Toxic protein expression in Escherichia coli using a rhamnose-based tightly regulated and tunable promoter system.
The refinement of tightly regulated prokaryotic expression systems that permit functional expression of toxic recombinant proteins is a continually evolving process. Unfortunately, the current best promoter options are either tightly repressed and produce little protein, or produce substantial protein but lack the necessary repression to avoid mutations stimulated by leaky expression in the absence of inducer. In this report, we present three novel prokaryotic expression constructs that are tightly regulated by L-rhamnose and D-glucose. These expression vectors utilize the Escherichia coli rhaT promoter and corresponding regulatory genes to provide titratable, high-level protein yield without compromising clone integrity. Together, these components may enable the stable cloning and functional expression of otherwise toxic proteins.
San Diego State University, CA, USA.
Matthew J
MJ
Giacalone
Angela M
AM
Gentile
Brian T
BT
Lovitt
Neil L
NL
Berkley
Carl W
CW
Gunderson
Mark W
MW
Surber
D003001:Cloning, Molecular / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D024861:Genetic Enhancement / Q000379:methods; D011401:Promoter Regions, Genetic / Q000235:genetics*; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000633:toxicity*; D012210:Rhamnose / Q000235:genetics*
D011994:Recombinant Proteins; D012210:Rhamnose