Expression temprature - (Apr/25/2006 )

Dear friends

I am trying to express a protein with 772 amino acids in pET28-a vector. I have tried 37 degree of centigrade but it wasn't successful.My protein is a hydrophobic one and I don't know if the lower temp. will help. What temp. should I try?

Cheers,
Nozhat

except temp. u can try it in different strain of E.coli, as well as late induction i.e, at OD 1.2,

Agree! Try room temp with gentle shaking. I sometimes grow them at 16 deg overnight. You can also try adding a little 1% sorbitol or ethanol upon induction using .1 to .5 mM IPTG. To find out best conditions, do a solubility test.

What I do is this: I start a 25 mL culture and let grow until OD reaches about .6 at 600 nm.

I pipet 5 mLs of this culture into test tubes. I mark them "A" - "B", etc. Then in "A" , I don't induce. "B" is induced with .1 mM IPTG, "C" is .1 mM IPTG plus 1% sorbitol, "D" is .1 mM IPTG with 1% ethanol, "E" is .5 mM IPTG, "F" is .5 mM IPTG plus 1% sorbitol etc until all done. Make sure you run sorbitol and no IPTG and Ethanol and no IPTG also.

Then, after three hours, take 1 mL and spin to pellet. Add 100 uL of 25mM Tris-Cl pH 7.5, 50mM NaCL and .2 ug/mL of Lysozyme. Incubate on ice for 30 minutes. Spin to pellet and remove supe. This is your soluble frac.

Resuspend the pellet in 50uL of 8M urea buffer and incubate 5 minutes at room temp. Spin 14Krpm for 10 minutes and carefully remove supe. This is your insoluble fraction.

For gel: 10 uL of soluble plus 1uL of 10X load dye. for insoluble fraction, use 2uL of supe plus 8 uL of water. Add 1 uL of load dye and run sample "A" as soluble/insoluble. Repeat for all samples.

I do this at first at 37 degrees, then 25 degrees, then 16 if necessary. It will certainly tell you how you need to grow your protein.

I follow the same protocol for all the proteins I've purified - over 100! And it works!

Good Luck!