Cloning And Analysis Of The Thermostable Domains In TAQ DNA Polymerase

After Taq DNA polymerase had been isolated and purified from the thermophilic bacteria (Thermus aquatics) in aquatic habitat by Randall K. Saiki and his colleagues in 1988, it was widely used in PCR techniques. High activity was still retained within Taq DNA polymerase after being treated with 95℃for hours, while no thermostability was observed within its homologous DNA polymerase from E.coli. This suggested that there be some thermostable tag(s) within the Taq DNA polymerase.In this experiment, several regions of Taq DNA polymerase gene were subcloned into pET-32a plasmids respectively. The recombinants were expressed, and the thermostatble domain H (399 bp) from Taq DNA polymerase was acquired with aid of thermo stability determination. DNA fragment of the thermostatble domain H was then fused with the non-thermostable gene IGF-II (insulin-like growth factor II) from Gymnocypris przewalskii. Results showed that H domain from Taq DNA polymerase was an efficient thermostable tag carrying recombinant of equal size in molecular weight. With aid of the tagged-H, the recombinant non-thermostable proteins of high purity (>90%) were acquired under treatment of 85℃for one hour.Immunization of mouse showed that there was no difference in antibody valence between the K50 recombinant (derived from Gymnocypris przewalskii) with H tag using thermo-purification and that using affinity chromatography (p>0.05). But very significant difference in antibody valence was found between the recombinant with H-tag and that without H-tag (p<0.01) (K50 and IGF-II were 6xHis-tagged; H tag was attached to K50 but not to IGF-II.). This suggests that H is a thermostable tag of high efficiency. With aim to prepare antigen derived from proteins independent on conformation, the thermo-purification could substitute the method of affinity chromatography based on 6xHis tag.In conclusion, both a thermostable tag of high efficiency and a simple economical purification with heat treatment were got in this experiment to prepare antigens with linear epitopes for acquiring monoclonal antibodies.