Human alphaB-crystallin. Small heat shock protein and molecular chaperone.
Human alphaB-crystallin. Small heat shock protein and molecular chaperone.
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DOI：
10.1074/jbc.272.4.2578
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发表时间：
1997-01
期刊：
影响因子：
--
通讯作者：
P. Muchowski;J. Bassuk;N. Lubsen;J. Clark
中科院分区：
文献类型：
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作者：
P. Muchowski;J. Bassuk;N. Lubsen;J. Clark
聚合酶链反应用于扩增编码人α B-晶状体蛋白的cDNA序列。将扩增的cDNA片段克隆到细菌表达载体pMAL-c2中，并表达为与麦芽糖结合蛋白（MBP）偶联的可溶性融合蛋白。经麦芽糖亲和层析和Xa因子裂解MBP后，通过阴离子交换层析将重组人α B-晶状体蛋白与MBP和Xa因子分离。采用SDS-聚丙烯酰胺电泳（PAGE）、Western免疫印迹分析、Edman降解、圆二色谱和尺寸排阻色谱对重组α B-晶状体蛋白进行表征。纯化的晶状体蛋白在SDS-PAGE上迁移至表观分子量（Mr约22，000），其对应于总的天然人α-晶状体蛋白，并在Western免疫印迹上被抗从透镜匀浆中纯化的人α B-晶状体蛋白的抗血清识别。化学测序，圆二色谱，和尺寸排阻色谱表明，重组晶体蛋白具有类似或相同的性质，其天然对应物。重组α B-晶状体蛋白和MBP-α B融合蛋白两者结合以形成高分子量复合物，所述复合物通过在37 ° C下抑制醇脱氢酶的聚集而显示出分子伴侣样功能，并且证明了α B-晶状体蛋白的C-末端结构域对于分子伴侣样活性的重要性。
The polymerase chain reaction was used to amplify a cDNA sequence encoding the human alphaB-crystallin. The amplified cDNA fragment was cloned into the bacterial expression vector pMAL-c2 and expressed as a soluble fusion protein coupled to maltose-binding protein (MBP). After maltose affinity chromatography and cleavage from MBP by Factor Xa, the recombinant human alphaB-crystallin was separated from MBP and Factor Xa by anion exchange chromatography. Recombinant alphaB-crystallin was characterized by SDS-polyacrylamide electrophoresis (PAGE), Western immunoblot analysis, Edman degradation, circular dichroism spectroscopy, and size exclusion chromatography. The purified crystallin migrated on SDS-PAGE to an apparent molecular weight (Mr approximately 22,000) that corresponded to total native human alpha-crystallin and was recognized on Western immunoblots by antiserum raised against human alphaB-crystallin purified from lens homogenates. Chemical sequencing, circular dichroism spectroscopy, and size exclusion chromatography demonstrated that the recombinant crystallin had properties similar or identical to its native counterpart. Both recombinant alphaB-crystallin and MBP-alphaB fusion protein associated to form high molecular weight complexes that displayed chaperone-like function by inhibiting the aggregation of alcohol dehydrogenase at 37 degrees C and demonstrated the importance of the C-terminal domain of alphaB-crystallin for chaperone-like activity.