Re: [ccp4bb] Cannot running NTA to purify the protein having His-tag?

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From: dirk {- dot -} kostrewa {- at -} PSI {- dot -} CH
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Subject: Re: Cannot running NTA to purify the protein having His-tag?
From: joost {- dot -} uitdehaag {- at -} ORGANON {- dot -} COM
Date: 2007-03-01

Hi,

We got some good experiences using the IBA streptag for baculo-expressed proteins. You'll have to redo the cloning, but it will be worth your while when you see the first purification step,

cheers

Joost

-----Original Message-----
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Alex Berndt
Sent: Wednesday, 28 February, 2007 20:21
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Cannot running NTA to purify the protein having His-tag?

sometimes the insect cell medium intereferes (for whatever reasons) with nta purifications when they ar employed as a first step in the purification scheme. i experienced that occasionally. this can easily be circumvented by doing an ion exchange step beforehand!

alternatively you might want to introduce a linker between your protein and the his-tag or create a 8xhis or 10xhis tag to enhance bing to the nta matrix. make sure you wash your cells from residual medium before you freeze your pellets.

alex

On 28 Feb 2007, at 19:18, Juergen Bosch wrote:

Ngo Duc Tri wrote:

Dear CCP4 users,

I'm purifying a kind of protease having His-tag. The protein is expressed in insect cells and broken by sonication.

I used NTA resin to purify this protein.

Buffer A is 50mM phosphate buffer pH 7.5 and 300mM NaCl. Buffer B is 50mM phosphate buffer pH 7.5, 300 mM NaCl and 300 mM Imidazole.

However, all proteins cannot bind to NTA resin. My protein is eluted in Flow-through. I also check the NTA resin with the control His-tag. The western blot also shows that my protein has His-tag.

Do you have any ideas about my problem? I'm really appreciate all of your advices how to solve this. Thank you very much!

My best regards,

TriNgo

Sungkyunkwan University

You His tag is most likely inaccessible, can you easily change the tag from e.g the N-terminus to the C-terminus ? Or if you have a structural homolog you could add the His tag into a loop, which is exposed.

Alternatively you can purify your protein under denaturing conditions using 8 M urea and refold it if you dare :-)

Juergen

--

Jürgen Bosch

University of Washington

Dept. of Biochemistry, K-426

1705 NE Pacific Street

Seattle, WA 98195

Box 357742

Phone: +1-206-616-4510

FAX: +1-206-685-7002

---

Alex Berndt

MRC Laboratory of Molecular Biology

Hills Road

Cambridge CB2 2QH

UK

mail : aberndt@mrc-lmb.cam.ac.uk

phone : +44 (0)1223 402113

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CCP4bb <-- 2007 <-- March 2007 <-- 01 March 2007

Previous message:
Subject: Re: process SeMet labelled data
From: dirk {- dot -} kostrewa {- at -} PSI {- dot -} CH
Date: 2007-03-01

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Subject: Very weird
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