Help with GST protein purification - (Mar/08/2011 )

Hi
I am currently working with GST-fusion protein (~61kDa ; GST+Protein). I have been purifing this protein since last year and still having degradation and low yield of the purified protein. The expression vector used to generate this fusion protein is pGEX 3x with GST tag (26 kDa)then transformed into E. coli BL21 expression strains.This fusion protein has been induced with 0.4mM IPTG and expressed for 5hrs at 28oC and Purified using GST affinity, HiTrap SP column and Gel Filtration. I used Factor Xa for cleavage. The final yield was really low about 10% of the starting product. Any one have any idea how to get better yield with low degradation. Actually I have to incubate this protein with factor Xa for cleavage at room temp for 20hrs.
Having probelm with degrading and low binding during affinity chromatography (using GSTtap/ Glutathione resin)

what buffer are you using for eluting your protein?

wasil on Tue Mar 8 10:12:37 2011 said:

I'm using 50mM Tris/150mM NaCl, pH 8.0.

do you remove bound material from the column after eluting your protein. if not then you lose capacity.

also, when you cleave the protein away from the tag the protein will pass through the matrix. after 20 hours most of the protein will be in the first few drops.

mdfenko on Wed Mar 9 21:24:40 2011 said:

I used 30mM reduced glutathione to remove remaining bound fusion protein, cleaved and GST tag on the column. Normally I will collect fraction from begining (From 1st drop until 10th Fraction (1.8ml each fraction)

This week I tried using 1ml resin incubated with 1/2 L cuture supernatant (filtered), about 1.6mg was roughly estimated was bound to the resin (1ml). In 1/2 L culture supernatant containing 7.5mg protein (roughly estimated from SDS-PAGE gel). Non was eluted out from the column using similar buffer as mentioned above.
The fusion protein was incubated with Factor Xa (50ul)+ 500ul digestion buffer + 2 or 5mM CaCl2. I am optimizing the digestion of this fusion protein usingdifferent tempt and incubation time. 37oC for 2 -4hr, room tempt for 14 to 20hrs. None gave me good yield. Protein degraded + uncleaved + less cleaved for on coloumn cleavage, but for in solution cleavage, protein precipitated + degraded + cleaved + uncleaved.