Effect of glycerol feed in methanol induction phase for hepatitis B surface antigen expression in Pichia pastoris strain KM71

This study describes expression of HBs Ag in methylotrophic yeast, Pichia Pastoris under alcohol oxidase promoter. A single copy number of HBs Ag gene was transformed into pichia strain of KM 71, a Mutˢ type, by using pA0815 pichia expression vector. The recombinant was cultivated in a shake flask either using methanol or a mixed feed of glycerol -methanol for induction. The HBs Ag gene integrity was justified using direct PCR method. The expressed products in the soluble cell extracts were analyzed by Western blot, SDS page, Bradford assay and ELISA tests. The recombinant HBs Ag was expressed successfully in Pichia pastoris strain KM71 at a high level of HBs Ag protein expression. Thus, an addition of glycerol in the ratio of glycerol per methanol 1/1 (g g-1) consistently produced 2-fold increment in both biomass accumulation and HBs Ag productivity

Wide dynamic range of surface-plasmon-resonance-based assay for hepatitis B surface antigen antibody optimal detection in comparison with ELISA

Limit of detection (LOD), limit of quantification, and the dynamic range of detection of hepatitis B surface antigen antibody (anti-HBs) using a surface plasmon resonance (SPR) chip-based approach with Pichia pastoris-derived recombinant hepatitis B surface antigen (HBsAg) as recognition element were established through the scouting for optimal conditions for the improvement of immobilization efficiency and in the use of optimal regeneration buffer. Recombinant HBsAg was immobilized onto the sensor surface of a CM5 chip at a concentration of 150 mg/L in sodium acetate buffer at pH 4 with added 0.6% Triton X-100. A regeneration solution of 20 mM HCl was optimally found to effectively unbind analytes from the ligand, thus allowing for multiple screening cycles. A dynamic range of detection of ∼0.00098–0.25 mg/L was obtained, and a sevenfold higher LOD, as well as a twofold increase in coefficient of variance of the replicated results, was shown as compared with enzyme-linked immunosorbent assay (ELISA). Evaluation of the assay for specificity showed no cross-reactivity with other antibodies tested. The ability of SPR chip-based assay and ELISA to detect anti-HBs in human serum was comparable, indicating that the SPR chip-based assay with its multiple screening capacity has greater advantage over ELISA

Two-phase fed-batch modification for 48 hour peak expression of hepatitis B surface antigen in Pichia pastoris shake flask system

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

Two-step purification strategy for enhanced recovery of recombinant hepatitis B surface antigen from Pichia pastoris

Univariate screening on factors affecting the purification performance of recombinant hepatitis B surface antigen (HBsAg) on ion exchange chromatography (IEC) and size exclusion chromatography (SEC) and the establishment of a two-step purification strategy were performed. Amongst four IEC adsorbents examined, the use of Q Sepharose XL IEC adsorbent under optimized conditions together with optimized SEC purification was able to efficiently purify HBsAg. An established purification strategy comprising the two techniques further demonstrated adaptability for scale-up operations with a final total purification factor (PF) of 94.82 ± 16.20, HBsAg purity of 95.48% and recovery yield of 78.07%

Multiple overlap extension PCR (MOE-PCR): an effective technical shortcut to high throughput synthetic biology

The current study describes multiple-overlap-extension PCR (MOE-PCR) as a simple and effective approach to assembling multiple DNA fragments with various sizes and features in a single in vitro reaction. In this research, 50 bp of homology in overlapping DNA fragments and a specific touchdown PCR program resulted in successful assembly of eight different DNA fragments using a single PCR protocol. The simplicity, speed, reliability and cost-effectiveness of MOE-PCR offers it as an attractive method for assembling and/or cloning single and multiple DNA fragments. Indeed, the method is a one-step approach that eliminates some of the routine steps such as ligation and complex enzymatic reactions as well as sequence limitations of the other methods. The applications of this relatively high fidelity method could be extended to the construction of chimeric recombinant sequences that can be widely used in metabolic engineering, functional analysis of genes and genetic elements, expression studies of multi-domain proteins, protein engineering and the most recent genome editing strategies which together with synthetic biology are revolutionizing the life sciences. We expect the technique to be used as a robust, reliable and fast method in synthetic biology

New expression vector for microalgae

The present invention relates to expression vectors for the improved production of recombinant proteins in microalgae through synthetic biology. The present invention also relates to the construction of a vector system which can be used for promoter analysis in gene expression