Cloning, Expression And Functional Identification Of Terminase From Pseudomonas Aeruginosa Bacteriophage PaP3

Terminase enzymes are found in many double-stranded DNA bacteriophage and eukaryotic DNA virus such as adenovirus and the herpesvirus group.These enzymes function to insert a viral genome into the confines of a preformed,empty precapsid.The bacteriophage terminase is a hetero-oligomer composed of two subunits,one is named as large subunit for its larger molecular weight,the other is named as small subunit.The large subunit possesses nuclease,ATPase catalytic activity which co-worked with the small one. Because the packaging paticipated by terminase is very unique,the research on the enzyme should be an ideal model of interacton between protein and protein,nucleotide and protein; besides,as a special protein in the virus or bacteriophage life period,the host shouldn't have any similar enzyme protein,so terminase would be a safe and specific drug target to control bacteriophage contamination and virus infection.In this study,we focus on the presumption and experimental identification of the terminase from bactriophage PaP3,the contents and results are as follows:1.Cloning,expression and renaturation of the putative terminase large subunit(tls) of PaP3.â‘ The putative tls gene was successfully amplified from genomic DNA of PaP3 by PCR.â‘¡The PCR product of putative tls gene was inserted into pQE31 vector and transformed into E.coli JM109.The recombinant pQE31-tls/JM109 expressed the fusion protein at a level about 30%of total cell proteins,and nearly all of it was in the inclusion-body.â‘¢In a 3.7L ferminator,the recombinant bactria were firstly cultured in the LB medium at pH 7.4,37℃,PO₂30%～40%for 4hr(OD₆₀₀≈2.5),then fed the organisms by continuous accelaration pattern,the organisms flow rate is 1.25ml/min,the volume is 150ml 20×LB,the ultimate yield reached 36.241g/L,and the fusion protein is at a level about 30% of total cell proteins.â‘£The inclusion body of fusion protein were extract from cultures of stably transformed E.coli JM109,and then dissolved in 8mol/L urea,the fusion H6-TLS was captured by Ni-NTA column and further purified by ion exchange chromatography.⑤The purified fusion protein was renatured through dialysis,then the consistency of the protein was analysised by BCA method.2.The experimental identification of putative terminase large subunit of PaP3:â‘ A target sequence containing the end(cosN) of PaP3 genome was amplified by PCR,then the PCR product was inserted into the pMD18-T vector,and contrasted a substrate plasmid pMD-cos which can be cutted by TLS;â‘¡Under the contidion of Mg²⁺,K⁺,pH8.0,the H6-TLS can partily cut the substrate plasmid pMD-cos,the result shows the H6-TLS has the activity of terminase large subunit.â‘¢After synthesization and label of the 20bp cosN oligonucleotide,the results of EMSA shows Hr-TLS can bind the cosN,which demonstrated H6-TLS's another function.â‘£Utilizing the traditional ATPase test method, H6-TLS didn't shows any ATPase activity.3.The scretion expression of terminase large subunit of bacteriophage PAP3:â‘ The tls gene was successfully amplified from genomic DNA of PaP3 by PCR.â‘¡The PCR product of tls was inserted into pET22b(+) vector and transformed into E.coli BL21.Under the culture condition of 1.2ug/ml chloromycetin,100ug/ml AMP,inducted by 0.05mol/L IPTG for 18hr at 25℃,the recombinant TLS-rH expressed at a level of 20.08%of total cell proteins,and nearly all of it was secreted into the supernatant.Then the fusion protein was purified by Ni-NTA affinity chromatography.â‘¢By the chemiluminesce method,the TLS-6H can hydrolysis ATP,which independent with DNA,indicating the ATPase activity of TLS-6H.â‘£The secrected fusion protein TLS-6H has higher nucleasing activity than 6H-TLS,and the best condition for the catalytic activity is pH8.0,50mmol/L Mg²⁺, 50mmol/L K⁺,37℃,lh,and it is not sitimulated by ATP and sperimide.5.The putative and experimental evidence for terminase small subunit(tss) of bacteriophage PaP3:â‘ By searching the encoding protein's conserved domain and the putative motif,and analyzing the gene organization of PAP3,the gene PaP3p01(orf1) was presumed as tss of PaP3.â‘¡The gene PaP3p01 was amplified from genome DNA of PAP3.â‘¢The PCR product of PaP3p01 was inserted into pQE31 vector and transformed into E.coli JM109.The recombinant pQE31-tss/JM109 expressed the fusion H6-TSS at a level about 25.8%,and all of it was soluble in the host endochylema.â‘£A 239bp DNA sequence including cosN and its upstream and downstream was cut from the PCR product of the genome end,and the 3' end of the 239bp sequence was labeled with biotin.⑤The results of EMSA showed that H6-TSS can bind the 239bp sequence but not another downstream 100bp sequence,it infers the fusion H6-TSS has the specifical DNA-binding ability,and it demonstrated the correctness of the presumption that PaP3p01 is the coding sequence of terminase small subunit of PaP3.In conclusion,the terminase's two subunits were presumpted and proved by experimental evidence,and some character of the enzymes had been researched.The results bring light to the annotation of the unkown genes of bacteriophage PaP3,also show that the specification of virus packaging protein.