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Insoluble Protein of Interest Resulted from Mammalian Gene Expression - Can Any

Gene Expression

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#1 It'sNotMe

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Posted 08 May 2013 - 02:30 AM

I am having a peculiar problem of protein of interest being detected in lysate pellet rather than supernatant after chemical lysis (M-PER Mammalian Protein Extraction Reagent). My initial thought was that the protein might have been membrane bound due to the presence binding domains at the C-terminal of the protein of interest. However, I have repeated the extraction using RIPA buffer which also resulted in protein detection in lysate pellet. Correct me if I'm wrong, eukaryotic expression does not result in inclusion bodies as prokaryotes, so it is safe to rule out aggregation and misfolding.

The following are the information of my experiment:
- Cell : CHO-S suspension cell
- Expression System: Invitrogen's Freestyle Max CHO Expression System
- Detection Method: Western Blot

Protein of Interest Properties:
- Tags: 6Xhis and GFP
- Molecular weight: 52kDa
- Theoretical pI: 8.68
- Aliphatic index: 68.88
- Grand average of hydropathicity (GRAVY): -0.415

I am doubtful whether the protein exhibits hydrophobicity with low solubility (contrary to the hydropathicity index) or is it being precipitated by the the lysis buffer. The lysate pellet is green due to the GFP tag, which is also confirmed by fluorescence microscopy with DAPI staining. I have tried sonication with the cell being suspended in M-PER (I also suspected incomplete cell lysis). I would like to repeat sonication by using different suspension buffer i.e. PBS. In the meantime I would eagerly welcome suggestions and insights for my problem.

#2 bob1

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Posted 08 May 2013 - 02:00 PM

There are definitely insoluble proteins produced by eukaryotic cells. Are you running on SDS-PAGE? If so - how are you denaturing the samples?

#3 It'sNotMe

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Posted 08 May 2013 - 08:35 PM

Thank you for your reply, that's good to know. For SDS-PAGE, I just resuspend the lysis pellet with sample buffer and boil it for 10 mins until it dissolved. I have indeed tried usual method by denaturing the pellet using 8M Urea mixed with column binding buffer (10mM Imidazole) and also with 6M Guanidine-HCL but the pellet did not dissolve. However when I sonicated the pellet in 8M urea the pellet dissolved. I sonicate for 5 mins. This quite peculiar for me. Can you please advise?

Edited by It'sNotMe, 08 May 2013 - 08:43 PM.


#4 bob1

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Posted 09 May 2013 - 12:52 AM

OK, for SDS-PAGE, try heating to 70 C for 15-20 min rather than boiling. For the purification stuff, you could try playing with the salt and detergent content of the buffer, but beyond that I don't know.





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