fructose, sucrose, citric acid, and glycerol, and nitrogen sources, i.e., (NH4)(2)HPO4, NH4Cl, yeast extract, casamino acids and peptone, pH, and...
Influence of nickel (II) and chromium (VI) on the laboratory scale rotating biological contactor
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High concentration of heavy metals is toxic for most microorganisms and cause strict damage in wastewater treatment operations and often a physico-chemical pretreatment prior to biological treatment is considered necessary. However, in this study it has been shown that biological systems can adapt to Ni (II) and Cr (VI) when their concentration is below 10 and 20 mg/L, respectively. The aim of this study was to evaluate the effect of Ni (II) and Cr (VI) on the lab-scale rotating biological contactor process...
Citation Formats
H. Selamoğlu, “high-level expression of hepatitis b surface antigen in pichia pastoris, its purification and immunological characterization,” M.S. - Master of Science, Middle East Technical University, 2009.