The secretion of active recombinant human gastric lipase by Saccharomyces cerevisiae.
The expression of the complete human gastric lipase (HGL) gene in Saceharomyces cerevisiae grown in defined medium resulted in the secretion of active recombinant HGL (rec.HGL) to levels of up to approximately 11 mg/liter. Of the total measurable HGL activity, 90% was detected by assaying intact cells, suggesting that the majority of rec.HGL produced was secreted but stayed attached to the cell wall. The remaining 10% was present in the growth medium and from this source active rec.HGL was purified 300-fold by a combination of hydrophobic interaction and ion-exchange chromatography. Rec.HGL migrated on reduced SDS-PAGE as three bands with estimated molecular masses of 47,45, and 43 kDa. All three forms cross-reacted with an antibody raised to natural HGL and their treatment with Endo H showed them to be N-linked glycosylation variants of a single polypeptide. The 47-kDa species was isolated using lentil lectin Sepharose 4B and shown to possess a specific activity comparable to that of the natural enzyme. Rec.HGL had an acid pH activity optimum using either tributyrin or olive oil as substrate and did not lose activity if incubated in the presence of pepsin at pH 2.0. These results demonstrate that HGL secreted by Saccharomyces cerevisiae retained those properties of the natural enzyme required for its use in the treatment of pancreatic insufficiency.
Crabbe
T
T
Celltech Therapeutics Ltd., Slough, United Kingdom.
Weir
A N
AN
Walton
E F
EF
Brown
M E
ME
Sutton
C W
CW
Tretout
N
N
Bonnerjea
J
J
Lowe
P A
PA
Yarranton
G T
GT
D002847:Chromatography, Agarose; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D005822:Genetic Vectors; D006031:Glycosylation; D006596:Hexosaminidases / Q000378:metabolism; D006801:Humans; D008049:Lipase / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000096:biosynthesis*; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D014170:Transformation, Genetic; D014280:Triglycerides / Q000737:chemistry
D011994:Recombinant Proteins; D014280:Triglycerides; D008049:Lipase; D006596:Hexosaminidases; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; C005830:tributyrin