Abstract
A 2.3 kb DNA fragment containing Pfu DNA polA gene was amplified by PCR from total DNA of Pyrococcus furiosus and cloned into a pGEM-T vector. The recombinant clone pT-pfu was digested with Nco I and Xho I and the fragment was inserted into an expression vector pET3d-X. The Pfu polA gene was expressed in Escherichia coli BL21 (DE3). The gene product (Pfu) was purified with heat denaturation, polyethylenemine (PEI) precipitation and Bio-rex 70 ion-exchange chromatography. The recombinant Pfu was verified by protein N-terminal sequencing. With the recombinant Pfu, large λ DNA fragments were successfully amplified in long-distance PCR.
Original language | English (US) |
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Pages (from-to) | 863-867 |
Number of pages | 5 |
Journal | Chinese Science Bulletin |
Volume | 43 |
Issue number | 10 |
DOIs | |
State | Published - May 1998 |
Keywords
- Expression
- Long-distance PCR
- Pfu enzyme
ASJC Scopus subject areas
- General