High-level expression of bovine beta-lactoglobulin in Pichia pastoris and characterization of its physical properties.
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.
Kim
T R
TR
Department of Food Science, Cornell University, Ithaca, NY 14853, USA.
Goto
Y
Y
Hirota
N
N
Kuwata
K
K
Denton
H
H
Wu
S Y
SY
Sawyer
L
L
Batt
C A
CA
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D002942:Circular Dichroism; D003001:Cloning, Molecular; D003460:Crystallization; D005285:Fermentation; D015870:Gene Expression; D007782:Lactoglobulins / Q000737:chemistry* / Q000235:genetics*; D009682:Magnetic Resonance Spectroscopy; D000072235:Mating Factor; D008969:Molecular Sequence Data; D010455:Peptides / Q000235:genetics; D010675:Pheromones / Q000235:genetics; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D017510:Protein Folding; D011993:Recombinant Fusion Proteins; D012441:Saccharomyces cerevisiae / Q000235:genetics; D017421:Sequence Analysis
D007782:Lactoglobulins; D010455:Peptides; D010675:Pheromones; D011993:Recombinant Fusion Proteins; D000072235:Mating Factor