protein expression in M15 PREP4

Michael R. Thompson mthompson at pbi.nrc.ca
Fri Nov 27 12:57:04 EST 1998


Precisely the same. 40 Kd protein with a fainter band just below. On a
repeat synthesis/purification we included a protease inhibitor cocktail
(Sigma cat # P-2714) in the extraction buffer, which pretty well eliminated
the second band. Our construct encoded a C-terminal His, vector pQE60, cell
line M15pRep4.

--
Regards, Mike

Dr. Michael R. Thompson         E-mail: mthompson at pbi.nrc.ca
110 Gymnasium Pl.                   Ph.: (306)975-5279
Saskatoon, Sk S7N 0W9         Fax: (306)975-4839
Canada
Ian McFarlane wrote in message ...
>Hi,
>
>We have expressed an N-terminal His tagged protein in M15 PREP4 E. Coli.
>from Qiagen. We get a nice induction with IPTG and the protein elutes from
>Ni2+ columns at around 200mM imidazole. The only fly in the ointment is a
>second band. The columns were loaded in 8M urea and washed clean followed
>by a renaturation step. The contaminant runs about 2kD lower than our
>target and the band is diffuse (appears to extend from the target protein
>down).
>
>I think it may be proteolysis products. Has anyone else out there
>experienced this sort of thing with the above strain?
>
>Thanks,
>
>Ian Mc





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