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...

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Bibliographic Details
Published in:Biotechnology letters 2006-07, Vol.28 (13), p.1019-1025
Main Authors: REYES-RUIZ, Jorge M, ASCACIO-MARTINEZ, Jorge A, BARRERA-SALDANA, Hugo A
Format: Article
Language:English
Subjects:
Animals
Base Sequence
Biological and medical sciences
Biotechnology
Capra hircus
Cattle
Enzymes
Fundamental and applied biological sciences. Psychology
Goats
Growth Hormone - genetics
Growth Hormone - metabolism
Growth hormones
Microbiology
Molecular Sequence Data
Mutagenesis
Mutagenesis, Site-Directed - methods
Pichia - genetics
Pichia - metabolism
Pichia pastoris
Polymerase Chain Reaction - methods
Protein Engineering - methods
Proteins
Recombinant Proteins - metabolism
Transfection - methods
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Summary: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.
ISSN:0141-5492
1573-6776
DOI:10.1007/s10529-006-9034-6