Over-expression of human sex steroid-binding protein (hSBP/hABP or hSHBG) in insect cells infected with a recombinant baculovirus. Characterization of the recombinant protein and comparison to the plasma protein.
Human sex steroid-binding protein (hSBP/hABP or hSHBG) was over-expressed in High Five and Sf9 cells adhered to plates and in suspension. The adherent cells expressed to levels of 2.3 mg/l and 1.4 mg/l after 4 and 6 days, respectively, while Sf9 cells grown in suspension yielded 4.67 mg/l after 6 days. Recombinant hSBP/hABP, purified to homogeneity by immunoadsorption, was found to fold similarly to native plasma hSBP/hABP and to display similar sequence epitopes after heat denaturation. The recombinant protein binds dihydrotestosterone, testosterone, and 17 beta-estradiol with KdS of 0.6, 2.4, and 14.2 nM, respectively, which are similar to plasma hSBP/hABP. The recombinant protein contains N-linked and O-linked oligosaccharide side-chains but the monomer exhibits a slightly lower molecular weight than plasma hSBP/hABP (40 kDa vs 44 kDa) which may be due to the absence of one N-linked side-chain or to shorter oligosaccharide side-chains. The partial N-terminal sequence LRPVLP(T)Q of recombinant hSBP/hABP is identical to plasma hSBP/hABP but appears to be less heterogeneous. These results indicate that recombinant baculovirus SBP represents a good model for investigating the structure of plasma hSBP/hABP. The expression system will allow the isolation of preparative amounts of SBP mutants generated by combinatorial site-directed mutagenesis to advance investigations on structure-function relationships and undertake crystallization trials for X-ray diffraction analyses.
Sui
L M
LM
Department of Biochemistry, University of Washington, Seattle 98195.
Wong
C
C
Petra
P H
PH
D000595:Amino Acid Sequence; D000818:Animals; D001667:Binding, Competitive; D002460:Cell Line; D000939:Epitopes / Q000032:analysis; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D017924:Nucleopolyhedroviruses / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D012738:Sex Hormone-Binding Globulin / Q000096:biosynthesis* / Q000737:chemistry / Q000502:physiology; D018411:Spodoptera
D000939:Epitopes; D011994:Recombinant Proteins; D012738:Sex Hormone-Binding Globulin