Cloning, functional expression, and characterization of recombinant pig liver esterase.
The N-terminal amino acid sequence of pig liver esterase (PLE) from a commercial sample was determined and shown to match closely to a published sequence encoding a proline-beta-naphthylamidase from pig liver. Next, mRNA isolated 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. Initial attempts to express the cDNA in Escherichia coli and Pichia pastoris with different expression vectors and secretion signal sequences failed. Only after deletion of the putative C-terminal sequence His-Ala-Glu-Leu, usually considered as an endoplasmic reticulum retention signal, could heterologous expression of PLE be readily achieved in the methylotrophic yeast P. pastoris. Recombinant PLE (rPLE) was secreted into the medium and exhibited a specific activity of approximately 600 Umg(-1) and a Vmax/Km value of 139 micromolmin(-1)mM(-1) with p-nitrophenyl acetate as a substrate. Activity staining of renatured sodium dodecylsulfate-polyacrylamide gels gave a single band with esterolytic activity for rPLE, whereas several bands are visible in crude commercial PLE preparations. This was confirmed by native gels, which also show that rPLE is active as a trimer. Biochemical characterization of the recombinant enzyme and comparison with properties of commercial PLE preparations as well as with published data confirmed that we expressed a single PLE isoenzyme which showed a high preference for proline-beta-naphthylamide. This is a substrate specificity for the so-called gamma subunit of PLE. The optimum pH value and temperature for the recombinant PLE were 8.0 and 60 degrees C, respectively. The determined molecular weight of the secreted enzyme was approximately 61-62 kDa, which closely matches the calculated value of 62.419 kDa. The active site residues are located at Ser203, His448, and Asp97, and the typical consensus sequence motif for hydrolases was found around the active site serine (Gly-Glu-Ser-Ala-Gly).
Lange
S
S
Institute for Technical Biochemistry, Stuttgart University, Allmandring 31, 70569 Stuttgart, Germany.
Musidlowska
A
A
Schmidt-Dannert
C
C
Schmitt
J
J
Bornscheuer
U T
UT
D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004950:Esterases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008099:Liver / Q000201:enzymology*; D008970:Molecular Weight; D010843:Pichia / Q000235:genetics; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020539:Sequence Analysis, Protein; D013552:Swine
D018076:DNA, Complementary; D012333:RNA, Messenger; D011994:Recombinant Proteins; D004950:Esterases