T4 DNA Ligase expression: working protocols?

[jenny_molloy]

Hi All

Does anyone have good working protocols for T4 DNA ligase expression?
@birhan_addisie is working on this

Jenny

[birhan_addisie]

Thanks @jenny_molloy.
I tried to express it using pOBL backbone in BL21 (DE3) induced with IPTG and here is the SDS PAGE.

I would appreciate if anyone have working protocols to share for T4 DNA ligase expression.

[Louise]

Hello,

We didn’t try it yet but I did some bibliography on it as we are looking to try too.
The problem may be in the induction. How long do you induce ? You can try lowering the temperature to 25°C or 18°C and induce overnight. In this case, we induce at a OD higher than usual, like 0.8-1.

The only paper I found its this one in vietnamese :EXPRESSION AND PURIFICATION OF RECOMBINANT T4 DNA LIGASE IN E. COLI | VNUHCM Journal of Science and Technology Development
They have a short explanation in english and they induce at low temperature.

I hope this help,
Louise

[birhan_addisie]

Thanks @Louise, The induction was overnight and that could be one of the reason. I will try at low temperature and see how it looks.

[eperkins]

My PhD lab (Ramsden lab) purified T4 DNA Ligase all the time, but it was using baculovirus and insect cell expression. If that is at all useful, I can as my old PI for a protocol. There’s no single paper that has the entire protocol that I could find, but Dale could probably help out.

[birhan_addisie]

Thanks @eperkins! Our plan is to express it in E.coli at the moment. I will let you know if we plan to use baculovirus and insect expression.

[SaneeDeLM]

hey @birhan_addisie any success with the expression using the E. coli system??

[birhan_addisie]

Hi @SaneeDeLM, yes it is working perfectly. We will share the protocols shortly.

[gianluca.centr]

Good morning people. We have been working on a T4 ligase purification protocol which has given us some encouraging results. We managed to produce discrete amounts of active material in E. coli.
We also performed some FRET-based activity assay conducted on short DNA duplexes. Next step will be to try to perform an actual ligase experiment.

I will keep you in the posted.

[Ahmed_Atef_2]

This is a great news, would you please share the map of your assembly?

[Ahmed_Atef_2]

This also extremely great news, the results is very promising, we struggle to produce successful producing clone since a while, would you please share your construct.

[SaneeDeLM]

@birhan_addisie This will be of great assistance