Codon Optimization, Cloning And Expression Of Recombinant Human Alpha Glucocerebrosidase

Abstract

The present research mainly deals with the codon optimization, cloning and expression of recombinant human alpha glucocerebrosidase( rhGC). Human alpha glucocerebrosidase was family of lysosomal hydrolases, it used to enzyme replacement therapy and leads to deficiency of lysosomal storage diseases(LSD). In this study Pichia.pastoris was used for the production of different strains of recombinant human α- glucocerebrosidase. Plasmid linearization and transformaton of P.pastoris expression of multicopy vectors , the whole expressed vectors  were amplified using PCR with primers of 2 different pairs.  Expression of rhGC gene of P.pastoris, we otimized the codon of PBIZα by denovo gene design and synthesis. After synthesized,  significantly  rhGC strains were divided into three different groups based on their methanol induced utilization capability in orbital thermoshaker (1300 rpm, 37 ºC for 1 hr). The diveded strains like mut⁺ (normal consumption), mut^s( slow consumption) and mut^-( disabled consumption), in this first two strains (mut⁺ , mut^s) are most commonly used for the codon optimization. Finally the research evaluates the stratagies of multiple GC gene copies were transformed by the multi copy clones of rhGC in P.pastoris.