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.
Microinjection is a powerful tool to deliver various substances, such as nucleic acids, proteins, peptides, RNA, and synthetic molecules into mammalian cells mechanically. Through microinjection, a controlled amount of protein can be delivered into the target cells to elucidate the specific functional
effects in vitro. In this study, a series of protein microinjection optimization was performed in human breast cancer cells. The presence of Maltose Binding Protein (MBP) was microscopically monitored through indirect immunofluorescence assay. The optimization experimentation gave a high success rate when MBP protein was used at the minimum concentration of 1.5 mg/ml and at the injection pressures of 50 and 70 hPa. The average success rate of injections was 49.2±4.15% and 50.8±4.6%, while the average cell survivability was 50.98±4.67% and 49.72±5.48% at 50 and 70 hPa, respectively. The optimization of the MBP concentration and injection pressures successfully allowed an efficient delivery of precise protein dosage into breast cancer cells without any adverse effect. This microinjection optimization can be a practical guideline in any downstream applications of protein functional work.