TKx1 protein production

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btsridhar
btsridhar's picture
TKx1 protein production

 Hello guys,

I am looking to produce a phosphorylated protein using the TKX1 cells. sometimes I am getting the protein, but some times I cannot. I have few questions like does IPTG concentration effects the protein production?. For extracting the protein I use Freeze thaw technique. after freeze thaw if the cell lysate is too viscous I go for sonication and give 10-25 pulses at 1 sec interval. These days, I am not going for the sonication because cell lystae is not viscous. My post doc is saying that sonication is the one making the difference. I cant figure it out. Another thing is abt IPTG. In the manual it says induce the protein production with 0.1mM IPTG, usually I do with 0.6mM, my post doc is saying to use 1mM. Does the IPTG concentration effects the protein production? any type of help is appreciated. 
Thanks
Sri

protoldo
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Hola, Yes IPTG is de inductor

Hola, Yes IPTG is de inductor of expression of the recombinant protein.There are some factors which affect to the expression : OD. of culture at the time of induction, concentration of IPTG, temperature, media composition, time of expression, etc. Before prepare your big culture, you could prepare some little cultures and make some assays of induction. People induces in LB at 0.6-0.8 OD units with 0.1-2mM IPTG and collect 2-4 hours after. If the induction is too strong could make to your protein to form inclusion bodies and make them insolubles, lowering temprature at 28ºC and with light induction increases the fraction of soluble protein, easier to purify, but the characteristics of the proteins play a role too in the solubility. The viscosity of your lysated bacteria is due to DNA, which sonicating is broken and shows the liquid consistence. Good luck

btsridhar
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 Hi Protoldo,

 Hi Protoldo,
Thanks for your suggestions. These things work in Bl21 cells, I dont have any problem in expressing the proteins in them. TKX1 cells are different, they phosphorylate the protein. Here i have to induce after O.D reaches 1, after reaching maximum O.D, I need to induce again with the TK induction media without tryptophan, which phosphorylates the protein. I want to know if anyone of you guys worked with TKX1 before? and need some tips. I will follow your suggestions, and really appreciate your help. Thanks for ur wishes :)
Sri