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why can't I get GST-fusion protein? - (Jun/08/2006 )

I used vector pGEX-6P-1 to express my GST fusion proetein, the host cell was BL21(DE3)pLySs, but the host only expressed GST,not GST-fusion protein,so what is the reason?Could you give me some suggestionS about this problem?Thank you in advance.


have you sequenced your construct?


QUOTE (aimikins @ Jun 8 2006, 07:12 PM)
have you sequenced your construct?

Likely it is an issue with your construct, but there is a possibility that your protein is cleaved near or at the junction where GST is fused. Have you run GST side by side to see if it is identical molecular weight?

What kind of protease inhibitors are you using? We use a combination of PMSF, leupeptin, benzamadine AND a protease inhibitor cocktail obtained from sigma. Also, ALL steps are performed at 4 degrees once the cells have been lysed. SOme proteins are more sensitive than others. For more sensitive proteins, we also shorten the dialysis times and freeze all proteins immediately after elution in the -80degrees to minimize degredation.

-Leigh CC-