A novel protein refolding protocol for the solubilization and purification of recombinant peptidoglycan-associated lipoprotein from Xylella fastidiosa overexpressed in Escherichia coli

详细信息	查看全文 | 推荐本文 |

• 作者：Clelton A. Santos^a ; Lilian L. Beloti^a ; Marcelo A.S. Toledo^a ; Aline Crucello^a ; Marianna T.P. Favaro^a ; Juliano S. Mendes^a ; André ; S. Santiago^a ; Adriano R. Azzoni^a ; ^b ; Anete P. Souza^a ; ^c ; ^{anete@unicamp.br}
• 关键词：Protein refolding ; Pal ; Outer membrane protein ; Peptidoglycan ; Xylella fastidiosa
• 刊名：Protein Expression and Purification
• 出版年：2012
• 期刊代码：184_10465928
• 类别：bio
• 出版时间：April, 2012
• 卷：82
• 期：2
• 页码：284-289
• 文件大小：557 K

摘要

Xylella fastidiosa is a Gram-negative xylem-limited plant pathogenic bacterium responsible for several economically important crop diseases. Here, we present a novel and efficient protein refolding protocol for the solubilization and purification of recombinant X. fastidiosa peptidoglycan-associated lipoprotein (XfPal). Pal is an outer membrane protein that plays important roles in maintaining the integrity of the cell envelope and in bacterial pathogenicity. Because Pal has a highly hydrophobic N-terminal domain, the heterologous expression studies necessary for structural and functional protein characterization are laborious once the recombinant protein is present in inclusion bodies. Our protocol based on the denaturation of the XfPal-enriched inclusion bodies with 8 M urea followed by buffer-exchange steps via dialysis proved effective for the solubilization and subsequent purification of XfPal, allowing us to obtain a large amount of relatively pure and folded protein. In addition, XfPal was biochemically and functionally characterized. The method for purification reported herein is valuable for further research on the three-dimensional structure and function of Pal and other outer membrane proteins and can contribute to a better understanding of the role of these proteins in bacterial pathogenicity, especially with regard to the plant pathogen X. fastidiosa.