Overproduction, purification and crystallization of TaqI restriction endonuclease.
Under phoA promoter control, TaqI endonuclease was overproduced to 5% of Escherichia coli cellular proteins. This was achieved by fusing the endonuclease gene to the first four codons of the alkaline phosphatase signal sequence. For maximal overproduction (30% of cellular proteins), a putative 14-bp hairpin within the endonuclease coding sequence was replaced with degenerate codons. In addition, TaqI methylase was required to protect host DNA. The endonuclease was purified in sufficient amounts for crystallization.
Barany
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Department of Microbiology, Hearst Microbiology Research Center, Cornell University Medical College, New York, NY 10021.
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004262:DNA Restriction Enzymes / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D004274:DNA, Recombinant; D015252:Deoxyribonucleases, Type II Site-Specific; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005786:Gene Expression Regulation; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010957:Plasmids; D011401:Promoter Regions, Genetic
D004274:DNA, Recombinant; D004262:DNA Restriction Enzymes; D015252:Deoxyribonucleases, Type II Site-Specific; C027046:TCGA-specific type II deoxyribonucleases