Hyperexpression and purification of Escherichia coli adenylate cyclase using a vector designed for expression of lethal gene products.
We describe the construction of a new generation of vectors (pRE) for the hyperexpression of lethal gene products such as adenylate cyclase in Escherichia coli. The pRE vectors are based on the lambda PL promoter and lambda cII ribosome binding site described by Shimatake and Rosenberg (Nature, 292, 128-132, 1981). They have a unique NdeI restriction endonuclease site 3' of the lambda cII ribosome binding site that includes the ATG initiation codon, multilinker cloning sites 3' to the NdeI site, and two lambda transcription terminators 5' and 3' of the lambda PL promoter to eliminate nonspecific transcription and reduce leaky PL transcription, respectively. For hyperexpression of adenylate cyclase, tight control of transcription was necessary since elevation of cAMP levels above the physiological range is lethal to E. coli. Lethality associated with the overproduction of adenylate cyclase was shown to be mediated through the cAMP receptor protein. We used this expression system to overproduce adenylate cyclase 7500 fold, corresponding to 30% of the total cellular protein. Under these conditions the enzyme precipitated with significant loss of activity. Reducing the rate and amount of adenylate cyclase expression to 16% of the total cell protein produced one fourth of the enzyme in a soluble form with high specific activity. The soluble adenylate cyclase was purified to near homogeneity.
Reddy
P
P
Laboratory of Biochemical Genetics, National Heart, Lung and Blood Institute, Bethesda, MD 20892.
Peterkofsky
A
A
McKenney
K
K
D000262:Adenylyl Cyclases / Q000235:genetics* / Q000302:isolation & purification; D010582:Bacteriophage lambda / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D003062:Codon; D000242:Cyclic AMP / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D015870:Gene Expression; D005804:Genes, Lethal; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D010957:Plasmids; D011401:Promoter Regions, Genetic / Q000235:genetics; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics; D015183:Restriction Mapping; D012270:Ribosomes / Q000378:metabolism; D014158:Transcription, Genetic
D003062:Codon; D012333:RNA, Messenger; D000242:Cyclic AMP; C037106:endodeoxyribonuclease NdeI; D015252:Deoxyribonucleases, Type II Site-Specific; D000262:Adenylyl Cyclases