N-terminus deletion affecting the preparation of soluble cyanobacterial glutaredoxin in Escherichia coli.
Glutaredoxin has been implicated in maintenance of a normal cellular thiol/disufide ratio and the regeneration of oxidatively damaged proteins. In order to obtain more information about these important regulatory proteins in cyanobacteria, we have previously cloned and expressed the first cyanobacterial glutaredoxin gene ssr2061 in Escherichia coli. In this work, the second glutaredoxin gene slr1562 was studied. About 90% of Grx2061 coded by ssr2061 was produced in a soluble form while 90% of Grx1562 coded by slr1562 was found in inclusion bodies. To improve the production of soluble Grx1562, we constructed two mutants: Grx1562NC with cysteines in conserved site substituted by serines, and Grx1562M with N-terminus hydrophobic region deletion. Only the latter mutant was successfully expressed in soluble form with increased glutaredoxin activity and showed less sensitivity in oxidative stress. Spectroscopic analysis shows that the structure of Grx1562M with less hydrophobic nature could give more opportunity for protein solubility and could improve the substrate catalytic efficiency. These results suggest that hydrophobic N-terminus determines the insolubility of Grx1562 and may provide another strategy for increasing expression level of soluble heterologous proteins in E. coli.
Li
Min
M
Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing, PR China.
Yang
Qing
Q
Gao
Yang
Y
Wu
Qingyu
Q
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002384:Catalysis; D002942:Circular Dichroism; D000458:Cyanobacteria / Q000235:genetics / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D054477:Glutaredoxins; D057927:Hydrophobic and Hydrophilic Interactions; D008969:Molecular Sequence Data; D009154:Mutation; D018384:Oxidative Stress; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D012995:Solubility; D013050:Spectrometry, Fluorescence
D001426:Bacterial Proteins; D054477:Glutaredoxins; D011994:Recombinant Proteins; D010088:Oxidoreductases