Name:

Dissociation constants

Category:

Binding

Property:

Dissociation Constant (Kd)

Units:

nM

Measurement Technique:

ELISA , , Site-specific mutagenesis , dideoxy sequencing

Buffers:

None

Temperature:

None

pH:

None

Protein Concentration:

None

Wavelength:

None

Ionic Strength:

None

Details:

Site-specific mutagenesis was carried out on a single-stranded template (pB0475) that had unique restriction sites distributed uniformly about every 15 codons throughout a synthetic hGH gene. Synthetic oligonucleotides (generally 20 to 40 bases long) that coded for the desired alanine (or other) substitution and altered the closest singly occurring restriction site, were used to primer heteroduplex synthesis.

Heteroduplexes were transformed into E. coli BMH 71-18 mutL and the mixture was grown in LB broth (Luria broth) plus ampicillin (50 μg/ml). The mutant sequence was enriched from a mini-lysate of DNA by restriction with the enzyme in which the restriction site was altered by the oligonucleotide. Residual undigested DNA was transformed directly into E. coli JM101, and the correct mutants were identified by dideoxy sequencing.

Mutant and wilt-type GH were secreted from E. coli W3110 grown in 20 ml of minimal media containing low phosphate for 24 hours at 37 celsius degree in 250-ml shake flasks; periplasmic extracts were prepared by osmotic shock. The growth hormones were purified uniformly to about 60% homogeneity after an (NH4)2SO4 precipitation (final concentration equal to 45% saturation) and their concentrations were determined to precision of SD ~5% by densitometric scanning of Coo-massie-stained SDS gels.

The dissociation constant for the soluble portion of the cloned liver hgH receptor was determined by competitive displacement of 125I-labeled hgH and Scatchard analysis. The standard deviations for Kd were at or generally below 25% of the values reported.