Overexpression, purification, and characterization of recombinant barley alpha-amylases 1 and 2 secreted by the methylotrophic yeast Pichia pastoris.
Overexpression, purification, and characterization of recombinant barley alpha-amylases 1 and 2 secreted by the methylotrophic yeast Pichia pastoris.
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甲基营养酵母毕赤酵母分泌的重组大麦 α-淀粉酶 1 和 2 的过表达、纯化和表征。
DOI：
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发表时间：
1996
影响因子：
1.6
通讯作者：
B. Svensson
中科院分区：
文献类型：
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作者：
N. Juge;J. Andersen;D. Tull;P. Roepstorff;B. Svensson
重组大麦α -淀粉酶同工酶1和2在毕赤酵母中分别以高达50和1 mg/l的水平分泌，与酿酒酵母的外源表达水平相比，大约增加了50倍。将编码同工酶1和同工酶2的cDNA克隆E和pM/C分别置于毕赤酵母AOX1基因调控序列的控制下，置于载体pHIL-D2上。这两种同工酶都能根据其自身的信号序列有效地分泌到培养基中，并且很容易通过β -环糊精- sepharose亲和层析纯化到均匀的定量产量。n端序列，pI和Mr表明发生了类似于本地的处理。然而，电喷雾电离质谱分析显示重组同工酶1的微观异质性。虽然45,452的一个重组同工酶1形式的Mr与序列计算的45,447的值非常吻合，但在纳米电喷雾电离/质谱/质谱系统上对endo Lys c产生的肽进行液相色谱/质谱分析后，发现了其他重组同工酶1形式在Thr410上糖基化，在His1上n -乙酰化，在Cys95上s -谷胱甘肽化，或c端截断-412RS， -411QRS。和-410等。重组酶与大麦芽α -淀粉酶对不溶性蓝淀粉、直链淀粉（聚合度为17）和2-氯-4-硝基苯- d -麦芽糖苷的酶活性以及对Ca2+的依赖性都非常相似。毕赤酵母由此产生高产重组α -淀粉酶，其结构和功能与从麦芽提取物中纯化的酶相似。这极大地促进了未来大麦α -淀粉酶的突变分析，从而探究其结构/功能关系。
Recombinant barley alpha-amylase isozymes 1 and 2 were secreted by Pichia pastoris at up to 50 and 1 mg/liter, respectively, representing approximately a 50-fold increase compared to the levels of the heterologous expression by Saccharomyces cerevisiae. The cDNA clones E or pM/C encoding isozymes 1 and 2, respectively, were placed under the control of regulatory sequences from the Pichia AOX1 gene in the vector pHIL-D2. Both isozymes were effectively secreted to the medium as directed by their own signal sequences and easily purified to homogeneity in quantitative yield by affinity chromatography on beta-cyclodextrin-Sepharose. The N-terminal sequence, pI, and Mr indicated that native-like processing took place. Electrospray ionization mass spectrometry, however, revealed microheterogeneity for recombinant isozyme 1. While Mr of one recombinant isozyme 1 form of 45,452 was in excellent agreement with a value of 45,447 calculated from the sequence, liquid chromatography/mass spectrometry of endo Lys C-generated peptides followed by tandem mass spectrometry on a nanoelectrospray ionization/mass spectrometry/mass spectrometry system identified additional recombinant isozyme 1 forms to be glycosylated on Thr410, N-acetylated on His1, S-glutathionylated on Cys95, or C-terminally truncated of -412RS, -411QRS, and -410LQRS. The recombinant enzymes and the alpha-amylases from barley malt closely resembled each other in enzymatic activity on insoluble Blue Starch, amylose of degree of polymerization 17, and 2-chloro-4-nitrophenyl beta-D-maltoheptaoside as well as in Ca2+ dependency of activity. Pichia pastoris thus produced in high yields recombinant alpha-amylase that is similar with respect to structure and function to the enzyme purified from malt extracts. This greatly facilitates future mutational analysis of barley alpha-amylase in order to probe structure/function relationships.