Synthesis of chloramphenicol acetyltransferase in a coupled transcription‐translation in vitro system lacking the chaperones DnaK and DnaJ

[摘要]

A trimeric enzyme chloramphenicol acetyltransferase (CAT₁) has been synthesized in the Zubay system genetically depleted from DnaK and DnaJ. Most of CAT formed in the system fail to assemble into an active trimer. Instead CAT is accumulated in either a GroEL-bound complex or as an inactive monomer. Addition of purified DnaK and DnaJ to the system prior to the start of protein synthesis leads to the increase of the specific activity of formed CAT. A portion of exogenous DnaK and DnaJ added to the system associate with nascent polypeptide chains in the ribosomes. DnaK also comigrates with 50S-ribosomal subunits.

[学科分类] 生物化学/生物物理

[关键词] Protein folding;Chaperone;Chloramphenicol acetyltransferase;DnaK;DnaJ;in vitro protein synthesis;CAT;chloramphenicol acetyltransferase;SDS;sodium dodecyl sulfate;PAGE;polyacrylamide gel electrophoresis;ECL;enhanced chemiluminescence