Protein aggregation kinetics in an Escherichia coli strain overexpressing a Salmonella typhimurium CheY mutant gene.
The tendency of recombinant protein in bacteria to partition into soluble and insoluble forms is attributed, in general, to a kinetic competition between protein folding and aggregation. However, little experimental work has actually been performed in vivo on the kinetics and mechanisms of protein folding and aggregation. Results are presented here from radiolabeling experiments which monitored the kinetics of recombinant protein aggregation in actively growing cultures. The strain used was an Escherichia coli strain overexpressing a Salmonella typhimurium CheY mutant gene. The rate of CheY aggregation was found to be time dependent in that the tendency of CheY to aggregate was greater for newly translated molecules, i.e., those translated within the previous several minutes, than for molecules translated less recently. CheY protein molecules that were translated less recently continued to aggregate for several hours but at a lower rate. The movement of soluble CheY to the insoluble form was enhanced at elevated growth temperatures and inhibited by the presence of chloramphenicol. The latter observation suggests that ongoing translation facilitates the movement of soluble CheY to the insoluble form. The implications of these results for the mechanism of protein aggregation in vivo, i.e., inclusion body formation, are discussed.
Klein
J
J
Department of Chemical Engineering, University of Delaware, Newark 19716, USA.
Dhurjati
P
P
D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism*; D002701:Chloramphenicol / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D029968:Escherichia coli Proteins; D005798:Genes, Bacterial; D002479:Inclusion Bodies / Q000378:metabolism; D007700:Kinetics; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism*; D000072236:Methyl-Accepting Chemotaxis Proteins; D009154:Mutation; D011487:Protein Conformation / Q000187:drug effects; D017510:Protein Folding; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012486:Salmonella typhimurium / Q000235:genetics*; D012995:Solubility; D013696:Temperature
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D000072236:Methyl-Accepting Chemotaxis Proteins; D011994:Recombinant Proteins; C000607606:cheY protein, E coli; D002701:Chloramphenicol