摘要：Many protocols for recombinant Taq DN A polymerase I (Taq DNA Pol I) expression in E.coli resulted forming inclusion body; and preparation o f recombinant Taq DNA polymerase included mechanical or heat releasing recombinant Taq DNA polymerase into cell lysate, which contained a vast number o f subcellular components, native protein, and DNA pollution. Methods for removal o f these contaminants cost much time and effort. Some fusion tags can be used to facilitate secretion o f recombinant protein, better folding and sim plified downstream process could be achieved. However, due to the covalent linkage o f a tag, a protease treatment needs to be introduced. In the current study, we found that Taq DNA polymerase I can secrete intoE.coli cell culture medium without the need for any fusion tags.Escherichia coli expression constructs composed o f Taq DNA Pol I-6^his tag or "Taq DNA PolI were made for Taq protein expression in BL21(DE3) cells. Taq DNA polymerase I can secrete into E.coli cell culture w ith relatively high purity, downstream purification employed the thermostable properties o f Taq DNA Pol I. For Taq DNA Pol I expression and purification, cells were induced w ith 1mM IPTG for 10 hours. Cell culture supernatant was collected by centrifugation, and then dialyzed; a period o f 10 min o f 94°C heating,10 min freezing at 00C and centrifugation was used for the purification o f Taq DNA polymerase I protein in the cell culture supernatant. In these procedures, the contaminant proteins were denatured and removed as a precipitate by centrifugation. W ith this method, the whole purification process could be achieved w ithin 4 hours. A total yield o f 103.3 mg o f nearly homogenous Taq DNA Pol I enzyme w ith an activity o f 1.3x106 units were obtained from 1 liter o f induced Escherichia coli cell culture medium.This newly described method represents a simple technique to produce Taq DNA Pol I from the induced cell culture supernatant o f Escherichia coli;the purified enzyme is nuclease-free and nucleic acid-free. In addition, by using extracellular Taq DNA Pol I production, the purification procedures were very simple, unlike the previous reporting methods, which need selective precipitation and column chromatography to remove protein and DNA contaminants; the method purification o f Taq DNA Pol I from the cell culture supernatant only requires dialysis and heating-freezing treatment, which is effective and low cost, making it suitable for general purification in laboratories or business production.
关键词：Taq DNA polymerase I;extracellular protein production;polymerase chain reaction
