Recombinant human bile salt-stimulated lipase: an example of defective O-glycosylation of a protein produced in milk of transgenic mice.
The expression of recombinant human bile salt-stimulated lipase (bssl) was targeted to the lactating mammary gland of transgenic mice. Expression of recombinant genes comprising bsslcDNA, or alternatively genomic bssl DNA, under control of regulatory elements derived from the murine whey acidic protein (wap) gene was achieved and evaluated. Constructs containing genomic bssl sequences mediated high levels (0.5-1 mg ml-1) of recombinant human BSSL in the milk. The recombinant BSSL produced was purified, biochemically characterized and compared to native BSSL and recombinant BSSL produced in mouse C127 and hamster CHO cells. Recombinant BSSL derived from transgenic mice showed a different migration and distribution after SDS-PAGE electrophoresis, lower apparent molecular mass on size-exclusion chromatography and no detectable interactions with a panel of lectins. These results indicate a significantly lower degree of O-glycosylation of recombinant BSSL in milk from transgenic mice than was found for the native enzyme or recombinant CHO- or C127 cell-produced BSSL. Despite these differences, mouse-milk-derived recombinant BSSL exhibited similar lipase activity, the same stability to low pH and similar sensitivity to elevated temperatures as the native enzyme. The observation that mouse-C127-cell-produced recombinant BSSL is heavily O-glycosylated makes species-related restrictions less attractive as an explanation for the reduced O-glycosylation.
Strömqvist
M
M
Astra Hässle AB, Umeå, Sweden.
Törnell
J
J
Edlund
M
M
Edlund
A
A
Johansson
T
T
Lindgren
K
K
Lundberg
L
L
Hansson
L
L
D000818:Animals; D002241:Carbohydrates / Q000032:analysis; D002478:Cells, Cultured; D006224:Cricetinae; D005260:Female; D005786:Gene Expression Regulation; D006031:Glycosylation; D006801:Humans; D008049:Lipase / Q000235:genetics* / Q000378:metabolism*; D008321:Mammary Glands, Animal / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D008808:Mice, Inbred CBA; D008822:Mice, Transgenic; D008892:Milk / Q000201:enzymology*; D008894:Milk Proteins / Q000235:genetics / Q000378:metabolism; D008895:Milk, Human / Q000201:enzymology; D008970:Molecular Weight; D012333:RNA, Messenger / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002787:Sterol Esterase
D002241:Carbohydrates; D008894:Milk Proteins; D012333:RNA, Messenger; D011994:Recombinant Proteins; C024471:whey acidic proteins; C054784:bile salt-stimulated lipase; D002787:Sterol Esterase; D008049:Lipase