One-step purification and characterization of human pancreatic lipase expressed in insect cells.
A cDNA clone encoding the sequence of human pancreatic lipase (HPL) was subcloned into the baculovirus transfer vector pVL1392 and used in co-transfection of Spodoptera frugiperda (Sf9) insect cells with wild-type Autographa californica nuclear polyhedrosis virus (AcNPV) DNA. A single recombinant protein (50 kDa) secreted by Sf9 cells was detectable in the culture medium 24 h post-infection using both anti-HPL polyclonal antibodies and potentiometric measurements of lipolytic activity. The expression level reached 40 mg/l of enzyme at 6 days. A single cation-exchange chromatography was sufficient to obtain a highly pure recombinant HPL as demonstrated by N-terminal sequencing, amino acid composition and carbohydrate analysis, as well as by mass spectrometry. These analyses revealed the production of mature protein with the correct processing of signal peptide and an homogenous glycosylation pattern. The kinetic properties of recombinant and native HPL were compared. Both enzymes showed similar profiles of interfacial activation, inhibition by bile salts and re-activation by colipase.
Thirstrup
K
K
Department of Protein Chemistry, Novo Nordisk, Novo Allé, Bagsvaerd, Denmark.
Carrière
F
F
Hjorth
S
S
Rasmussen
P B
PB
Wöldike
H
H
Nielsen
P F
PF
Thim
L
L
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae; D002240:Carbohydrate Sequence; D002460:Cell Line; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D008049:Lipase / Q000235:genetics / Q000302:isolation & purification*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009036:Moths; D010179:Pancreas / Q000201:enzymology; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification; D014162:Transfection
D011994:Recombinant Proteins; D008049:Lipase