Cloning the cDNA for horse growth hormone and expression in Escherichia coli.
A 514 bp cDNA transcript coding for 78% of horse (Equus caballus.) GH has been cloned and sequenced. The deduced amino acid sequence corresponded precisely to that previously obtained by protein sequencing and, in addition, provided new sequence information for the signal peptide. The missing 3' fragment of the cDNA was reconstructed using synthetic oligonucleotides and site-specific directed mutagenesis. The complete cDNA sequence was then inserted into an expression vector (PIN-III-lppp-5) which utilizes a bacterial signal peptide to secrete the expressed product into the periplasmic space of Escherichia coli. Western blot analysis of cell lysates and periplasmic fractions prepared from cells harbouring this construct revealed significant quantities of immunoreactive GH and indicated that the bacterial signal peptide was successfully cleaved from the fusion protein on secretion. Recombinant-derived horse GH, recovered by osmotic shock from the periplasm, was active in a heterologous radioimmunoassay and a horse liver radioreceptor assay and resulted in a recovery of 0.5-2 mg GH/l cell culture. An apparent limitation on the secretion rate of horse GH in E. coli, possibly involving a block to translocation across the cytoplasmic membrane, prevented higher levels of expression being obtained.
Stewart
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Thoroughbred Breeders' Association Equine Fertility Unit, AFRC Institute of Animal Physiology and Genetics Research, Babraham, Cambridge.
Tuffnell
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PP
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods; D004247:DNA / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D013006:Growth Hormone / Q000032:analysis / Q000235:genetics*; D006736:Horses; D008969:Molecular Sequence Data; D008970:Molecular Weight; D015345:Oligonucleotide Probes; D010957:Plasmids; D011863:Radioimmunoassay; D011869:Radioligand Assay; D011994:Recombinant Proteins / Q000032:analysis; D015183:Restriction Mapping; D014158:Transcription, Genetic
D015345:Oligonucleotide Probes; D011994:Recombinant Proteins; D013006:Growth Hormone; D004247:DNA