Nucleic Acids Research, 1989, Vol. 17, No. 24 10473-10488
© 1989
Articles |
Hyperexpression and purification of Escherichia coli adenylate cyclase using a vector designed for expression of lethal gene products
Laboratory of Biochemical Genetics, National Heart, Lung and Blood Institute 1Laboratory of Molecular Genetics, National Institute of Neurological and Communicative Disorders and Stroke Bethesda, MD 20892, USA
*To whom correspondence should be addressed
Received June 22, 1989. Revised October 23, 1989. Accepted October 23, 1989.
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 
PL promoter and cII ribosome binding site described by Shimatake and Rosenberg (Nature, 292, 128–132, 1981). They have a unique Ndel restriction endonuclease site 3' of the cII ribosome binding site that includes the ATG initiation codon, multilinker cloning sites 31 to the Ndel site, and two transcription terminators 51 and 31 of the 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.
+Present address: Center for Advanced Research in Biotechnology, National Institute of Standards and Technology, 9600 Gudelsky Drive, Rockville, MD 208050, USA
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