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Protein Eng

Title:		High-level expression of bovine beta-lactoglobulin in Pichia pastoris and characterization of its physical properties
Author(s):		Kim TR; Goto Y; Hirota N; Kuwata K; Denton H; Wu SY; Sawyer L; Batt CA;
Address:		"Department of Food Science, Cornell University, Ithaca, NY 14853, USA"
Journal Title:		Protein Eng
Year:		1997
Volume:		10
Issue:		11
Page Number:		1339 - 1345
DOI:		10.1093/protein/10.11.1339
ISSN/ISBN:		0269-2139 (Print) 0269-2139 (Linking)
Abstract:		"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"
Keywords:		"Amino Acid Sequence Animals Base Sequence Cattle Circular Dichroism Cloning, Molecular Crystallization Fermentation *Gene Expression Lactoglobulins/*chemistry/*genetics Magnetic Resonance Spectroscopy Mating Factor Molecular Sequence Data Peptides/genetic;"
Notes:		"MedlineKim, T R Goto, Y Hirota, N Kuwata, K Denton, H Wu, S Y Sawyer, L Batt, C A eng Research Support, Non-U.S. Gov't England 1998/03/26 Protein Eng. 1997 Nov; 10(11):1339-45. doi: 10.1093/protein/10.11.1339"