Effect of IPTG amount on apo- and holo- forms of glycerophosphate oxidase expressed in Escherichia coli.
Escherichia coli has proved to be a successful host for the expression of many heterologous proteins, and much efforts have been made toward improving recombinant protein expression including the usage of strong promoters and co-expression with chaperones. But little attention was paid on the relation between expression level and function of the target protein. Glycerophosphate oxidase (GPO) is a protein with FAD cofactor (without free cysteine and disulfide bonds).It was observed that the specific activity of GPO dramatically decreased with the increase of inducer IPTG. In addition, the stability of it decreased correspondingly. The structural difference of samples expressed under varying IPTG was investigated using size-exclusion and reverse-phase high performance liquid chromatography, together with CD spectrum. It was found that the conformation of peptide and organization of subunits were not affected. The loss of specific activity and stability were correlated to incomplete attachment of FAD onto GPO. These results revealed that synthesis speed should be controlled either by reduction of IPTG amount or using weak promoters in the production of GPO.
Zheng
Chunyang
C
Center for structural and molecular biology, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, PR China.
Zhao
Zhanyong
Z
Li
Yan
Y
Wang
Lin
L
Su
Zhiguo
Z
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D002845:Chromatography; D002942:Circular Dichroism; D003001:Cloning, Molecular; D016984:Enterococcus faecium / Q000201:enzymology* / Q000235:genetics; D004795:Enzyme Stability; D004926:Escherichia coli; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D005993:Glycerolphosphate Dehydrogenase / Q000235:genetics / Q000378:metabolism; D020035:Holoenzymes / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D007544:Isopropyl Thiogalactoside / Q000378:metabolism; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013057:Spectrum Analysis
D001426:Bacterial Proteins; D020035:Holoenzymes; D011994:Recombinant Proteins; D005182:Flavin-Adenine Dinucleotide; D007544:Isopropyl Thiogalactoside; D005993:Glycerolphosphate Dehydrogenase