DnaK and DnaJ facilitated the folding process and reduced inclusion body formation of magnesium transporter CorA overexpressed in Escherichia coli

作者：Chen Y; Song JM; Sui SF; Wang DN^*

来源：Protein Expression and Purification, 2003, 32(2): 221-231.

DOI：10.1016/S1046-5928(03)00233-X

• magnesium transporter
• membrane protein folding
• inclusion bodies
• chaperones
• co-expression

Overexpression of CorA, the major magnesium transporter from bacterial inner membrane, in Escherichia coli resulted in the synthesis of 60 mg of protein per liter of culture, most of which however was in the form of inclusion bodies. The levels of inclusion body formation were reduced by lowering the cell culture temperature. To dissect CorA inclusion body formation and the folding process involved, we co-expressed the protein with various chaperones and other folding modulators. Expression of DnaK/DnaJ (Hsp70) prevented inclusion bodies from forming and resulted in the integration of more CorA into the membrane. GroEL/GroES (Hsp60/Hsp10) were less effective at reducing CorA inclusion body formation. Co-expression with either Ffh/4.5S-RNA, the signal recognition particle, or SecA, the ATPase that drives protein insertion into the membrane, had little effect on CorA folding. These results indicate: (1) that CorA inclusion bodies form immediately after synthesis at 37 degreesC, (2) that CorA solubility in the cytosol can be increased by co-expressing a chaperone system, (3) membrane targeting is probably not a rate-limiting factor, and (4) that membrane insertion becomes a limitation only when large amounts of soluble CorA are present in the cytosol. These co-expression systems can be used for producing other membrane proteins in large quantities.

• 出版日期2003-12
• 单位清华大学