PCR directed preparation and single step purification of highly active histidine-tagged restriction endonuclease HgiBI (GGWCC).
The polymerase-chain-reaction technique is used to produce fusion proteins via deletion of any intervening piece of DNA. Here a stretch of six histidine codons is fused to the 3'-terminus of any defined gene using a standard plasmid vector or a derivative thereof. The advantage over existing methods is that no other amino acids besides the six histidines are added to the protein terminus and only one oligonucleotide needs to be synthesized as special primer. Genes of interest must only be cloned in the correct orientation into a universal multilinker. Using just one specific primer derived from the 3'-terminus of the gene and one standard primer derived from the six histidine codons the fusion is performed by amplifying the entire vector system as described for inverse PCR. As an example, we report on the modification and purification of the restriction endonuclease HgiBI (GGWCC). Enzymatically active protein was obtained in a single step purification under nondenaturating conditions with a purity greater than 95% according to polyacrylamide gel electrophoresis.
Blum
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E
Institut für Mikrobiologie und Molekularbiologie, Justus-Liebig-Universität Giessen, Germany.
Horst
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G
Kröger
M
M
D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods; D003062:Codon; D015252:Deoxyribonucleases, Type II Site-Specific / Q000096:biosynthesis* / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D005822:Genetic Vectors; D006639:Histidine; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016133:Polymerase Chain Reaction / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D015183:Restriction Mapping; D017384:Sequence Deletion; D016324:Sequence Tagged Sites
D003062:Codon; D011993:Recombinant Fusion Proteins; D006639:Histidine; D015252:Deoxyribonucleases, Type II Site-Specific; C038308:GGWCC-specific type II deoxyribonucleases