Automated purification of His6-tagged proteins allows exhaustive screening of libraries generated by random mutagenesis.
In the course of site-directed mutagenesis or directed evolution experiments, large numbers of protein variants are often generated. To characterize functional properties of individual mutant proteins in vitro, a rapid and reliable protein purification system is required. We have developed an automated method for the parallel purification of 96 different protein variants that takes about two hours. Using a 96-well format, the whole process can be performed automatically by a pipetting robot. Coupled with a suitable assay, again using a 96-well format, all variants can be functionally characterized within a few hours. The protein purification procedure described here is based on the interaction between His6-tagged proteins and Ni-NTA-coated microplates. Typical yields are 3-8 pmol purified protein/well, which is sufficient to analyze most enzymatic activities. Using this procedure, we have purified and characterized variants of the restriction endonuclease EcoRV, which were produced in an effort to enhance the selectivity of this enzyme. For this purpose, three amino acid residues were randomized in a region known from the co-crystal structure to be located at the protein-DNA interface. From a library of about 1200 variants, predominantly single and double mutants, more than 1000 variants were purified and characterized in parallel, which corresponds to an almost complete screening of the library.
Lanio
T
T
Justus-Liebig-Universität, Giessen, Germany.
Jeltsch
A
A
Pingoud
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A
D001331:Automation; D004269:DNA, Bacterial / Q000235:genetics; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D004798:Enzymes / Q000737:chemistry / Q000302:isolation & purification; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D015723:Gene Library; D005798:Genes, Bacterial; D006639:Histidine / Q000032:analysis*; D018189:Immunomagnetic Separation; D008863:Microspheres; D016296:Mutagenesis; D010957:Plasmids; D015202:Protein Engineering / Q000295:instrumentation / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification*; D012641:Selection, Genetic; D012685:Sepharose; D013997:Time Factors
D004269:DNA, Bacterial; D004798:Enzymes; D011993:Recombinant Fusion Proteins; D006639:Histidine; D012685:Sepharose; D015252:Deoxyribonucleases, Type II Site-Specific; C027440:GATATC-specific type II deoxyribonucleases