Overexpression of 630 aa protein - (Aug/12/2006 )

I have been trying to overexpress a protein of 630 aa length in pET26b. I am getting 3 new bands compared to the uninduced, one of which appears at the correct position and others of lower MW. But none of them can be said to be overexpressing as the bands are quite faint. I have only tried 0.5 and 1 mM IPTG of which 1mM is only positive. Can I go to higher conc. of IPTG. Can Chloramphenicol be useful in this case. How it should be used?
Hope someone can help.

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Chloramphenicol inhibits protein synthesis. It is used 2 increase copy no. of the plasmid.
U can try upto 2mM IPTG.
The OD of culture at time of induction also matters. Best is generally 0.8 to 1.0.
Also try shortening incubation after induction as ur protein may be degrading fast.

Hope this helps

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Addition of the antibiotic chloramphenicol to bacterial culture medium can be used to amplify the copy number of plasmids. Chloramphenicol inhibits bacterial protein synthesis and thus bacteria chromosomal replication

well have you a tag to your protein?
I'm thinking of degradation of your protein that gives you these faint products.

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you could also try lowering your induction temperature which may result in higher yields of full length protein (if required)

could try inducing with 2mM IPTG overnight at 4 degrees for example

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