Over-expression and refolding of beta-subunit from the chloroplast ATP synthase.
We established a bacterial system for high-level over-expression of the spinach chloroplast atpB gene which encodes the ATP synthase beta subunit. Upon induction, atpB was expressed as at least 50% to 70% of total cell protein. Although the over-expressed beta polypeptide formed insoluble inclusion bodies, more than fifty percent of it was restored to a functional form by solubilizing the inclusion bodies with 4 M urea and slowly removing the urea by stepwise dialysis. The resulting beta subunit exhibited specific and selective nucleotide binding properties identical to those of the native beta subunit.
Chen
Z
Z
Department of Biochemistry, University of Kansas, Lawrence 66045.
Wu
I
I
Richter
M L
ML
Gegenheimer
P
P
D001483:Base Sequence; D002736:Chloroplasts / Q000201:enzymology*; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D010944:Plants / Q000201:enzymology; D011485:Protein Binding; D011487:Protein Conformation; D006180:Proton-Translocating ATPases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry*
D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; D006180:Proton-Translocating ATPases