We are expressing a fluorescent prokaryotic protein, and have had lots of trouble expressing the full length protein in E.coli without inclusion body formation (tried different expression parameters with no luck)
After resorting to insect cell expression, the protein was still found in the insoluble fraction of the lysate!
Methods of lysing for both expression systems included french press or sonication.
This is a cytoplasmic sensory HK. Can anyone advise me about why this is happening and if there are any solutions?
have you tried lysing the cells with a mild detergent mix such as DDM or even one of the commercial lysis reagents such as bug buster, if you have a fluorescent protein you can then use FSEC to monitor it to see if its in solution. If the protein is truly insoluble typically trying temp drops (22oc) cell lines (Sf9, 21 and Tni) additives and cofactor addition media (we use ESF 921) or switching promotor away from polH to a softer one such as p10. Other wise you will be looking at trying to optimise the construct (truncations, addition of solubilising proteins etc....)
Good luck
Certain forms of protein glycosylation or photoreactive amino acids are formed. Iclusion bodies are due to background infection. Keep your sample at -80 0C. VPLs (virus-like particles) may also appear. Undigested RNAse may be there.
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