Purification of recombinant Pfu DNA polymerase using a new JK110 resin

Zhanghui Sun; Jin Cai

Korean Journal of Chemical Engineering, Vol.23, No.4, 607-609, July, 2006

DOI10.1007/BF02706802 Export Citation

Purification of recombinant Pfu DNA polymerase using a new JK110 resin

Zhanghui Sun, and Jin Cai^†

Institute of Bioengineering, College of Material & Chemical Engineering, Zhejiang University, Hangzhou 310027, China

E-mail:

The purification of recombinant Pfu DNA polymerase expressed in Escherichia coli was studied. The lysed supernatant was heated to 75 °C to denature E. coli protein, followed by chromatography on JK110 and Sephadex G-75. The purified protein had comparable activity to the commercially obtained Pfu in both DNA polymerase and PCR amplification. The final product had a specific activity of 17,600 U/mg and 149,600 U of Pfu DNA polymerase was obtained from 500ml culture. JK110 has worked well in our study and appears to be a new method of choice for purification of Pfu DNA polymerase.

Keywords:Pfu;Polymerase;Pyrococcus fuiosus;Purification;JK110

[References]

Ausubel FM, Kingston RE, Seidman JG, Struhl K, Brent R, Moore DD, Smith JA, Short protocols in molecular biology, 4th ed, John Wiley & Sons, Inc. US (2004)
Chen Y, Xu Z, Shen W, Lin J, Cen P, Korean J. Chem. Eng., 22(1), 121 (2005)
Cline J, Braman JC, Hogrefe HH, Nucleic Acids Res., 24, 3546 (1996)
Dabrowski S, Kur J, Protein Expr. Purif., 14, 131 (1998)
Lu CL, Erickson HP, Protein Expr. Purif., 11, 179 (1997)
Lundberg KS, Dan DS, Gene, 108, 1 (1991)
Mathur, Eric J, US patent, 6, 489150 (2002)
Mroczkowski BS, Huvar A, Lernhardt W, Misono K, Nielson K, Scott B, J. Biol. Chem., 269, 13522 (1994)
Pavlov AR, Pavolva NV, Sergei AK, Trends Biotechnol., 22, 253 (2004)
Sambrook J, Russell D, Molecular cloning: a laboratory manual, 3rd ed., Cold Spring Harbor Lab (CSHL) Press, New York (2001)
Studier FW, Moffatt BA, J. Mol. Biol., 189, 113 (1986)
Uemori T, Ishino Y, Toh H, Asada F, Kato I, Nucleic Acids Res., 21, 259 (1993)
Yao SJ, Guan YX, Yu LH, Korean J. Chem. Eng., 20(1), 93 (2003)

[Referenced By]

[1] Ausubel FM, Kingston RE, Seidman JG, Struhl K, Brent R, Moore DD, Smith JA, Short protocols in molecular biology, 4th ed, John Wiley & Sons, Inc. US (2004)

[2] Chen Y, Xu Z, Shen W, Lin J, Cen P, Korean J. Chem. Eng., 22(1), 121 (2005)

[3] Cline J, Braman JC, Hogrefe HH, Nucleic Acids Res., 24, 3546 (1996)

[4] Dabrowski S, Kur J, Protein Expr. Purif., 14, 131 (1998)

[5] Lu CL, Erickson HP, Protein Expr. Purif., 11, 179 (1997)

[6] Lundberg KS, Dan DS, Gene, 108, 1 (1991)

[7] Mathur, Eric J, US patent, 6, 489150 (2002)

[8] Mroczkowski BS, Huvar A, Lernhardt W, Misono K, Nielson K, Scott B, J. Biol. Chem., 269, 13522 (1994)

[9] Pavlov AR, Pavolva NV, Sergei AK, Trends Biotechnol., 22, 253 (2004)

[10] Sambrook J, Russell D, Molecular cloning: a laboratory manual, 3rd ed., Cold Spring Harbor Lab (CSHL) Press, New York (2001)

[11] Studier FW, Moffatt BA, J. Mol. Biol., 189, 113 (1986)

[12] Uemori T, Ishino Y, Toh H, Asada F, Kato I, Nucleic Acids Res., 21, 259 (1993)

[13] Yao SJ, Guan YX, Yu LH, Korean J. Chem. Eng., 20(1), 93 (2003)