Optimization of Physical Parameters for the Enhanced Expression of Recombinant Chemokine Receptors D6 and DARC in Pichia Pastoris

D6 and DARC proteins had been reported as decoy chemokine receptors in cancer study. In this study, electrotransformation of Pichia pastoris GS115 was carried out to allow integration of linearized recombinant yeast expression vectors into yeast genome. Upon antibiotic screening and PCR analysis to confirm the integration of gene of interest in the yeast genome, the positive clones were plated at increasing ZeocinTM concentrations. Clones able to confer resistance to ZeocinTM concentration of up to 2 mg/mL were selected for recombinant protein expression. Two types of clones were constructed, using pPICZ and pPICZα vectors. Initial profile of recombinant protein expression showed low yields of heterologous proteins from pPICZα clones. Hence, only pPICZ clones, which expressed recombinant protein intracellularly were selected for further expression studies. Optimizations of selected physical parameters were performed and the intracellular expression of soluble recombinant proteins was found to increase dramatically with an increase of 1012% for recombinant D6 and 452% for recombinant DARC proteins. The optimized parameters are: buffered methanol-complex medium (BMMY) at pH 6.0, inoculum size at OD600 of 2.5 (for D6) and 1.0 (for DARC), induction with 1.0% (vol/vol) of methanol once every 24 h and incubation at 16°C for 48 h.