人乳铁蛋白阳离子多肽重组Taq DNA聚合酶的研究
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摘要
本文报道了人乳铁蛋白阳离子多肽(HL-N)替换Taq DNA聚合酶N-末端结构域,构建新型重组Taq酶的研究结果.利用合成寡核苷酸,成功获得具DNA合成功能的HL-N重组Taq酶.对该重组酶的DNA合成加工、血清耐受性等测试显示,HL-N重组Taq DNA聚合酶的DNA扩增速度、扩增物产量、以及对反应体系中血清浓度的耐受性都得到显著提高.
Taq DNA polymerase consists of isolated functional domains responsible for binding of template DNA and catalysis of DNA synthesis,respectively.In the present study,a recombinant Taq DNA polymerase was constructed by replacing the N-terminal 289 amino acids of Taq DNA polymerase with cationic peptide of human lactoferrin(NL-N).Some enzymatic properties of the chimerical recombinant DNA polymerase,such as the processivity,5′→3′exonuclease activity,and tolerance to serum concentration in PCR reaction system,were characterized experimentally and the testing data showed that the modification of DNA-binding domain with NL-N improved the polymerase in several aspects.
Taq DNA polymerase consists of isolated functional domains responsible for binding of template DNA and catalysis of DNA synthesis,respectively.In the present study,a recombinant Taq DNA polymerase was constructed by replacing the N-terminal 289 amino acids of Taq DNA polymerase with cationic peptide of human lactoferrin(NL-N).Some enzymatic properties of the chimerical recombinant DNA polymerase,such as the processivity,5′→3′exonuclease activity,and tolerance to serum concentration in PCR reaction system,were characterized experimentally and the testing data showed that the modification of DNA-binding domain with NL-N improved the polymerase in several aspects.
引文
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[9]Wang Y,Prosen D E,Mei L,et al.A novel strategy to engineer DNA polymerases for enhanced processivity and improved performance in vitro[J].Nucleic Acids Res.2004,32(3):1197.
[10]Uehara-Ichiki T,Shiba T,Matsukura K,et al.Detection and diagnosis of rice infecting viruses[J].Front Microbiol,2013,4:289.
[11]程玉鹏,李慧玲,高宁,等.Taq DNA polymerase结构改造与功能改进及其在医药领域的应用前景[J].现代生物医学进展,2008,18(10):1975.
[12]van Berkel P H,Geerts M E,van Veen H A,et al.N-terminal stretch Arg2,Arg3,Arg4and Arg5of human lactoferrin is essential for binding to heparin,bacterial lipopolysaccharide,human lysozyme and DNA[J].Biochem J,1997,328(1):145.
[13]Huo L,Zhang K,Ling J,et al.Antimicrobial and DNA-binding activities of the peptide fragments of human lactoferrin and histatin 5against Streptococcus mutans[J].Arch Oral Biol,2011,56(9):869
[14]Vieille C 1,Zeikus G J.Hyperthermophilic enzymes:sources,uses,and molecular mechanisms for thermostability[J].Microbiol Mol Biol Rev,2001,65(1):1.
[15]Hunter H N,Demcoe A R,Jenssen H,et al.Human lactoferricin is partially folded in aqueous solution and is better stabilized in a membrane mimetic solvent[J].Antimicrob Agents Chemother,2005,49(8):3387.
[16]Pavlov A R,Belova G I,Kozyavkin S A,et al.Helix-hairpin-helix motifs confer salt resistance and processivity on chimeric DNA polymerases[J].Proc Natl Acad Sci USA,2002,99(21):13510.
[2]Terpe K.Overview of thermostable DNA polymerases for classical PCR applications:from molecular and biochemical fundamentals to commercial systems[J].Appl Microbiol Biotechnol,2013,97(24):10243.
[3]Kaltenboeck B,Wang C.Advances in real-time PCR:application to clinical laboratory diagnostics[J].Adv Clin Chem,2005,40:219.
[4]Kim Y,Eom S H,Wang J,et al.Crystal structure of Thermus aquaticus DNA polymerase[J].Nature.1995,376(6541):612.
[5]Lawyer F C,Stoffel S,Saiki R K,et al.Isolation,characterization and expression in Escherichia coli of the DNA polymerase gene from Thermus aquaticus[J].J Biol Chem,1989,264(11):6427.
[6]Erlich H A,Gelfand D,Sninsky J J.Recent advances in the polymerase chain reaction[J].Science,1991,252(5013):1643.
[7]Lawyer F C,Stoffel S,Saiki R K,et al.High-level expression,purification,and enzymatic characterization of full-length Thermus aquaticus DNA polymerase and a truncated form deficient in 5′to 3′exonuclease activity[J].Genome Res,1993,2(4):275.
[8]Merkens L S,Bryan S K,Moses R E.Inactivation of the 5′→3′exonuclease of Thermus aquaticus DNA polymerase[J].Biochim Biophys Acta,1995,1264:243.
[9]Wang Y,Prosen D E,Mei L,et al.A novel strategy to engineer DNA polymerases for enhanced processivity and improved performance in vitro[J].Nucleic Acids Res.2004,32(3):1197.
[10]Uehara-Ichiki T,Shiba T,Matsukura K,et al.Detection and diagnosis of rice infecting viruses[J].Front Microbiol,2013,4:289.
[11]程玉鹏,李慧玲,高宁,等.Taq DNA polymerase结构改造与功能改进及其在医药领域的应用前景[J].现代生物医学进展,2008,18(10):1975.
[12]van Berkel P H,Geerts M E,van Veen H A,et al.N-terminal stretch Arg2,Arg3,Arg4and Arg5of human lactoferrin is essential for binding to heparin,bacterial lipopolysaccharide,human lysozyme and DNA[J].Biochem J,1997,328(1):145.
[13]Huo L,Zhang K,Ling J,et al.Antimicrobial and DNA-binding activities of the peptide fragments of human lactoferrin and histatin 5against Streptococcus mutans[J].Arch Oral Biol,2011,56(9):869
[14]Vieille C 1,Zeikus G J.Hyperthermophilic enzymes:sources,uses,and molecular mechanisms for thermostability[J].Microbiol Mol Biol Rev,2001,65(1):1.
[15]Hunter H N,Demcoe A R,Jenssen H,et al.Human lactoferricin is partially folded in aqueous solution and is better stabilized in a membrane mimetic solvent[J].Antimicrob Agents Chemother,2005,49(8):3387.
[16]Pavlov A R,Belova G I,Kozyavkin S A,et al.Helix-hairpin-helix motifs confer salt resistance and processivity on chimeric DNA polymerases[J].Proc Natl Acad Sci USA,2002,99(21):13510.