inclusions from pET vector expresion

jkreps at biochem.umass.edu jkreps at biochem.umass.edu
Tue May 9 07:44:17 EST 1995


>Dear protein expression in bugs sufferers
>
>I'm after ideas. We have a viral non-structural protein expressed nicely using 
>a pET vector. Problem is we get quite variable lysis of the bugs. I'll give 
>some details of the way we do the expression and if anyone has improvements 
>I'd love to hear about it.
>
>1   we use 200 ml LB cultures. We use pGP-1 and heat induction to induce 
>expression of the pET vector.
>2   So, 1/40 dilution of overnight in LB, grow at 28 oC, 3 h
>3   induce at 42 oC 1 h 
>4   grow 37 oC 3 h
>5   pellet bugs
>6   resuspend in 50 ml: 50mM Tris, pH 8, 1 mM edta, 0.1 M NaCl, 0.1 mM PMSF, 
>200 ug/ml lysozyme, 20 ug/ml DNAase, 20 ug/ml RNAase. Incubate room temp 30 
>min to 3 h.
>7   sonicate
>8   pellet and resuspend in 5 ml wash buffer: 50 mM Tris pH 8, 0.1 mM NaCl, 10 
>mM EDTA, 1 % triton x100
>9   sonicate again, pellet and wash once or twice more
>
>
>Problem is that after all this a huge gluggy mess of bug bits makes getting at 
>the inclusions impossible/difficult.
>
>Anyone do freeze/thaw to crack open the cells? Any good? How do you get 
>solubilised bugs or smash them up so small they are no longer a problem? Are 
>we trying to play with too great a volume of bugs?
>
>Cheers
>
>Tony Lough
>HortResearch
>Mt Albert Research Centre
>Auckland, NZ
>
>
Tony,

I have only ever worked with the pGEX system and soluble proteins but here
is what has worked for me (and an undergraduate lab course).

1.  Follow your induction protocol.
2.  Pellet the E. coli (assume a 50 ml culture, OD600 ~ .45), resuspend in 3
ml ice cold PBS, add 150 ul 20% triton X100.
3.  Sonicate well, sometimes the triton X100 leads to excessive foaming
which inhibits sonication.  I sonicate for 30 sec with 1 min. cooling
periods, repeat 4 to 8 times.
4.  For soluble proteins, pellet cell debris and purify from the
supernatant.  Cliff Carpenter in our lab has used this system with two
putative movement proteins from Turnip Crinkle Virus and found that the
proteins were always in the pellet.  He tried some variations on the above
theme with some success.  You should email me directly if you need more info.

Sincerely,

Joel A. Kreps
Postdoc, Anne Simon's lab
UMass at Amherst, MA USA




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