Pfu DNA Polymerase Quick Expression Guide

Hi community! The information included in this topic derives from literature based protocols gathered by Reclone Science Team, and this space is intended for everyone to ask questions, share comments and discuss whether there can be improvements in any step of the protocol or any other issue! Feel free to share your own experience/protocol/advice

Pfu DNA Polymerase

E. coli BL21 overexpression guide



Pfu DNA polymerase is an enzyme derived from Pyrococcus furiosus, it’s widely used for High Fidelity Polymerase Chain Reaction since it has greater thermostability and exonuclease activity (3’ to 5’) than TaqPol.

Pfu DNA polymerase gene encodes a polypeptide of 775 amino acids with predicted molecular weight of 90-109 kDa.

Gene can be found at


Disclaimer: the below reported expression conditions represent a summary of non-exhaustive literature searches but represent a most likely to work scenario.

Many comprehensive protein expression reviews are available: 

Pfu DNA polymerase




E. coli strain


Expression vector


Growth media

LB (containing 100 µg/ml ampicillin and 35 µg/ml chloramphenicol)

Purification method

Denaturation of non-thermostable proteins and retrieval of Pfu from supernatant


No tag


IPTG 0.5 mM

expression conditions pre/post induction

Best conditions at OD600=0.5, 37°C  for 5 hours, 200 rpm.


Additional notes:

Depending on the application, DNA contaminants can be removed by benzonase or DNAse treatment.  Primers for gene amplifying can be found in the article mentioned below.



Full protocol at “A simple method for in-house Pfu DNA polymerase purification for high-fidelity PCR amplification”


Google Drive folder:


Hi Tommy – Thanks very much for the summary and the really helpful links.
@jenny_molloy : When we discussed the expression guides (for RTX specifically) is this an example of what you were looking for as the distilled output?