Construction, Expression and Characterization of Multi Cassettes Encoding Indonesian Small Hepatitis B Surface Antigen (s-HBsAg) in Methylotropic Yeast Pichia pastoris

Abstract

A B S T R A C T Hepatitis B is the most common liver infection worldwide. The recombinant VirusLike Particles (VLP) of small Hepatitis B surface antigen (s-HBsAg) vaccine provides excellent protection against Hepatitis B Virus (HBV). In this study, we have generated a construct of Pichia pastoris recombinant which comprises multi expression cassettes encoding s-HBsAg which belongs to HBV B3 genotype predominantly found in Indonesia. An expression cassette containing a synthetic codon optimized open reading frame of HBV B3 genotype/adw serotype s-HBsAg was cloned into pAO815 to generate pAOHBs1 in Escherichia coli. The expression cassette from pAOHBs1 was subsequently isolated and ligated with recombinant previously obtained, generated recombinant plasmid containing 2, 3 and 4 direct repeats of HBsAg expression cassette. The resulted recombinant containing 4 expression cassettes was integrated to P. pastoris genome. Positive integrants and stability during cultivation were identified by PCR and qPCR. The expression of s-HBsAg was induced by methanol and analyzed by SDS PAGE and western blotting. Nucleotide sequencing analysis showed that the inserted fragment encodes amino acids with 100% similarity compared to that designed for HBV B3 genotype. The PCR and qPCR analysis showed that stable P. pastoris integrated with 4 s-HBsAg expression cassette was successfully obtained with methanol utilizing slow phenotype. A protein band with apparent molecular weight which similar to s-HBsAg size was detected based on a SDS PAGE and western blot analysis. A stable integrant of P. pastoris containing four expression cassettes of HBV B3 genotype s-HBsAg capable of producing a vaccine candidate against Hepatitis B was generated.

Integration Stability of sHBsAg-Multi Expression Cassettes in Pichia pastoris GS115 during Methanol Induction

“…This stage aims to induce P. pastoris to produce sHBsAg protein using 2% methanol. The concentration has been showed by Giri-Rachman et al (2015) to have positive effect on growth and sHBsAg protein expression level. Besides, Santoso et al (2012) showed that 2.5% methanol can increase human erythropoietin gene expression in P. pastoris.…”

“…P. pastoris GS115 transformant culture, pAO815 plasmid harbouring single copy and four copies of sHBsAg expression cassette (pAO-HBs1 and pAO-HBs4) was obtained from previous study (Giri-Rachman et al 2015). Yeast DNA purification kit were purchased from Epicentre.…”

“…Therefore, production of sHBsAg protein that matches Indonesia's HBV genotype is much needed. A study from Giri-Rachman et al 2015 showed that predominant HBV genotype in Indonesia is B3.…”

“…In previous study, four copies of sHBsAg expression cassette from HBV genotype B3 has been constructed using pAO815 expression vector (Giri-Rachman et al 2015). Expression cassettes were integrated to P. pastoris GS115 using gene replacement method.…”