Protein Expression & Isolation & Purification
Through expression , we can explore the function and the mechanism of regulation of gene expression , as well as the protein encoded by cloned gene expression can be used for the study of structure and function .
E . Coli is the most widely used protein expression systems and the expression level of exogenous gene product is much higher than other gene expression systems .
The amount of targeted protein expressed is even more than 80 % of the total bacterial proteins .
In this study , a plasmid carrying the gene of the target protein in E . coli BL21 , and at 37 C , IPTG induced overexpression of carries six consecutive histidine residues recombinant chloramphenicol acetyltransferase protein that available one kind by covalent conjugation of nitrilotriacetic acid ( NTA ) nickel ions ( Ni2 + ) immobilized chromatographic media to be purified , in fact boil metal affinity chromatography ( MCAC ) .
The degree of purification of the protein can be analyzed by polyacrylamide gel electrophoresis .
Reagents and Equipment ReagentLB broth : Trytone 10g , yeast extract 5g , NaCl 10g ; add up to 1000ml with distilled water ; ampicillin : 100mg / mLSampleBuffer : 100 mM NaH2PO4 , 10 mMTris , 8M Urea , 10 mM2 - ME , pH8.0 ( 4 ) Washing Buffer：100 mM NaH2PO4 , 10 mM Tris , 8 M Urea , pH6.3 ( 5 ) Elution Buffer : 100 mM NaH2PO4 , 10 mMTris , 8M Urea , 500 mM Imidazole , pH8.0 ( 6 ) IPTG EquipmentShaker , centrifuges , chromatography column ( 1'10 cm ) .
Operating method .
Induction of Chloramphenicol acetyltransferase recombinant protein .
Inoculate E . coli BL21 strain containing the recombinant protein of chloramphenicol acetyltransferase in 5mL LB liquid medium ( containing 100ug / mL ampicillin ) , 37 C shaking and culture overnight .
Transfer 1mL overnight cultures in 100 mL ( containing 100ug / mL ampicillin ) LB liquid medium ; 37 C shake culture until OD600 = 0.6 - 0.8 .
Take 10ul samples for SDS - PAGE analysis .
Add IPTG to a final concentration of 0.5 mmol / l , culture 1 - 3h at 37 C .
12,000rpm centrifugation for 10 min , discard the supernatant ; store cell precipitate at - 20 C or - 70 C .
To be continued . . .
