5 replies to this topic

[biovinz]

Dear All,
         I am trying to purify a GST-tagged protein. My protein size is around 37 kDa. After IPTG induction, most of the protein expressed in insoluble. how can i get soluble protein? I already tried different temperatures ( 18,25,30 and 37 C) with different induction time(2hr, 3hr and overnight) and different IPTG concentration (0.1, 0.5, 1mM) and different sonication time(10 sec, 20sec, 30 sec, 40 sec 4cycles). I request you to please help me.Looking for your reply.
                   Thanks in Advance...


VINOD

[prabhubct]

how you know your protein is in inclusion bodies. did you add loading dye to bacterial pellet, boil it & load on PAGE?
you can use protocol for purification of protein from inclusion bodies.

[HOYAJM]

You could try a cleavable Thioredoxin (Trx) tag. You just clone your gene in frame with the tag and you can cleave it off later. The Trx tag is supposed to be pretty good for increasing solubility of unstable proteins. We use pET32a-c vectors that contain Trx, S, His6 tags that are all cleavable.

[Pangea]

SDS-Page check. İn Frame check.

Edited by Pangea, 03 January 2013 - 10:47 AM.

[biovinz]

Dear Veteran, yes i run sds-page for soluble and insoluble protein..after that i confirmed most of the protein expressed in insoluble.If you know any good protocol for GST-fusion protein purification from insouble protein, pls let me know...Thank you for ur reply..

[biovinz]

Dear Enthusiast, Thank you for your reply..my lab don't have pET32a vector but we have pET28a vector..i tried pET28a vector also but after IPTG induction didnt expressed..so i changed to pGEX4Ta vector..