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

• Published in: The Journal of steroid biochemistry and molecular biology Vol. 68; no. 3; pp. 119 - 127
• Main Authors: Sui, L.M, Lennon, J, Ma, C, McCann, I, Woo, I, Pétra, P.H
• Format: Journal Article
• Language: English
• Published: Oxford Elsevier Ltd 01.02.1999; Elsevier Science
• Subjects: 17β-Estradiol; Amino Acid Sequence; Analytical, structural and metabolic biochemistry; Biological and medical sciences; Cloning, Molecular - methods; Deglycosylated SHBG; Dihydrotestosterone - metabolism; DNA, Complementary; Fundamental and applied biological sciences. Psychology; Glycosylation; Humans; Kinetics; Mating Factor; Peptides - genetics; Pheromones; Pichia - genetics; Pichia - metabolism; Pichia pastoris; Protein hormones. Growth factors. Cytokines; Protein Sorting Signals - genetics; Proteins; Recombinant Proteins - biosynthesis; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; Saccharomyces cerevisiae; SBP; Sequence Deletion; Sex hormone binding globulin; Sex Hormone-Binding Globulin - biosynthesis; Sex Hormone-Binding Globulin - genetics; Sex Hormone-Binding Globulin - metabolism; Sex steroid-binding protein; SHBG; Testosterone; Yeast expression; SBP; SBP, plasma sex steroid-binding protein; SHBG, sex hormone binding globulin; DHT, 5α-dihydrotestosterone; 17β-Estradiol; fdphSBP, fully-deglycosylated Pichia-expressed hSBP; ABP, androgen binding protein; E 2, 17β-estradiol; Testosterone; pdphSBP, partially-deglycosylated Pichia-expressed hSBP; BMGY, buffered minimal glycerol-complex medium; wphSBP, wild type Pichia-expressed hSBP; BMMY, buffered methanol-complex medium; hSBP, human SBP; PAGE, polyacrylamide gel electrophoresis; Sex hormone binding globulin; Deglycosylated SHBG; SHBG; T, testosterone; Sex steroid-binding protein; Yeast expression; Yeast; Androgen; Estrogen; Sex hormone binding protein; Glycosylation; Ovarian hormone; Pichia; Fungi; Characterization; Side chain; Structure activity relation; Ascomycetes; Testicular hormone; Sex steroid hormone; Thallophyta
• DOI: 10.1016/S0960-0760(99)00024-2
• ISSN: 0960-0760

Wild type, partially and fully-deglycosylated human sex steroid-binding protein (SBP or SHBG) cDNAs lacking the native eucaryotic signal sequence were cloned into a yeast expression vector containing the Saccharomyces cerevisiae α-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 NH 2QSAHDPPAV- indicating that cleavage of the α-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 5α-dihydrotestosterone and steroid binding specificity were found to be identical to that of the human plasma protein indicating that the missing N-terminal segment NH 2-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.