Isolation and renaturation of bio-active proteins expressed in Escherichia coli as inclusion bodies.
Over-expression of recombinant proteins in Escherichia coli often results in the formation of insoluble and inactive material as inclusion bodies. Within the last years specific methods and strategies have been developed to produce bio-active proteins from these inclusion bodies. These methods include (i) isolation and purification of inclusion bodies, (ii) solubilization and reduction of the insoluble material, and (iii) renaturation of the proteins including formation of native disulphide bonds. Inclusion bodies are isolated from E. coli cells after desintegration of cells by mechanic forces. Due to their stability, inclusion bodies may be purified by washing with detergent solutions or low concentrations of denaturant. High concentrations of urea and guanidine hydrochloride are used in combination with reducing reagents such as 2-mercaptoethanol or dithiothreitol to reduce and solubilize proteins from inclusion bodies. After purification of solubilized materials, proteins are renatured and native disulphide bonds are formed by either air oxidation or glutathione reoxidation starting from reduced material, or by disulphide interchange starting from mixed disulphides containing peptides. Final yield of renatured proteins is raised by low concentrations of proteins and by adding low concentrations of denaturant during renaturation.
Fischer
B
B
AFRC Institute of Food Research, Department of Protein Engineering, Reading, England.
Sumner
I
I
Goodenough
P
P
D000818:Animals; D004926:Escherichia coli / Q000378:metabolism* / Q000648:ultrastructure; D006801:Humans; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification*
D011994:Recombinant Proteins