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consistent expression of overlarge proteins

bacterial expression

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#1 ocram



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Posted 02 November 2015 - 08:00 AM

I have recently joined a new lab and started to express bacterial recombinant proteins again.  I have expressed three, all in the same vector, a modified pET vector with simple His tag in BL21(DE3) and all three proteins are too large, by between 5 -15kDa.  The sequences are fine, (both of the empty vector and recombinants) their mobility is not affected by running on Tris-glycine or Tricine gels, but I am able to purify them on HisTrap and they give a signal with anti-His monoclonal on Western.  The problem is I am not able to further purify them either by IEX or size exclusion and their size bothers me.  How can I be sure these are really my proteins - any suggestions..??

#2 labtastic



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Posted 02 November 2015 - 08:12 AM

Are you saying the proteins migrate on SDS-PAGE gels ~5-15kDa larger than their predicted size? If so, I wouldn't worry about that. SDS-PAGE is not always a great indicator of protein size...it is not unusual for things to run faster or slower than expected. 


If you want to be absolutely sure these are your proteins, run them out on a gel, cut the bands out and send for MS/MS sequencing. Or, if you have a specific activity assay of some kind then you can do that of course.


Why can't you do ion exchange or size exclusion? If you explain more then we might be able to help troubleshoot more effectively.

Edited by labtastic, 02 November 2015 - 09:13 AM.

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