Construction of an overproducing strain, purification, and biochemical characterization of the 6His-Eco29kI restriction endonuclease.
We constructed a strain of Escherichia coli overproducing 6His-tagged Eco29kI by placing the coding sequence under control of a strong bacteriophage T5 promoter. The yield of 6His-Eco29kI restriction endonuclease expression could be increased to about 20% of the total cellular protein, but inclusion bodies formed consisting of insoluble 6His-Eco29kI protein. We developed a fast and effective protocol for purification of the homogeneous enzyme from both soluble and insoluble fractions and established their identity by catalytic activity assay. The isolated enzymes were tested for recognition specificity and optimal reaction conditions as a function of NaCl and KCl concentrations, temperature, and pH compared with the native Eco29kI restriction endonuclease. The 6His-tagged enzyme retained the specificity of the native protein but had an altered optimum of its catalytic reaction.
Nikitin
Dmitri
D
Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, Prospekt Nauki, 5, Pushchino, Moscow region 142290, Russia. dvnikitin@rambler.ru
Mokrishcheva
Marina
M
Denjmukhametov
Marat
M
Pertzev
Alexander
A
Zakharova
Marina
M
Solonin
Alexander
A
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D004587:Electrophoresis, Agar Gel; D004926:Escherichia coli / Q000235:genetics; D006639:Histidine / Q000378:metabolism*; D002479:Inclusion Bodies / Q000737:chemistry; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012995:Solubility
D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; D006639:Histidine; C035337:CCGCGG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific