[ccp4bb] N-terminal cleavage after thrombin cleavage?

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CCP4bb <-- 2008 <-- March 2008 <-- 25 March 2008

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From: AMIT amitd453 {- at -} GMAIL {- dot -} COM
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Subject: N-terminal cleavage after thrombin cleavage?
From: HTC htc {- dot -} ccp4 {- at -} GMAIL {- dot -} COM
Date: 2008-03-25

Dear all,

Sorry for the non-CCP4 question.

We recently crystallized a protein expressed from pGEX4T-1 vector. The GST tag is cleaved by thrombin before crystallization. The cleavage site of thrombin is Leu-Val-Pro-Arg

Gly-Ser, thus Gly and Ser should remain at the N-terminus of the protein. Strangely, from the 2.5A map, we observed some unexplainable electron density before the most N-terminal glycine residue (http://www.dddc.ac.cn/group/htc/NT_map.jpg). Has anyone encountered similar situation with thrombin cleavage? Could it be some kind of modification or additional residues resulted from unspecific cleavage of thrombin? The crystallization liquor contains MES, PEG6000, Tris, DTT, NaCl and EDTA.

Any suggestion is highly appreciated!

Thanks in advance!

--------------
Tiancen Hu
Shanghai Institute of Materia Medica
Chinese Academy of Sciences
Rm. 2107, #555, ZuChongzhi Rd.
Shanghai 201203
P.R. China
Phone: +86-21-50806600 ext. 2107

CCP4bb navigation

CCP4bb <-- 2008 <-- March 2008 <-- 25 March 2008

Previous message:
Subject: Re: Peltier Crystallisation Incubators
From: Artem Evdokimov artem {- at -} XTALS {- dot -} ORG
Date: 2008-03-25

Next message:
Subject: Re: Peltier Crystallisation Incubators
From: AMIT amitd453 {- at -} GMAIL {- dot -} COM
Date: 2008-03-26