Development of metal-affinity partitioning of Hepatitis B core antigen from unclarified bacteria feedstock

Hepatitis B core antigen (HBcAg) is one of the most frequently studied viral-like-particle (VLP) for the display of foreign epitopes and has a great potential in the development of diagnostic reagent and vaccine for hepatitis B infection. Various researches have been reported to purify HBcAg such as sucrose gradient ultracentrifugation and chromatography. However, these conventional methods are tedious, time consuming, and expensive to use due to multiple purification steps. Furthermore, the yield obtained was reported to be very low. As a result, different methods have been investigated to improve the yield and purity of HBcAg. Therefore, aqueous two-phase system (ATPS), which consists of two types of polymers or polymer with a type of salt, can be seeked as an alternative method for HBcAg recovery. In this study, immobilised metal affinity partitioning (IMAP) was incorporated in ATPS to improve the overall recovery of his-tagged HBcAg from unclarified Escherichia coli (E. coli) feedstock. Modified polymer involved in IMAP was synthesised prior to ATPS purification of his-tagged HBcAg. The effects of activation duration, epichlorohydrin concentration, boron trifluoride ethyletherate (BFEE) concentration, sodium hydroxide (NaOH) concentration, iminodiacetic acid concentration and type of metal ions were evaluated for the optimal production of modified PEG. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), the Bradford assay and densitometric analysis were used to analyse and quantify the samples obtained from ATPS. Parameters such as the mode for phase inversion and separation, molecular weight (MW) of PEG, phase-forming salts, tie-line length (TLL), phase volume ratio (VR), neutral salt addition, system pH and biomass concentration on the partitioning of his-tagged HBcAg were evaluated in this study. The antigenicity and the morphology of the purified HBcAg were analysed using enzyme-linked immunosorbent assay (ELISA) and transmission electron microscope (TEM), respectively. Based on the phase diagram, PEG 6000-IDA-Cu(II)- Na2SO4 system was an ideal system to use in ATPS. However, when the unclarified homogenate was added to the system, it was shown that PEG 2000-IDA-Cu(II)-KPB was the optimal system to use as it partitioned the highest amount of his-tagged HBcAg to the PEG rich top phase. The optimal recovery of his-tagged HBcAg was achieved using PEG 2000-IDA-Cu(II)-KPB system with phase inversion by using a vortex mixer for 5 mins, phase separation by centrifugation for 3 mins at 2000 xg, TLL 3 and a phase volume ratio of 2.3, pH 8, without NaCl addition and loaded with 5 % (w/v) unclarified homogenate. The purity of HBcAg obtained was about 94 % with a purification factor of 3.0 and a recovery yield of approximately 91 %. Based on the ELISA results, the antigenicity of the ATPS purified his-tagged HBcAg was still preserved and it was comparable to the his-tagged HBcAg purified using sucrose-gradient ultracentrifugation. When the purified his-tagged HBcAg were viewed using TEM, they were still able to form icosahedral particles with a diameter of about 28 to 32 nm. Therefore, this study showed that IMAP incorporated in ATPS is an efficient method to recover his-tagged HBcAg from unclarified E. coli homogenate.