Leu-enkephalin purification from E. coli cells carrying the plasmid with fused synthetic leu-enkephalin gene

Chemically synthesized leu-enkephalin gene was fused to a large Eco RI-Barn HI fragment of pBR322 along with a Eco RI fragment of Ch4A phage DNA carrying the promoter and most of the E.coli β-galactosidase gene.The resulting recombinant DNA was used to transform E. coli cells. Transformants were scr...

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Bibliographic Details
Published in:Nucleic acids research 1980-12, Vol.8 (24), p.6163-6174
Main Authors: Shemyakin, M.F., Chestukhin, A.V., Dolganov, G.M., Khodkova, E.M., Monastyrskaya, G.S., Sverdlov, E.D.
Format: Article
Language:English
Subjects:
Chromatography, Gel
Chromatography, Paper
DNA, Recombinant - metabolism
Endorphins - isolation & purification
Enkephalin, Leucine
Enkephalins - biosynthesis
Enkephalins - isolation & purification
Escherichia coli
Escherichia coli - genetics
Escherichia coli - metabolism
Molecular Biology
Plasmids
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Summary:Chemically synthesized leu-enkephalin gene was fused to a large Eco RI-Barn HI fragment of pBR322 along with a Eco RI fragment of Ch4A phage DNA carrying the promoter and most of the E.coli β-galactosidase gene.The resulting recombinant DNA was used to transform E. coli cells. Transformants were screened for Tc-sensitivity, Am-resistance, and β-galactosidase constitutional synthesis. Restriction endonuclease analysis combined with DNA sequencing of the plasmid DNAs revealed a complete nucleotide leu-enkephalin sequence and Eco RI lac-operon fragment in two possible orientations. Radioimmunoassay for leu-enkephalin activity in BrCN-treated bacterial extracts showed that in vivo leu-enkephalin is synthesized only in strains carrying plasmids with the proper lac-fragment orientation. About 5.104 molecules of the former are synthesized per single E. coli cell. One of the clones was used for leu-enkephalin purification. Using 100 g of cells it is possible to obtain about 2 mg of practically pure leu-enkephalin.
ISSN:0305-1048
1362-4962
DOI:10.1093/nar/8.24.6163