Cloning, functional expression, and characterization of recombinant pig liver esterase

The N-terminal amino acid sequence of pig liver esterase (PLE) from a comme rcial sample was determined and shown to match closely to a published seque nce encoding a proline-beta -naphthylamidase from pig liver. Next, mRNA iso lated from pig liver was transcribed into cDNA and primers deduced from the N-terminal sequence were used to clone the 1698 base pairs of PLE cDNA. In itial attempts to express the cDNA in Escherichia coli and Pichia pastoris with different expression vectors and secretion signal sequences failed. On ly after deletion of the putative C-terminal sequence His-Ala-Glu-Leu, usua lly considered as an endoplasmic reticulum retention signal, could heterolo gous expression of PLE be readily achieved in the methylotrophic yeast P. p astoris. Recombinant PLE (rPLE) was secreted into the medium and exhibited a specific activity of approximately 600 U mg(-1) and a V-max/K-m value of 139 mu mol min(-1) mM(-1) with beta -nitrophenyl acetate as a substrate. Ac tivity staining of renatured sodium dodecylsulfate-polyacrylamide gels gave a single band with esterolytic activity for rPLE, whereas Several bands ar e visible in crude commercial PLE preparations. This was confirmed by nativ e gels, which also show that rPLE is active as a trimer, Biochemical charac terization of the recombinant enzyme and comparison with properties of comm ercial PLE preparations as well as with published data confirmed, that we e xpressed a single PLE isoenzyme which showed a high preference for praline- beta -naphthylamide. This is a substrate specificity for the so-called gamm a subunit of PLE. The optimum pH value and temperature for the recombinant PLE were 8.0 and 60 degreesC, respectively. The determined molecular weight of the secreted enzyme was approximately 67 - 62 kDa, which closely matche s the calculated value of 62.419 kDa. The active site residues are located at Ser(203), His(448), and Asp(97), and the typical consensus sequence moti f for hydrolases was found around the active site serine (Gly-Glu-Ser-Ala-G ly).