Have you removed the introns from the gene?

Dear Haoran Z Du

in my experience the expression yields for large proteins (>100-150KDa) in E.coli is generally low, and often protein degradation in subdomain is observed due to the presence of proteases in he E.coli citoplasm and secretion ability of the E.coli in the periplasm is limited to lower MW (eg you can easly produce a Fab antibody in the periplasm while is very difficult to obtain a full lenght mab)

If you gene is derived from an eucariotic organism you can try to perform codon optimization to improve the expression yield but i think that mammalian cells as Expi293 are more able to work with so high MW.

best

Manuele

To express a large fusion protein (e.g., 223 kDa) in bacteria, use a robust expression system like E. coli. Optimize codon usage, employ strong promoters, and consider split-intein or dual-vector systems. Include solubility tags, use chaperones, and optimize growth conditions to enhance protein folding and expression yield.