High-level expression of bovine beta-lactoglobulin in Pichia pastoris and characterization of its physical properties.
Protein Eng
; 10(11): 1339-45, 1997 Nov.
Article
em En
| MEDLINE
| ID: mdl-9514124
Bovine beta-lactoglobulin (BLG) variant A has been expressed in the methylotropic yeast Pichia pastoris by fusion of the cDNA to the sequence coding for the alpha-mating factor prepro-leader peptide from Saccharomyces cerevisiae. P. pastoris Mut+ transformants were obtained by single cross-over integration of the BLG-containing vector into the AOX1 locus. In a fed-batch fermenter, a cell density of approximately 300 mg/ml was achieved by controlled glycerol feeding for a total of 24 h. After 72 h of methanol induction, the secreted BLG reached levels of > 1 g/l. The secreted protein could be purified to homogeneity by ion-exchange chromatography. Amino-terminal sequencing of the secreted BLG revealed that the Glu-Ala spacer repeats inserted between the mature protein and the alpha-factor prepro-leader were still present. The purified protein was characterized by a number of methods, including CD spectroscopy, guanidine-HCl unfolding, crystallization and two-dimensional 1H-NMR spectroscopy. By all of these measures, the physical characteristics of recombinant BLG were indistinguishable from those of the native purified bovine BLG, making it useful as a model for protein folding and other biophysical studies.
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Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Pichia
/
Expressão Gênica
/
Lactoglobulinas
Tipo de estudo:
Prognostic_studies
Limite:
Animals
Idioma:
En
Revista:
Protein Eng
Ano de publicação:
1997
Tipo de documento:
Article