Molecular Cloning and Chartacterization DNA Polymerase I from thermophilic Geobacillus SBS 4S strain

Thermostable DNA polymerases are extensively used in biotechnology and life science applications. DNA Polymerase I was isolated from a hyperthermophile bacteria Geobacillus SBS 4S. Primers were designed using the template sequence of DNA Polymerase I gene of Geobacillus kaustophilus HTA26 strain. Nco 1 and Hind III sites were introduced on the forward and reverse primers respectively. Polymerase I gene of 2.6 Kb was cloned in pTZ57/ RT vector. Cloned gene of Polymerase I was restricted with Nco 1 and Bam H1, and ligated to pET 22b vector. Nco 1 site was used to insert twenty two N-terminal aminoacids (pelB) leader sequence at the start of the gene, which lead the recombinant protein in the periplasmic space, which increases the half life of recombinant protein. pelB fused with DNA Polymerase I produces soluble protein, which was detected after sonication. Sequencing shows that DNA polymerase I consists of 2499 bp with encodes for 832 amino acids, showed 99 % similarity with Geobacillus Kaustophillus . The expression of pelB fused DNA Polymerase I was optimized at different concentrations of IPTG and lactose. Highest expression was observed with 0.5mM IPTG and 20mM lactose. After Harvesting and sonication of BL21 codon plus cells, Polymerase I was produced in the soluble fraction. The supernatant containing the protein of interest, was separated after centrifugation at 10,000 rpm for 20 min. The protein was purified by ammonium sulphate precipitation and cation exchange column. The activity of purified DNA Polymerase I was checked by PCR reaction.

Sequencing shows that DNA polymerase I consists of 2499 bp with encodes for 832 amino acids, showed 99 % similarity with Geobacillus Kaustophillus .
The expression of pelB fused DNA Polymerase I was optimized at different concentrations of IPTG and lactose. Highest expression was observed with 0.5mM IPTG and 20mM lactose. After Harvesting and sonication of BL21 codon plus cells, Polymerase I was produced in the soluble fraction. The supernatant containing the protein of interest, was separated after centrifugation at 10,000 rpm for 20 min. The protein was purified by ammonium sulphate precipitation and cation exchange column.
The activity of purified DNA Polymerase I was checked by PCR reaction. Background DNA polymerases are key enzymes that are involved into DNA replication and repair processes that occur in all living organisms (1). Thermostable DNA polymerases are produced by thermophilic bacteria are valuable source of DNA polymerases. Thermophilic and hyperthermophilic enzymes have more importance over mesophilic enzymes, the most important application of thermostable enzyme is DNA polymerase.The most important application of DNA polymerase is in PCR. In prokaryotes there are three main types of DNA polymeases, Polymerase I, II and III. DNA polymerase III is the enzyme involved in DNA synthesis, whereas Pol I, and II has polymerase as well as exonuclease activity.
Synthesis of DNA by DNA polymerase occur in four steps. In the first step polymerase enzyme binds 3 to the template primer dimer. In the second step dNTPS binds to enzyme template primer complex, in the third step phosphodiester bond is formed as a result of nucleophilic attack, In the final step a conformational change cause elimination of pyrophosphate moiety (2).

Methods
Samples of Geobacillus SBS 4S was taken from Prof. Dr. Naeem Rashid (School of Bioloical Sciences).
Total DNA was isolated from bacterial cell, using classical method of Sambrook and Russels.(6) All the chemicals and kits. PCR ampilifaction kit was from Thermofisher Scientific. For plasmid DNA extraction "Vivantis Nucleic Acid Extraction" kit was used. InsTAclone TM PCR cloning kit was of vivantis. EcoRI, HindIII and BamH1was from Thermofisher Scientific. Thermo Scientific GeneJet TM MiniprepKit.

PCR amplification and primer designing
The sequence of DNA Polymerase I of Geobacillus kaustophilus was retrieved from NCBI database (GenBank: JYBP01000003.1). Primers were designed manually and checked on oligo nucleotide property calculator. Restriction sites were introduced at the start and end of the gene. One forward (Pol-F), one reverse primer (Pol-R) and one internal primer (Pol-I) were designed (Table 1).
PCR optimized with 0.15mM MgCl 2 , 0.25 mM DNTPS, 100 pmoles of forward (Pol-F) and reverse primer (Pol-R) and 5U of Taq DNA polymerase (Fermentas). PCR was performed with initial denaturation at 94 o C for 3 min, denaturation at 94 o C for 30sec, annealing at 55 o C for 90 sec, extension at 72 o C for 1min and final extension at 72 o C for 10min. The PCR product was extracted by Vivantis Quick Gel Extraction kit. The PCR samples was visualized on agarose gel and "Vivantis Nucleic Acid Extraction" kit was used to extract PCR product from agarose gel.

Cloning and Transformation of DNA polymerase I in pTZ57R/T vector
After Gene clean purified gene (2.6 kB) sample was cloned into cloning vector using ligation kit (InsTAclone TM PCR cloning kit). PCR product (2.6kB) Vector pTZ57R/T were mixed with 10 X ligation buffer (3ul) and incubated at 22 o C for overnight in water bath. DH 5 α competent cells were

Cloning and Transformation of DNA polymerase I in pET 22b vector
Pol-pTZ57R/T vector was restricted with NcoI first, in 50 ul reaction mixture, with 25 U of Nco1 and 1 µg of Pol-pTZ57R/T vector and incubated at 37 0 C for 3hrs. The reaction mixture was kept at -20 0 C overnight. Next day 25 U of BamH1 was added and incubated at 37 0 C for 3 hr. Sample was visualized on the agarose gel. Polymerase I gene insert (2.6 kb) was ligated to pET22b vector in a 30 µl reaction with gene to vector ratio of 3:1 and 100 U of T4 DNA ligase and incubated at 22 o C overnight.
Presence of insert (pol) into pET 22b vector was confirmed with double digestion with NcoI and Enzyme activity PCR amplification of the polymerase I gene was done to check the activity of the enzyme (pol-1) by using Pol-F and Pol-R primers. Reaction mixture was prepared in three PCR tubes. Own heat treated crude enzyme extract was added in one tube, purified protein was added to second tube & commercial taq polymerase (Thermofisher) was added to the third tube. The conditions used for PCR were initial denaturation at 94 0 C for 3 minutes, denaturation at 94 0 C for 30 sec, annealing at 55 0 C for 1 minute, extension at 72 0 C for 1minute and final extension at 72 0 C for 10 minutes. PCR product was analysed on 1% agarose gel. The heat treated lysate was further purified by passing through cation exchange column, desired protein was eluted at 1M NaCI concentration. Polymerase I was precipated at 40% saturation of ammonium sulfate.

Results And Discussion
On FPLC column polymerase I was eluted with 1M concentration of NaCI, 5.86mg of protein was 8 loaded on ion exchange column and 1.952 mg (figure 5a).
DNA polymerase I has 5'-3' polymerase activity and 3'-5' exonuclease activity. It fills the gaps which arises during DNA replication, repair and recombination. Sequencing results shows ten silent mutations in DNA polymerase I gene of Geobacillus SBS 4S (figure 2s).
Polymerase I from Geobacilus sp. was found to be 99KDa protein on SDS-PAGE, Taq DNA polymerase was cloned and expressed in E.coli. and molecular weight determination on SDS-PAGE showed a 94 KDa protein The purified enzyme retained the activity comparable to commercial Taq DNA polymerase (7).  (8). Domain C is most important domain that is further divided into three subdomains designated as thumb, palm and fingers with highly conserved motifs A, B and C respectively (9). The palm subdomain has catalytic center and contains conserved carboxylate residues while subdomains fingers and thumb are involved into binding of DNA and incoming dNTPs 9 during replication (10). Ten silent mutations were observed in polymerase I gene with one silent mutation in the C-terminal domain.
Cloning of DNA polymerase I from a thermophilic Rhodothermus marinus was done in E.coli. Sequence analysis of protein showed mutation at 756 position where phenylalanine was substituted by tyrosine.
DNA polymerase from Pyrococcus KOD1 strain encode for 1671 amino acids polymerase I gene was expressed in E.coli (12). DNA polymerase from Geobacillus sp. SBS 4S was sequenced and purified for the first time. After heat treatment, ammonium sulphate precipitation and FPLC the protein was 99 % pure and was successfully used in a PCR reaction.     The supernatant and pellet checked after heat treatment at 60oC. Lane 1: without heat treatment. Lane 2 & 3 supernatant and pellet after heat treatment at 60oC.

Supplementary Files
This is a list of supplementary files associated with this preprint. Click to download.