Hello,

1/ Do you have the plasmids in E. coli cells designed for protein overexpression and not cloning?

2/ Check the clones by sequencing (if e. g. a mutation introducing a stop codon is early in the gene, you will get no expression of the desired protein).

3/ The protein may be unstable in your expression strain. Try several protein expression strains.

4/ Sometimes, when overexpressing a protein, no extra band is apparent in the IPTG+ lane in total lysate but when you affinity purify it you still get a reasonable yield. If the sequencing validates your clone, I suggest to at least once attempt to purify it.

There are a number of possible reasons why the E. coli cell containing the recombinant cell may not express the particular protein upon induction with IPTG. These include incorrect promoter and/or RBS selection, incorrect gene sequence, incorrect codon usage, improper growth conditions and/or media, incorrect induction conditions, and incorrect post-translational modifications. Additionally, the protein may be expressed at very low levels which may make it difficult to detect.

Dr. Bandla answer can help you. These few points may also help you in resolving your issue.

Check

1.Your gene of interest is in frame

2. sequence the clone u have to check whether the gene of interest is correct, not any point mutation and still in frame. These factors can affect protein expression u trying to express.

3. whether protein have long stretch of rare codons? this may result in truncated or non-functional protein

4. host you using. leaky expression could have adverse effect on cell's growth

5. growth conditions and culture condition you using.

Here are some possible explanations:

To troubleshoot the problem, you can try the following:

These video playlists might be helpful to you:

https://www.youtube.com/playlist?list=PLzBiAPKi8vONSqjcweFWHMRy13y3484bE

https://www.youtube.com/playlist?list=PLzBiAPKi8vOMheKwb0mu1dcZRaeGUFSlw

https://www.youtube.com/playlist?list=PLzBiAPKi8vOPbE7Db9mKwloe34E8rXXEU