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Protein Expresion and Isolation from Lactobacillus - Insoluble protein

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#1 silverphoenix



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Posted 12 March 2015 - 04:08 PM

Hi :)


I'm trying to express and purify proteins in Lactobacillus. My protein is his-tagged and the correct size is detected by Western Blot however, the protein is in the insoluble fraction. I used the B-Per Complete Protein Extraction Reagent (which is a Detergent in Tris buffer with Lysozyme and Universal Nuclease), and I supplemented the lysis buffer with lysozyme (10mg/ml) + mutanolysin (10U/ml); incubated at 3hours with gentle mixing at 37C. I've tried different growth temperatures (room temperature, 30C and 37C) and different time points (8, 16, and 24hrs). I've tried solubilizing the inclusion bodies, however only a small fraction of the protein is soluble.


Any suggestions would be greatly appreciated.

Thanks a lot :)



#2 labtastic



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Posted 14 March 2015 - 06:17 AM

Is it transmembrane? Sounds like your protein is soluble in SDS (for the western) but not the extraction kit detergent. Maybe you need to screen a few different detergents. What are you using to solubilize inclusion bodies? Are you sure your protein is in inclusion bodies (i.e., if you isolate the IB's and run on a gel, is your protein there?)


Consider expression at 18C overnight.


Though honestly your problem probably lies in the protein itself. Make more constructs. Truncate residues from the N and C terminus (look at predicted topology...secondary structure, transmembrane domains, signal peptides, etc). It is standard practice in our lab to make a minimum of 5 constructs for each new protein we work with, and depending on how that goes, as many as 15-20. Add cleavable solubilization tags to your protein at (SUMO, MBP, GST, GFP, etc).


Or try a different ortholog of your protein instead. Orthologs can have substantially different expression patters, even if they are 80-90% similar in sequence.

Edited by labtastic, 14 March 2015 - 06:27 AM.

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