Taq DNA聚合酶是分子生物学研究中最常用的热稳定DNA聚合酶之一, 与其他热稳定DNA聚合酶具有相似的特征, 其纯化策略不但有潜在的应用前景, 也对同类聚合酶的分离具有指导意义。已报道的适宜大量制备Taq酶的方案所需成本较高, 而文章介绍了一种利用国产阳离子交换树脂廉价制备Taq酶的方案。在本方案中, 采用热变性、(NH4)SO4沉淀与724离子交换层析分离大肠杆菌表达的Taq酶, 约18 g Na型树脂干粉一次可回收比活约8 131.98 U/mg、总酶活2.2×105 U、近27.07 mg Taq酶。纯化的产率可达48.92%, 纯化倍数约59.35。所制酶SDS-PAGE电泳只检测到94 kDa单一蛋白条带, 未检测到DNA核酸酶污染, 与商品酶的PCR扩增能力无区别。此纯化方法成本低, 适合实验室一般性的制备和生产应用。
Taq DNA polymerase is one of the most commonly thermostable DNA polymerases in molecular biological researches, which shares its basic characters with others of the family, thereby its purifying strategy could be used not only in itself production but also in the extraction of the others as a reference. At present, the protocols reported for large scale preparation of Taq DNA are high cost, so a cheaper method was described here. In this protocol, by heat de-naturation, ammonium sulfate precipitation and cation exchange chromatography of 724 resin, about 18 g powder of Na form resin could recover about 27.07 mg of Taq enzyme. The total activity and specific activity were approximately 2.2×105 U and 8131.98 U/mg. The total yield was about 48.92% with 59.35 of purification folds. Analysis of quality of purified enzyme indicated that only one protein 94 kDa was identified against SDS-PAGE and the remnant of DNA nuclease was not detected. For PCR reaction, The amplification ability of purified Taq polymerase was not different from that of the commercially available ones. This method reported in the present study is effective and low cost, making it suitable for general purification in laboratories or business production.
[1] Kaledin AS, Slyusarenko AG, Gorodetski? SI. Isolation and properties of DNA polymerase from extreme thermo-phylic bacteria Thermus aquaticus YT-1. Biokhimiia, 1980, 45(4): 644-651.
[2] Chien A, Edgar DB, Trela JM. Deoxyribonucleic acid polymerase from the extreme thermophile Thermus aquaticus. J Bacteriol, 1976, 127(3): 1550-1557.
[3] Mullis KB, Faloona FA. Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. Methods Enzymol, 1987, 155: 335-350.
[4] Engelke DR, Krikos A, Bruck ME, Ginsburg D. Purification of Thermus aquaticus DNA polymerase expressed in Escherichia coli. Anal Biochem, 1990, 191(2): 396-400.
[5] Lawyer FC, Stoffel S, Saiki RK, Chang SY, Landre PA, Abrarnson RD, Gelfand DH. 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. Genome Res, 1993, 2(4): 275-287.
[6] Lawyer FC, Stoffel S, Saiki RK, Myambo K, Drummond R, Gelfand DH. Isolation, characterization, and expression in Escherichia coli of the DNA polymerase gene from Thermus aquaticus. J Biol Chem, 1989, 264(11): 6427-6437.
[7] Abu Al-Soud W, Rådström P. Capacity of nine thermostable DNA polymerases to mediate DNA amplification in the presence of PCR-inhibiting samples. Appl Environ Microbiol, 1998, 64(10): 3748-3753.
[8] Pavlov AR, Pavlova NV, Kozyavkin SA, Slesarev AI. Recent developments in the optimization of thermostable DNA polymerases for efficient applications. Trends Biotechnol, 2004, 22(5): 253-260.
[9] Rittié L, Perbal B. Enzymes used in molecular biology: a useful guide. J Cell Commun Signal, 2008, 2(1-2): 25-45.
[10] Grimm E, Arbuthnot P. Rapid purification of recombinant Taq DNA polymerase by freezing and high temperature thawing of bacterial expression cultures. Nucleic Acids Res, 1995, 23(21): 4518-519.
[11] Pluthero FG. Rapid purification of high-activity Taq DNA polymerase. Nucleic Acids Res, 1993, 21(20): 4850-4851.
[12] Yang ZG, Ding YM, Zhang YH, Liu FH. Rapid purification of truncated Taq DNA polymerase Stoffel fragment by boiling lysis of bacterial expression cultures. Biotechnol Appl Biochem, 2008, 50(2): 71-75.
[13] Gräslund T, Nilsson J, Lindberg AM, Uhlén M, Nygren PÅ. Production of a thermostable DNA polymerase by site-specific cleavage of a heat-eluted affinity fusion protein. Protein Expr Purif, 1997, 9(1): 125-132.
[14] Melissis S, Labrou NE, Clonis YD. One-step purification of Taq DNA polymerase using nucleotide-mimetic affinity chromatography. Biotechnol J, 2007, 2(1): 121-132.
[15] Brandis JW, Johnson KA. High-cell density shake-flask expression and rapid purification of the large fragment of Thermus aquaticus DNA polymerase I using a new chemically and temperature inducible expression plasmid in Escherichia coli. Protein Expr Purif, 2009, 63(2): 120-127.
[16] Kim YJ, Lee HS, Bae SS, Jeon JH, Lim JK, Cho Y, Nam KH, Kang SG, Kim SJ, Kwon ST, Lee JH. Cloning, purification, and characterization of a new DNA polymerase from a hyperthermophilic archaeon, Thermococcus sp. NA1. J Microbiol Biotechnol, 2007, 17(7): 1090-1097.
[17] Moreno R, Haro A, Castellanos A, Berenguer J. High-level overproduction of His-tagged Tth DNA polymerase in Thermus thermophilus. Appl Environ Microbiol, 2005, 71(1): 591-593.
[18] Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem, 1976, 72(1-2): 248-254.
[19] Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 1970, 227(5259): 680-685.
[20] Roa
