Derivation of a growth hormone gene cassette for goat by mutagenesis of the corresponding bovine construct and its expression in Pichia pastoris.
Recombinant bovine growth hormone (rbGH), a 191-aa polypeptide that affects animal growth and lactation, has been used for several years to increase milk production in dairy cattle. It has also been used in goats (Capra hircus) instead of their own hormone (chGH), which is still not available in the market. Since both hormones differ in only one amino acid residue, a strategy based on PCR mediated site-directed mutagenesis, was used to convert the bGH expression cassette harbored by an integration plasmid for Pichia pastoris into a chGH. Transformation by homologous recombination of Pichia pastoris GS115 strain with the linearized new plasmid resulted in transformants that, upon fermentation and induction with methanol, secreted a band with the expected size and immunoreactivity for GH. Production of total proteins secreted into culture medium (50 ml) was 20 microg/ml, of which 60% was chGH as judged by densitometry in SDS-PAGE. Its biological activity was confirmed in vitro when 3T3 pre-adipocytes exposed to the induced culture medium differentiated into adipocytes in cell culture.
Reyes-Ruíz
Jorge M
JM
Laboratorio de Ingeniería y Expresión Genéticas, Departamento de Bioquímica, Facultad de Medicina, Universidad Autónoma de Nuevo León, Col. Mitras Centro, 64460 Monterrey, NL, México.
Ascacio-Martínez
Jorge A
JA
Barrera-Saldaña
Hugo A
HA
D000818:Animals; D001483:Base Sequence; D002417:Cattle; D006041:Goats; D013006:Growth Hormone / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D016133:Polymerase Chain Reaction / Q000379:methods; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000378:metabolism; D014162:Transfection / Q000379:methods
D011994:Recombinant Proteins; D013006:Growth Hormone