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