Your protein is probably in the form of inclusion bodies. Look in your insoluble fraction after sonicated. You can try to do a Western Blot if you find an antibody on the Nephrin protein. See if you can get more soluble protein by screening the culture conditions (induction temperature, time...) otherwise you will have to do refolding steps to obtain your protein

Nicolas Poirier I have the antibody for the nephrin protein. I will perform a Western blot to detect whether the protein is expressed.

Thank you.

Sebastian Schmitt

1. The plasmid I purchased was cloned with the GOI using the Golden Gate method, and I double-checked it by sequencing. The sequence matched the expected GOI, with no frameshifts or mutations. Additionally, I performed PCR targeting the GOI in that plasmid template, and the amplicon size matched the expected size of the GOI.

2. For the codon-optimized construct, I did not verify which codons are preferred and which are not.

3. The temperature of expression that I tested was 20 and 25 degrees Celsius, for up to 20 hours post-IPTG induction.

It's probably misfolded, nephrin requires multiple disulfide bonds for proper folding. Have you considered periplasmic expression? Ideally, I'd try insect cells, it has other PTMs like phosporylation and glycosylation that you'd never achieve with bacterial system. From what I've seen people express only single nephrin domains in bacteria.