Protein Glycosylation/Thrombin cleavage of GST fusion prote - Proteomics (Feb/16/2002 )

jlmcmurry

Junior Member

I am having trouble with the Amersham GST fusion protein system. I am
expressing a peptide fused to GST with a thrombin cleavage site. After
cleavage (on the GST column) the peptide eluate seems to precipitate. A
second purification step is necessary. The peptide does not bind to SP
sepharose. In previous preps, all worked well. My questions, for anyone out
there who has had solubility or cleavage problems with pGEX systems are:

1) does glycerol or triton inhibit thrombin?
2) how did you solve your problem?

I'd love to hear from anyone who has dealt with a similar situation, either by
posting here or email. Thanks.

Jonathan McMurry

Total Posts: 2 | Joined Jan. 2001 | Posted on: 5:16 am on Jan. 9, 2001 | IP

zlx

Junior Member

I used to express a 15KDa protein using pGEX-2T, the expressed protein could
be easily cleaved off both in eluates and in situ in the presence of Triton
X-100(0.1%) and very low level of glycerol . But some cleaved peptides
absorbed on the gel when using in situ cleavage.

Lixin Zhang

Total Posts: 1 | Joined Jan. 2001 | Posted on: 4:23 pm on Jan. 12, 2001 | IP

rtandon

Junior Member

Hi ,
I have two questions, independent of each other-

1-Can anybody tell me how to do the thrombin cleavage in GST fusion proteins,
pl. tell me about the difficulties if any... their solutions and suggestions
Thanks in anticipation,
RTandon
2- I have expressed a protein in E. coli. which is approximately 10-15 kDa bigger
in size than in the mammalian system by cDNA expression. I am looking for the
possibility of glycosylation as a result of post translational modification. How to
check if protein has been glycosylated.....I would invite all suggestions and
protocols in this regard.

Total Posts: 3 | Joined Jan. 2001 | Posted on: 12:14 pm on Feb. 16, 2001 |
IP

rtandon

Hi ,
I have two questions, independent of each other-

1-Can anybody tell me how to do the thrombin cleavage in GST fusion proteins,
pl. tell me about the difficulties if any... their solutions and suggestions

2- I have expressed a protein in E. coli. which is approximately 10-15 kDa bigger
in size than in the mammalian system by cDNA expression. I am looking for the
possibility of glycosylation as a result of post translational modification. How to
check if protein has been glycosylated.....I would invite all suggestions and
protocols in this regard.
Thanks in anticipation,
RTandon

-rtandon-

Amersham web site has a really good protein purification handbook that tells you general information in regards to GST fusion proteins, thrombin cleavage and things to watch for. The handbook is called "The recombinant Protein Handbook: Protein amplification and simple purification" . There's a number 18-1142-75, edition AA maybe you could put in that number for searching their site in order to pull it up. You can print the whole manual out ~ 80pages. Another good source is Application note pamphlet that they have, number 18-1146-70 AA. tiltled Efficient rapid protein purification and on-column cleavage using GSTrap FF columns.

Hope this will help you.

Catherine Nguyen
Thanh.t.nguyen@uth.tmc.edu

-godivess-

I have trouble with purifying thrombin cleaved GST fusion protien. After using thrombin to cleaved my fusion protein, I rerun into Glutathione Sepharose 4B prepack column. The elute that I collect should be the protein without GST. However, when I ran the SDS PAGE, I still saw the GST band and the GST fusion protein bands. I already exchange the buffer using the desalted column before reran the GS4B column.
Could anyone tell me how to solve this problem.

-wtsam-