Abstract: The recombinant structural protein SRT-ELP36 consisted of repetitive squid ring teeth protein segment and cationic elastin-like polypeptide sequence (PAATAVSHTTHHAP-VPGVG(VPGKG)₅) can be used to fabricate protein fibers with extraordinary mechanical properties and biocompatibility. In order to improve the production of recombinant protein SRT-ELP36, the pET-25b(+) vector carrying SRT-ELP36 expression cassette was transformed into Escherichia coli, and the fermentation process was optimized in shake flasks and bioreactors. In comparison with the recombinant E. coli BLR(DE3), BL21(DE3) grew more rapidly with a higher expression level of SRT-ELP36. Thus, batch cultivation of recombinant BL21(DE3) in shake flasks was optimized to achieve the highest cell density OD₆₀₀ of 22.3 and SRT-ELP36 production of 0.60 g/L in TB (Terrific Broth) medium with 5% volume ratio of medium to flask after 0.5 mmol/L IPTG induction at 37 ℃ at the end of exponential growth phase. Subsequently, the fed-batch fermentation process in a 5 L bioreactor was developed: induction with 0.5 mmol/L IPTG at the OD₆₀₀ of 35, a high OUR level up to (180±5) mmol/(L·h). With the highest OD₆₀₀ of 88, the highest volumetric and specific production of SRT-ELP36 of 1.85 g/L and 70 mg/g was obtained, respectively. Based on the cultivation conditions optimized in the 5 L bioreactor, high cell density fermentation of recombinant BL21(DE3) was successfully carried out in a 50 L bioreactor with the highest OD₆₀₀ of 103. SRT-ELP36 volumetric and specific production were increased to 2.22 g/L and 90 mg/g, respectively, which was the highest expression level of Squid ring teeth protein as reported to date and providing the foundation for eventual application of SRT-ELP36 in industrial fabrication of mechanically strong fibers.