Purification and interfacial behavior of recombinant human gastric lipase produced from insect cells in a bioreactor.
Recombinant human gastric lipase (rHGL) (EC 3.1.1.3) was produced on a large scale (5-13 mg/liter) from recombinant baculovirus-infected insect cells using a bioreactor apparatus. Here an improved procedure is described for purifying rHGL involving the use of cation exchange chromatography followed by immunoaffinity column methods, which gives a total yield of 62% and a purification factor of 464, using 10% isopropanol in all the purification buffers. The presence of isopropanol was necessary to preserve the stability of the enzyme during the chromatographic separation steps. The specific activity of rHGL on tributyroylglycerol (700 U/mg) was lower than that of native HGL (nHGL) (1080 U/mg). The rHGL interfacial adsorption kinetics were studied by recording the changes in the surface pressure with time in the presence or absence of an egg phosphatidycholine monomolecular film spread at the air/water interface at various initial surface pressures. The surface behavior of rHGL was similar to that of nHGL. It can be concluded that the lipid binding affinity of rHGL is identical to that of the native lipase and, consequently, that the presence of detergents and lipids in the insect cell culture media did not affect the interfacial behavior of the purified rHGL. It will be therefore possible to specifically study the binding step of HGL mutants to a lipid monolayer.
Laboratoire de Lipolyse Enzymatique, UPR 9025, de l'IFR-1 du CNRS, 31 Chemin Joseph-Aiguier, Marseille Cédex 20, 13402, France.
S
S
Canaan
L
L
Dupuis
M
M
Rivière
K
K
Faessel
J L
JL
Romette
R
R
Verger
C
C
Wicker-Planquart
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D019149:Bioreactors; D002460:Cell Line; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D003956:Dialysis; D005753:Gastric Mucosa / Q000201:enzymology*; D015870:Gene Expression; D006801:Humans; D008049:Lipase / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D009036:Moths; D010713:Phosphatidylcholines; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D012997:Solvents
D010713:Phosphatidylcholines; D011994:Recombinant Proteins; D012997:Solvents; D008049:Lipase