Expressed protein ligation-mediated template protein extension

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• 作者：Ayako Kamei^a ; Paul S. Hauser^a ; Jennifer A. Beckstead^a ; Paul M.M. Weers^b ; Robert O. Ryan^a ; ^{rryan@chori.org}
• 关键词：Expressed protein ligation ; Apolipoprotein A-I ; Milano ; Protein stability ; Intein
• 刊名：Protein Expression and Purification
• 出版年：2012
• 期刊代码：184_10465928
• 类别：bio
• 出版时间：June, 2012
• 卷：83
• 期：2
• 页码：113-116
• 文件大小：446 K

摘要

Expressed protein ligation (EPL) was performed to investigate sequence requirements for a variant human apolipoprotein A-I (apoA-I) to adopt a folded structure. A C-terminal truncated apoA-I, corresponding to residues 1-172, was expressed and isolated from Escherichia coli. Compared to full length apoA-I (243 amino acids), apoA-I(1-172) displayed less 伪-helix secondary structure and lower stability in solution. To determine if extension of this polypeptide would confer secondary structure content and/or stability, 20 residues were added to the C-terminus of apoA-I(1-172) by EPL, creating apoA-I_Milano(1-192). The EPL product displayed biophysical properties similar to full-length apoA-I_Milano. The results provide a general protein engineering strategy to modify the length of a recombinant template polypeptide using synthetic peptides as well as a convenient, cost effective way to investigate the structure/function relations in apolipoprotein fragments or domains of different size.