米曲霉酰基辅酶A结合蛋白的重组表达、纯化及特性分析

Recombinant expression, purification, and characterization of an acyl-CoA binding protein from Aspergillus oryzae.

作者信息

Hao Qing, Liu Xiaoguang, Zhao Guozhong, Jiang Lu, Li Ming, Zeng Bin

机构信息

Key Laboratory of Industrial Fermentation Microbiology, National Engineering Laboratory for Industrial Enzymes, College of Biotechnology, Ministry of Education, Tianjin University of Science & Technology, Tianjin, 300457, China.

College of Chemical Engineering and Materials Science, Tianjin University of Science & Technology, Tianjin, 300457, China.

出版信息

Biotechnol Lett. 2016 Mar;38(3):519-25. doi: 10.1007/s10529-015-2003-1. Epub 2015 Dec 16.

DOI:10.1007/s10529-015-2003-1

PMID:26677124

Abstract

OBJECTIVES

To characterize biochemically the lipid metabolism-regulating acyl-CoA binding protein (ACBP) from the industrially-important fungus Aspergillus oryzae.

RESULTS

A full-length cDNA encoding a candidate ACBP from A. oryzae (AoACBP) was cloned and expressed in Escherichia coli as a maltose-binding protein (MBP) fusion protein. The MBP-AoACBP protein was purified by an amylose resin chromatography column. SDS-PAGE showed that MBP-AoACBP has an estimated molecular weight of 82 kDa. Microscale thermophoresis binding assay showed that the recombinant AoACBP displayed much greater affinity for palmitoyl-CoA (K d = 80 nM) than for myristoyl-CoA (K d = 510 nM), thus demonstrating the preference of AoACBP for long-chain acyl-CoA.

CONCLUSION

The data support the identification of AoACBP as a long-chain ACBP in A. oryzae.

摘要

目的

从具有重要工业价值的米曲霉中对调节脂质代谢的酰基辅酶A结合蛋白（ACBP）进行生化特性分析。

结果

克隆了编码米曲霉候选ACBP（AoACBP）的全长cDNA，并在大肠杆菌中作为麦芽糖结合蛋白（MBP）融合蛋白进行表达。通过直链淀粉树脂色谱柱纯化MBP-AoACBP蛋白。SDS-PAGE显示MBP-AoACBP的估计分子量为82 kDa。微量热泳结合试验表明，重组AoACBP对棕榈酰辅酶A（Kd = 80 nM）的亲和力比对肉豆蔻酰辅酶A（Kd = 510 nM）的亲和力大得多，从而证明了AoACBP对长链酰基辅酶A的偏好。