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Pfu DNA Polymerase
E. coli BL21 overexpression guide
Introduction
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 https://benchling.com/openbioeconomy/f/lib_LngJ7WMH-dna-polymerase-sequences-to-freegenes/seq_tMfpgmip-pfu-optimized-and-domesticated/edit
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: https://doi.org/10.3389/fmicb.2014.00172
|
Pfu DNA polymerase |
|
|
Length |
Truncated |
|
E. coli strain |
BL21 |
|
Expression vector |
pSE420 |
|
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 |
|
Tag |
No tag |
|
Inducer |
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.
References
Full protocol at “A simple method for in-house Pfu DNA polymerase purification for high-fidelity PCR amplification” https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6635313/pdf/IJM-11-181.pdf
Google Drive folder: https://drive.google.com/drive/folders/1oTNJW1PBmNSSfKehA3p2-JcA52UenyYw