Resolution of the steroid-binding and dimerization domains of human sex hormone-binding globulin by expression in Escherichia coli

Biochemistry ◽  
1995 ◽  
Vol 34 (10) ◽  
pp. 3231-3238 ◽  
Author(s):  
Catherine Hildebrand ◽  
Wayne P. Bocchinfuso ◽  
David Dales ◽  
Geoffrey L. Hammond
Keyword(s):  
Escherichia Coli ◽  
Sex Hormone ◽  
Sex Hormone Binding Globulin ◽  
Hormone Binding ◽  
Expression In Escherichia Coli ◽  
Steroid Binding

Rabbit sex hormone binding globulin: primary structure, tissue expression, and structure/function analyses by expression in Escherichia coli

1997 ◽  
Vol 153 (3) ◽  
pp. 373-384 ◽  
Author(s):  
W M Lee ◽  
A S T Wong ◽  
A W K Tu ◽  
C-H Cheung ◽  
J C H Li ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Binding Affinity ◽  
Reproductive Tract ◽  
Dissociation Constants ◽  
Sex Hormone ◽  
Sex Hormone Binding Globulin ◽  
Hormone Binding ◽  
High Affinity ◽  
Steroid Binding

Abstract Sex hormone binding globulin (SHBG) is a homodimeric plasma protein found in mammals that binds sex steroids with high affinity and regulates their bioavailability. The protein is identical in structure and properties to the androgen binding protein (ABP) found in the male reproductive tract. We have isolated a 1245-base pair rabbit SHBG cDNA encoding a reading frame for a signal peptide followed by a protein of 367 amino acids, which shares 79·0, 68·1 and 63·2% amino acid identity with the corresponding human, rat and mouse proteins respectively. Northern blot and hot-nested PCR analyses indicated that rabbit SHBG is produced from a 1·6 kilobase mRNA in the liver of both sexes and in the testis. The rabbit SHBG cDNA was inserted into pGEX-1λT for expression of a glutathione S-transferase/SHBG fusion protein in Escherichia coli. The bacterial product bound 5α-dihydrotestosterone (DHT) in the same manner as the corresponding protein in serum. The dissociation constants (Kd) for rabbit and human SHBGs produced in E. coli were 11·1 ± 1·1 nm and 2·1 ± 0·6 nm respectively, and rabbit SHBG formed a less stable protein-steroid complex (t½=5 min) than human SHBG (t½>60 min). Unlike human SHBG, rabbit SHBG does not bind estradiol with high affinity. To aid in the identification of differences in the sequences of rabbit and human SHBG, which determine species differences in steroid-binding affinity and specificity, chimeras containing the 5′-terminal half of SHBG from one species and 3′-terminal half of SHBG from the other species were constructed and expressed. It was found that the chimeric proteins assumed similar steroid-binding affinity and specificity as the wild-type proteins when the amino (N)-terminal half of SHBG was derived from the same species. Replacement of the carboxyl (C)-terminal half of rabbit SHBG by the corresponding region of the human molecule increased the integrity of its steroid-protein complex. This supports the concept that amino acids within the N-terminal half of SHBG constitute the steroid-binding domain while the C-terminal half of the molecule may provide structural stability to the protein and its steroid-binding site. Journal of Endocrinology (1997) 153, 373–384

scholarly journals Resolution of the Human Sex Hormone-binding Globulin Dimer Interface and Evidence for Two Steroid-binding Sites per Homodimer

2001 ◽  
Vol 276 (37) ◽  
pp. 34453-34457 ◽  
Author(s):  
George V. Avvakumov ◽  
Irina Grishkovskaya ◽  
Yves A. Muller ◽  
Geoffrey L. Hammond
Keyword(s):  
Binding Sites ◽  
Sex Hormone ◽  
Sex Hormone Binding Globulin ◽  
Hormone Binding ◽  
Dimer Interface ◽  
Steroid Binding

The cDNA-deduced primary structure of human sex hormone-binding globulin and location of its steroid-binding domain

FEBS Letters ◽  
1987 ◽  
Vol 215 (1) ◽  
pp. 100-104 ◽  
Author(s):  
G.L. Hammond ◽  
D.A. Underhill ◽  
C.L. Smith ◽  
I.S. Goping ◽  
M.J. Harley ◽  
...  
Keyword(s):  
Primary Structure ◽  
Binding Domain ◽  
Sex Hormone ◽  
Sex Hormone Binding Globulin ◽  
Hormone Binding ◽  
Steroid Binding

Resolution of a Disordered Region at the Entrance of the Human Sex Hormone-binding Globulin Steroid-binding Site

2002 ◽  
Vol 318 (3) ◽  
pp. 621-626 ◽  
Author(s):  
Irina Grishkovskaya ◽  
George V Avvakumov ◽  
Geoffrey L Hammond ◽  
Yves A Muller
Keyword(s):  
Binding Site ◽  
Sex Hormone ◽  
Sex Hormone Binding Globulin ◽  
Hormone Binding ◽  
Steroid Binding

Time-resolved immunofluorometric assay of sex-hormone binding globulin.

1988 ◽  
Vol 34 (1) ◽  
pp. 63-66 ◽  
Author(s):  
S Niemi ◽  
O Mäentausta ◽  
N J Bolton ◽  
G L Hammond
Keyword(s):  
Binding Capacity ◽  
Reference Intervals ◽  
Sex Hormone ◽  
Sex Hormone Binding Globulin ◽  
Hormone Binding ◽  
Time Resolved ◽  
Interassay Coefficient ◽  
The Mean ◽  
Steroid Binding ◽  
The Eu

Abstract A time-resolved immunofluorometric assay (trlFMA) for human sex-hormone binding globulin (SHBG) is described in which antibody-coated tubes or microliter strip-wells and a europium (Eu) chelate-labeled monoclonal antibody are used. The trlFMA sensitivity is similar to that of other SHBG immunoassays, and other analytical variables compare favorably with an SHBG immunoradiometric assay (IRMA) kit and a steroid binding capacity assay: the interassay coefficient of variation (CV) is less than 8% and the intra-assay CV is less than 6% for concentrations between 6 and 200 nmol/L. The reference intervals (means +/- SD) for SHBG concentrations (nmol/L) in serum from 10 men, 10 women, and 10 pregnant women were 23 +/- 12, 65 +/- 39, and 439 +/- 122, respectively. In 14 hirsute women the mean +/- SD serum SHBG concentration (37 +/- 21 nmol/L) was significantly lower (P less than 0.01) than the mean for an age-matched, nonhirsute female comparison group. The trlFMA is technically simple, requires no centrifugation or separation reagent, and takes a counting time of only 1 s. In addition, the Eu-label is nontoxic, presents no waste-disposal problems, and has a long shelf life.

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