Preparation of normal DNA polymerase

Question

i don't need Taq polymerase. Just need normal DNA polymerase. So the main idea is preparation polymerase directly from Lactobacillus without using cloned vector. My idea is electrophoresis using agarose gel and calculating the isoelectric point of Polymerase based on protein sequence to know where they will migrate after electrophoresis. Is it possible for this idea ?

Answer 1

This is a very ambitious project for a DIY-er. To explain why, I'm going to use the example of DNA polymerase I from E. coli.

DNA polymerase I is the most abundant DNA polymerase in the E. coli cell, but nevertheless is still only present at around 300 copies per cell

Ishihama Y et al. (2008) Protein abundance profiling of the Escherichia coli cytosol. BMC Genomics. 9:102.

This was the first DNA polymerase that was ever purified, in the laboratory of Arthur Kornberg

(Enzymatic Synthesis of Deoxyribonucleic Acid : I. PREPARATION OF SUBSTRATES AND PARTIAL PURIFICATION OF AN ENZYME FROM ESCHERICHIA COLI Lehman, MJ et al. (1958) J. Biol. Chem. 33:163-170.).

In this paper a method of purification is described that starts with 60 litres of culture. There is also this statement:

"One kilo of E. coli yields less than 10 mg of the purified enzyme."

I'm guessing that you have no experience of protein purification, so I'll just tell you that this is very discouraging: these days, with overexpression, people expect to get mg quantities of their protein from just a few grams of bacterial cells.

This is a clear illustration of why it is pretty much unthinkable for you to set out to purify an enzyme like this using small-scale methods, and explains why techniques of overexpression and the use of affinity tags have revolutionised protein purification.

And it gets worse: even if you did manage to purify some DNA polymerase I from a bacterial source you would find that it is actually not very good at making DNA. This is because as well as extending primers in a 5'>3' direction, it is is very good at degrading DNA in the same direction. In other words as the enzyme moves along a template molecule, copying it, it will degrade any existing DNA that it meets "ahead" of its direction of travel. This is why the most commonly-used form of this enzyme in research is the "Klenow fragment", a fragment of the polymerase that lacks the 5'>3' exonuclease activity. This fragment was originally made by treating DNA polymerase I with the protease subtilisin, but it is now expressed from an engineered polA gene. Klenow fragment polymerase was used in the original PCR experiments (it had to be added afresh at every cycle).

I don't know how much money you have to spend on your projects, but in fact these enzymes can now be bought relatively cheaply. I don't think that you can expect to make them for yourself, without this becoming the actual project.

Finally, if you can get hold of a strain of E. coli with an expression plasmid for Taq polymerase then you really could make your own very easily - I've done this myself (but I no longer have the strain) and the heat stability of the enzyme makes the purification trivial. It would be much easier to use your own Taq with a heated water bath than to try to make DNA polymerase I. But obviously it depends upon what you are trying to do: Taq polymerase may be unsuitable for other reasons.