Reversion Analysis For Five Mutation Sites Altered Polymerase Function In The Large Fragment Of Taq DNA Polymerase

Taq polymerase I (Thermus aquaticus polymerasel ) is an important tool in the polymerase chain reaction (PCR), whose molecular weight is 93.9kDa. It belongs to the family of DNA polymerase I. Taq polymerase I has three structural domains: polymerase domain, 5'-3' exonuclease domain and 3'-5' exonuclease domain. The polymerase domain includes: palm domain, finger domain and thumb domain. The function of the palm domain is to catalyze the phosphoryl transfer reaction. The interaction between incoming nucleoside triphosphate and paired template base is in finger domain. The thumb domain works on positioning the duplex DNA and on processing translocation. The action mechanism of Taq polymerase is a two-metal ion catalysis mechanism. The conformational changes from open state to close state correspond to DNA polymerization. Taq polymerase I has 5'-3'exonuclease activity, but has not 3'-5'exonuclease proof-reading activity. Therefore, an error-process happens during Taq polymerase amplification. Through the deletion of 5'nuclease domain, the function of Taq polymerase has been efficiently improved. The fidelity is increase two folders. Large fragment Taq polymerase can amply GC rich template , and 35kb long run PCR. It is also regarded as ideal tool for DNA sequencing . In order to determine the specific active site and to improve the function, one inactive large fragment Taq DNA polymerase-LFTM5, is studied in this paper.LFTM5, 1682bp, was cloned into vector pPCR-Script, which was then transformed into E.coli DH5a. However, it can not express for the promoter interruption. Both expression and clone vector is reselected. pLiv-SelectII is used as clone, sequencing and mutation vector. pLiv-SelectII is 2.8kb. Blunt end restriction digestion site HpaI is for the ligation of pfu PCR production. The length of pLiv-SelectII/LFTM5 is 4.4kb that is ideal mutation template. There are universal sequencing primers beside MCS. Target was sequenced by LI-COR DNA sequence system and related analysis software. The result of blast shows that there are five mutant sites in LFTM5: K230N, A383T, F389Y, R399H and F491L. 30 reverse mutants are created. The rate of Y 389Fappears in active re-mutants is100%, and this indicates that F389 is a necessary active site .It is immutable. N230K and H399R have same frequency in active mutants 66%. T383A and L491F are same rate in active mutants 50%. This result suggest that K230 and R399 are more important than A383 and F491. Alignment with structurally functional site of other polymerase reveals that: F389Y in the O-helix of finger domain, is a conserved site interacting DNA in closed form; K230N between H1-helix and H2-helix of thumb domain is a related conserved site interacting with DNA in both open and closed form, and it is allowable substituted site. R399Hin O1-helix of finger domain interacting DNA in closed form is an allowable substituted site. A383T in O-helix of finger domain is not conserved site which is unnecessary for activity of polymerase.F491 in Q-helix of finger domain is not conserved site which is unrelated to activity of polymerase. However, the result of fidelity examination shows that errors of mutant A383T is 6-7 folds higher than wild type Taq DNA polymerase. It indicates that A383 is a critical site of fidelity, therefore, any substitution of this site would deduce fidelity.Target protein expression by pET System. The pET System is the most powerful system yet developed for the cloning and expression of recombinant proteins in E. coli. Target genes are cloned in pET plasmids under control of strong bacteriophage T7 transcription and (optionally) translation signals; expression is induced by providing a source of T7 RNA polymerase in the host cell. T7 RNA polymerase is so selective and active that, when fully induced, almost all of the cell's resources are converted to target gene expression; the desired product can comprise more than 50% of the total cell protein a few hours after induction. Although this system is extremely powerful, it is also possible to attenuate expression levels simply by lowering the concentration of inducer. Decreasing the expression level may enhance the soluble yield of some target proteins. Another important benefit of this system is its ability to maintain target genes transcriptionally silent in the uninduced state. Target genes are initially cloned using hosts that do not contain the T7 RNA polymerase gene, thus eliminating plasmid instability due to the production of proteins potentially toxic to the host cell . Once established in a non-expression host, target protein expression may be initiated either by infecting the host with λCE6, a phage that carries the T7 RNA polymerase gene under the control of the λ pL and pI promoters, or by transferring the plasmid into an expression host containing a chromosomal copy of the T7 RNA polymerase gene under lacUV5 control. In the second case, expression is induced by the addition of IPTG to the bacterial culture. Although in some cases (e.g., with innocuous target proteins) it may be possible to clone directly into expression hosts, this approach is not recommended as a general strategy. pET15b is expression vector. LFTM5 is inserted between NdeI and BamHI. 6-His peptide locates upstream N-terminal of the target gene for protein purification. Protein introduced by 0.5mM IPTG is faster than introduced by 0.3 IPTG. Finally, protein is yielded in 0.5mM IPTG 30℃ shaking culturing 5h.Protein purification by both His-tag affinity and heat purification. The procedure of His-tag affinity includes: binding, washing, elution and dialysis. Some protein will lose during purification. Therefore, protein collection by His-tag is less than by heating purification. However, the protein quality by His-tag affinity is higher than that by heating purification. PCR reaction is related to the purifying quality of enzyme protein. The unclean DNA and RNA in enzyme protein can disturb PCR reaction, result in irregular amplification and smear in electrophoresis. In addition, lower density of enzyme results in non PCR reaction or smear. Higher density of enzyme can suppress PCR reaction or smear.