Title Solubilities and Activities of Chloramphenicol Acetyltransferase and β-Lactamase Overproduced by the T7 Expression System in Escherichia coli
Author Kim, Han Bok
Address Department of Biochemistry, Hoseo University
Bibliography Korean Journal of Microbiology, 31(4),274-278, 1993
Key Words chloramphenicol acetyltransferase, β-lactamase, inclusion body, T7 expression system
Abstract Overproduced proteins in many cases result in forming insoluble inclusion bodies, and their formation might be due to high concentration of protein. To investigate how proteins become insoluble, chloramphenicol acetyltransferase (CAT) and β-lactamase were overproduced, and their solubilities and activities were determined. CAT was accumulated from 9 to 45% of total cellular protein in a fully soluble form without inclusion body formation. CAT specific activity was shown to be proportional to the amount of the protein produced. Moderately produced β-lactamase by the phase T7 expression system at 30℃ comprised only mature forms in a soluble form. However, overproduced β-lactamase at 37℃ became insoluble. Most precursor forms of β-lactamase in the cytoplasm were insoluble, whereas majority of the mature forms in the periplasm space were soluble. Also, chaperone GroE proteins which assist proper protein folding and translocation did not increase β-lactamase solubility significantly under the experimental condition. It seems that the formation of inclusion bodies in the cell is related to the nature of protein itself rather than just to high concentration of protein
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