Heterologous expression of wild type and deglycosylated human sex steroid-binding protein (SBP or SHBG) in the yeast, Pichia pastoris. Characterization of the recombinant proteins.
Wild type, partially and fully-deglycosylated human sex steroid-binding protein (SBP or SHBG) cDNAs lacking the native cucaryotic signal sequence were cloned into a yeast expression vector containing the Saccharomyces cerevisiae alpha-factor for extracellular secretion. Following transformation into Pichia pastoris, the wild type and all constructed mutants were successfully expressed. The levels were lower for the deglycosylated mutants indicating that oligosaccharide side chains may play a role in SBP secretion. Under fermentation conditions, the wild type protein was expressed at a level of 4 mg/l while the fully-deglycosylated mutant T7A/N351Q/N367Q was expressed at about 1.5 mg/l. The latter was purified from several fermentation runs and was found to be completely deglycosylated, electrophoretically homogeneous and fully active. The aminoterminus was found to have the sequence NH2QSAHDPPAV- indicating that cleavage of the alpha-factor occurred at the A(+7)-Q(+8) peptide bond. The molecular mass of the subunit was determined to be 39,717.8 Da, which is in complete agreement with the amino acid sequence of the T7A/N351Q/N367/Q mutant. The equilibrium constants for the dissociation of 5alpha-dihydrotestosterone and steroid binding specificity were found to be identical to that of the human plasma protein indicating that the missing N-terminal segment NH2-LRPVLPT and the removal of oligosaccharide side chains do not affect the stability and active conformation of the protein. In conclusion, the data presented reveal that the SBP mutant T7A/N351Q/N367/Q is the protein of choice for solving the three-dimensional structure.
Sui
L M
LM
Department of Biochemistry, University of Washington, Seattle 98195, USA.
Lennon
J
J
Ma
C
C
McCann
I
I
Woo
I
I
Pétra
P H
PH
D000595:Amino Acid Sequence; D003001:Cloning, Molecular / Q000379:methods; D018076:DNA, Complementary; D013196:Dihydrotestosterone / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D007700:Kinetics; D000072235:Mating Factor; D010455:Peptides / Q000235:genetics; D010675:Pheromones; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D021382:Protein Sorting Signals / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D017384:Sequence Deletion; D012738:Sex Hormone-Binding Globulin / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism
D018076:DNA, Complementary; D010455:Peptides; D010675:Pheromones; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D012738:Sex Hormone-Binding Globulin; D013196:Dihydrotestosterone; D000072235:Mating Factor