scholarly journals Overproduction of the cyclic AMP receptor protein of Escherichia coli and expression of the engineered C-terminal DNA-binding domain

1986 ◽  
Vol 236 (3) ◽  
pp. 643-649 ◽  
Author(s):  
A M Gronenborn ◽  
G M Clore
Keyword(s):  
Escherichia Coli ◽  
Dna Binding ◽  
Cyclic Amp ◽  
Binding Domain ◽  
Vector System ◽  
Receptor Protein ◽  
Cell Protein ◽  
Scale Production ◽  
Dna Binding Domain ◽  
Cyclic Amp Receptor Protein

Overproduction of the cyclic AMP receptor protein (CRP) from Escherichia coli, up to 25% of the soluble cell protein, has been achieved in an inducible host-vector system under transcriptional control of the lambda promoter PL. This system is ideally suited for large scale production and purification of CRP. In addition, a structural gene for the DNA-binding domain of CRP has been constructed. To this end the nucleotide sequence coding for the C-terminus was fused to the sequence coding for the first 10 N-terminal amino acids and cloned into suitable vectors. Good expression was achieved using the lambda PL promoter. The gene product, beta CRP, is recognized by anti-CRP antibodies.

scholarly journals 19F n.m.r. studies of conformational changes accompanying cyclic AMP binding to 3-fluorophenylalanine-containing cyclic AMP receptor protein from Escherichia coli

1992 ◽  
Vol 287 (2) ◽  
pp. 627-632 ◽  
Author(s):  
M G Hinds ◽  
R W King ◽  
J Feeney
Keyword(s):  
Escherichia Coli ◽  
Dna Binding ◽  
Cyclic Amp ◽  
Conformational Changes ◽  
Direct Evidence ◽  
Chemical Shifts ◽  
Binding Domain ◽  
Hinge Region ◽  
Receptor Protein ◽  
Intact Protein

A fluorine-containing analogue of the cyclic AMP (cAMP) receptor protein (CRP) from Escherichia coli was prepared by biosynthetic incorporation of 3-fluorophenylalanine (3-F-Phe). 19F n.m.r. studies on this protein have provided direct evidence for cAMP-induced conformational changes not only within the cAMP-binding domain but also within the hinge region connecting the cAMP-binding domain to the DNA-binding headpiece. At 313 K, the 19F n.m.r. spectrum of [3-F-Phe]CRP showed five signals corresponding to the five phenylalanine residues as expected for a symmetrical dimer. Proteolysis of [3-F-Phe]CRP with subtilisin produced a fragment (the alpha-fragment) containing the cAMP-binding domain. The alpha-fragment contains all the phenylalanines except for Phe-136, a residue located in the hinge region. By comparing the 19F spectra of [3-F-Phe]CRP and its alpha-fragment, the signal for Phe-136 was assigned. The chemical shifts of the corresponding signals in the two spectra are similar, indicating that the alpha-fragment retains the structure it has in the intact protein. The largest cAMP-induced shift was observed for the signal from Phe-136 providing direct evidence for a conformational change in the hinge region. However, whereas binding of a single cAMP molecule to a CRP dimer is known to be sufficient to activate the DNA binding, the n.m.r. data indicate that the hinge region does not have the same conformation in both subunits when only one cAMP molecule is bound.

The lacI Gene as a Target for Mutation in Transgenic Rodents and Escherichia coli

Genetics ◽  
1998 ◽  
Vol 148 (4) ◽  
pp. 1441-1451
Author(s):  
Johan G de Boer ◽  
Barry W Glickman
Keyword(s):  
Escherichia Coli ◽  
Dna Repair ◽  
Dna Binding ◽  
Transgenic Animals ◽  
Single Copy ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Large Database ◽  
Deletion Mutations ◽  
Laci Gene

Abstract The lacI gene has been used extensively for the recovery and analysis of mutations in bacteria with various DNA repair backgrounds and after exposure to a wide variety of mutagens. This has resulted in a large database of information on mutational mechanisms and specificity of many mutagens, as well as the effect of DNA repair background on mutagenicity. Most importantly, knowledge about the mutational sensitivity of the lacI gene is now available, yielding information about mutable nucleotides. This popularity and available knowledge resulted in the use of the lacI gene in transgenic rodents for the study of mutagenesis in mammals, where it resides in ~40 repeated copies. As the number of sequenced mutations recovered from these animals increases, we are able to analyze the sites at which mutations have been recovered in great detail and to compare the recovered sites between bacteria and transgenic animals. The nucleotides that code for the DNA-binding domain are nearly saturated with base substitutions. Even after determining the sequences of ~10,000 mutations recovered from the animals, however, new sites and new changes are still being recovered. In addition, we compare the nature of deletion mutations between bacteria and animals. Based on the nature of deletions in the animals, we conclude that each deletion occurs in a single copy of the gene.

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