Efficient cloning of a mutant adenylate-kinase-encoding gene from Escherichia coli

doi:10.1016/0378-1119(89)90246-1

Gene

Volume 80, Issue 1, 1 August 1989, Pages 21-28

https://doi.org/10.1016/0378-1119(89)90246-1 Get rights and content

Abstract

An optimized system has been developed for the transfer of a mutant gene from the Escherichia coli chromosome to a plasmid carrying the wild type (wt) allele.The wt allele was first cloned into a low-copy-number, self-transmissible plasmid with a single Eco RI, Hind111,and Bam HI site. The plasmid was then transferred to a mutant strain that had been previously transformed with a high-copy-number plasmid carrying the recA⁺ gene to allow efficient homologous recombination.A 15% frequency of homogenotization was obtained during cloning of an adk gene that encodes a temperature-sensitive adenylate kinase (AK).The mutant AK had decreased mobility on sodium dodecyi sulfate-polyacrylamide gels compared with the wt enzyme.This was due to a point mutation that changed leucine-107 in the wt enzyme to glutamine-107 in the mutant enzyme as determined by nucleotide sequencing.

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Cited by (5)

Use of adenylate kinase as a solubility tag for high level expression of T4 DNA ligase in Escherichia coli
2015, Protein Expression and Purification
Citation Excerpt :
Its functions may also include coordinating macromolecular synthesis and the production of adenosine tetraphosphate [17,18]. Our previous work showed that not only AK from E. coli could be expressed in soluble and active form at high level with little toxicity to the host cells [19], but also the enzyme could be easily purified via affinity elution off Blue Sepharose beads with a transition state substrate analog, Ap5A [20–23]. Here we explore the use of AK from E. coli as a solubility tag for high-level bacterial expression of T4 DNA ligase.
The discovery of T4 DNA ligase in 1960s was pivotal in the spread of molecular biotechnology. The enzyme has become ubiquitous for recombinant DNA routinely practiced in biomedical research around the globe. Great efforts have been made to express and purify T4 DNA ligase to meet the world demand, yet over-expression of soluble T4 DNA ligase in E. coli has been difficult. Here we explore the use of adenylate kinase to enhance T4 DNA ligase expression and its downstream purification. E. coli adenylate kinase, which can be expressed in active form at high level, was fused to the N-terminus of T4 DNA ligase. The resulting His-tagged AK-T4 DNA ligase fusion protein was greatly over-expressed in E. coli, and readily purified to near homogeneity via two purification steps consisting of Blue Sepharose and Ni–NTA chromatography. The purified AK-T4 DNA ligase not only is fully active for DNA ligation, but also can use ADP in addition to ATP as energy source since adenylate kinase converts ADP to ATP and AMP. Thus adenylate kinase may be used as a solubility tag to facilitate recombinant protein expression as well as their downstream purification.
PKILLIN: A versatile positive-selection cloning vector based on the toxicity of Killin in Escherichia coli
2014, Gene
Citation Excerpt :
A single colony containing pKILLIN-AK was verified for the expression of His-tagged AK upon IPTG induction using 12% SDS-PAGE and Coomassie Blue staining. All bacteria cells, including XL-1 containing pKILLIN, were first grown in LB until OD600 reaching 0.7 when IPTG induction (2 mM) began and lasted for 6 h before harvesting cells for protein extraction as described previously (Liang and Glaser, 1989). In addition, an AK enzyme activity assay was performed to confirm the expression following the method described previously (Huss and Glaser, 1983).
The invention of DNA cloning over 40 years ago marked the advent of molecular biology. The technique has now become a routine practice in any modern biomedical laboratory. Although positive-selection of recombinants in DNA cloning seems to be superior to blue/white selection based on the disruption of the lacZ gene, it is rarely practiced due to its high background, lack of multiple cloning sites, and inability to express the genes of interest or purify the protein products. Here we report the creation of a new positive-selection cloning vector dubbed pKILLIN, which overcomes all of the above pitfalls. The essence behind its high cloning efficiency is the extreme toxicity and small size of the toxic domain of killin, a recently discovered p53 target gene. Insertion inactivation of killin within the multiple cloning site via either blunt- or sticky-end ligation not only serves as a highly efficient cloning trap, but also may allow any cloned genes to be expressed as His-tagged fusion proteins for subsequent purification. Thus, pKILLIN is a versatile positive-selection vector ideal for cloning PCR products, making DNA libraries, as well as routine cloning and bacterial expression of genes.
High level expression and purification of recombinant proteins from Escherichia coli with AK-TAG
2016, PLoS ONE
Protein folding pathways of adenylate kinase from E. coli: Hydrostatic pressure and stopped-flow studies
2001, Biochemistry
Assignment of the nucleotide binding sites and the mechanism of substrate inhibition of Escherichia coli adenylate kinase
1991, Proteins: Structure, Function, and Bioinformatics

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Efficient cloning of a mutant adenylate-kinase-encoding gene from Escherichia coli

Abstract

J. Mol. Biol.

Gene

Gene

Cell

Gene

Plasmid

J. Mol. Biol.

J. Biol Chem.

Biochem. Biophys. Res. Commun.

Cell

Plasmid

Gene

Gene Anal. Techn.

Cloning and sequencing of the adenylate kinase gene (adk) of Escherichia coli

Nucleic Acids Res.

‘Western blotting’: electrophoretic transfer of proteins from sodium dodecyi sulfate-polyacrylamide gels to unmodified nitrocellulose and radiographie detection with antibody and radioiodinated protein A

Anal. Biochem.

Selection for the transfer of phenotypically nonselectable chromosomal mutations to re combinant plasmids through introduction of an altered restriction site

Gene

Recombination deficient mutants of E. coli and other bacteria

Annu. Rev. Genet

Revised interpretation of the origin of the pSClOl plasmid

J. Bacteriol.