Kim T R, Goto Y, Hirota N, Kuwata K, Denton H, Wu S Y, Sawyer L, Batt C A
Department of Food Science, Cornell University, Ithaca, NY 14853, USA.
Protein Eng. 1997 Nov;10(11):1339-45. doi: 10.1093/protein/10.11.1339.
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.
牛β-乳球蛋白（BLG）变体A已通过将其cDNA与编码来自酿酒酵母的α-交配因子前原导肽的序列融合，在甲基营养型酵母毕赤酵母中表达。通过将含BLG的载体单交换整合到AOX1基因座中获得了毕赤酵母Mut+转化体。在分批补料发酵罐中，通过控制甘油补料共24小时，细胞密度达到约300mg/ml。甲醇诱导72小时后，分泌的BLG水平达到>1g/l。分泌的蛋白质可通过离子交换色谱法纯化至同质。对分泌的BLG进行氨基末端测序表明，插入成熟蛋白和α-因子前原导肽之间的Glu-Ala间隔重复序列仍然存在。通过多种方法对纯化的蛋白质进行了表征，包括圆二色光谱、盐酸胍变性、结晶和二维1H-NMR光谱。通过所有这些测量，重组BLG的物理特性与天然纯化的牛BLG的物理特性没有区别，使其成为蛋白质折叠和其他生物物理研究的有用模型。
