Molecular cloning of a human gastric lipase and expression of the enzyme in yeast.
The molecular cloning of a cDNA coding for human gastric lipase and its expression in yeast is described. A lipase present in human gastric aspirates was purified and its N-terminal amino-acid sequence was determined. This was found to be homologous with the N-terminal sequence of rat lingual lipase. A cDNA library was constructed from mRNA isolated from human stomach tissue and probed with cloned rat lingual lipase DNA. One clone, pGL17, consisting of approximately 1450 base-pairs, contained the entire coding sequence for a human gastric lipase. The amino-acid sequence from the isolated protein and the DNA sequence obtained from the cloned gene indicated that human gastric lipase consists of a 379 amino acid polypeptide with an unglycosylated Mr of 43,162. Human gastric lipase and rat lingual lipase amino-acid sequences were closely homologous but were unrelated to porcine pancreatic lipase apart from a 6 amino-acid sequence around the essential Ser-152 of porcine pancreatic lipase. A yeast expression plasmid containing the phosphoglycerate kinase promoter and terminator sequences together with the human gastric lipase gene was constructed. Yeast transformed with this vector synthesised the lipolytically active enzyme.
Bodmer
M W
MW
Angal
S
S
Yarranton
G T
GT
Harris
T J
TJ
Lyons
A
A
King
D J
DJ
Pieroni
G
G
Riviere
C
C
Verger
R
R
Lowe
P A
PA
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D004274:DNA, Recombinant; D005088:Exocrine Glands / Q000201:enzymology; D006801:Humans; D008049:Lipase / Q000096:biosynthesis / Q000235:genetics*; D008970:Molecular Weight; D009693:Nucleic Acid Hybridization; D010179:Pancreas / Q000201:enzymology; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D012689:Sequence Homology, Nucleic Acid; D013270:Stomach / Q000201:enzymology*; D013552:Swine; D014170:Transformation, Genetic
D004274:DNA, Recombinant; D012333:RNA, Messenger; D008049:Lipase