Expression, purification and characterization of ubiquitin-specific protease 1 (UBP1) for hydrolysis of ubiquitin-fused hGH expressed in recombinant E. coli

Abstract

This research was focused on expression, purification and characterization of ubiquitinspecific protease 1 (UBP1) expressed in recombinant E. coli and P. pastoris. Various systems were constructed by fusing polycationic fusion tails or fusion partners to the C- or N-terminus of the product protein. In particular, UBP1 containing 6 histidine residues at the N-terminal end showed best results in terms of expression level and purification efficiency. UBP1 expressed in P. pastoris was secreted to the medium and enzyme activity was detected after 48 hours induction with methanol. The N-terminal 6×His-tagged UBP1 was overproduced in recombinant E. coli using high cell density cultivation technology and purified using immobilized metal affinity chromatography (IMAC). The molecular weight of UBP1 was found to be 83,500 daltons. The optimum temperature and pH for the enzyme reaction when ubiquitin-hGH (human growth hormone) was used as a substrate were 40℃ and pH 8.0, respectively.