Some basic questions about cell fractionation and incl.bodies - (Jan/21/2008 )

Hi there,

I expressed my protein of interest in E. coli BL21 and Rosetta and wanted to see if it is soluble or not. I did a cell fractionation today and ran the fractions on an SDS-PAGE gel. I came across two questions:

1. I sociated the cell suspension (in Tris-HCl buffer pH 7.9), centrifuged the samples (5 min, 2000 g) and the Cell free extract was not clear. I centrifuged further. Same result.
Is this normal?

So I took the supernatant and did the ultra centrifugation.
I ran the high speed supernatant and HS pellet fractions on SDS-PAGE and most of my protein seems to go to HSP.

2. Does this necessarily mean the protein is going to inclusion bodies? If so, could I ever get an enzyme activity after purification and refolding? May the band come from left over unlysed cells or whatever in my CFE?

Thank you for your help and to answer that question: I induce with 1 mM IPTG and let the cultures grow for 4 h at 30 °C.

chalet2

P.s.: The protein has a eukaryotic signal sequence, could this be a problem?

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Dear friend

so much writing so i didnt understand what u did.

Proteins solubility test- 1) as u started, sonicate ur cells in lysis buffer, centrifuge at 10000xg for 20 min. the sup containing all

ur soluble proteins (the sup is with a little color). take sup sample for analyzing.

Resuspend the pellet with the same buffer and take sample for analyzing. this sample containing ur insoluble proteins

(inclusion bodies).

Test samples on SDS-PAGE and look for ur protein.

If ur protein appears in the insoluble fraction, u can try adding detergents in the lysis buffer (tween, triton, sarkosyl)

helping solublize the protein. Most likely if ur protein is in inclusion body form, u will need to do purification under denaturing

conditions with UREA then refold the protein.

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First how long and how strong did you sonicate? Sonication can in some strange cases leed to insolubility of the protein.

to 1): 2000g is not very much. It could be that you see an emulsion/suspension of insoluble lipids (membranes, etc.) still in supernatant. So with 2000g for 5 min I would not necessarily expect a clear supernatant. It also be, that all inclusion bodies are in the pellet. IB can be smal enough to withstand 2000g centrifugation and stay in the supernatant.

to 2): it does not necessarily mean that they are in inclusion bodies but it is likely. But the protein can also be associated with membranes and other cell organelles which are in the pellet. the eucaryotic signal sequence could favor this.

Before thinking of trying to refold (it sounds easy, but it is usually pretty laborious and time consuming) I would try to clone different constructs: e.g without signal peptide, diffenent tags (MBP or GST might increase solubility).

The signal sequence in general might not harm the protein but it might also not be functional. I don´t know what it is for (secretion, posttranslational modifications like cleavage, etc) but be aware thet Ecoli might not be able to process the protein porperly. As long as it is still soluble, you should be ok.

There are for example some growth factors which have a signal sequence targeting the protein in mammalian cells to endosomes with a specific protease to cleave that pepide in acidic enviroment. because of the lack of the acidic enviroment and the protease, the growth factors are insoluble in Ecoli, but when you express them without the peptide, they are soluble. The signal peptide in this case is anyhow not needed in Ecoli. You might consider this also.

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