Abstract: This paper describes the development of a new recombinant expression and purification strategy for lysyl endopeptidase (Lys-C) in order to improve the efficiency of natural Lys-C production chain. An artificial pro-peptide (MGSK) and the 6×His tag were fused to the N-terminus and C-terminus of the Lys-C mature peptide gene sequence from Lysobacter enzymogenes , respectively, and then the codon-optimized sequence was inserted into plasmid pET-28a. Lys-C was efficiently expressed in the recombinant strain JM109DE3_P _T7 -LysC and controlled by the IPTG-inducible promoter P_T7. The inclusion bodies were collected by high-density fermentation in a bioreactor, after which they were solubilized to obtain the Lys-C denaturing solution, and DTT was removed by Sephadex G25 chromatography. Then, the pre-pro peptide (pre-N-pro) was added to Lys-C refolding solution to assist in the folding of the mature protein. Further, the large volume Lys-C refolding solution was concentrated using tangential flow filtration, followed by a multi-step purification process including Ni NTA-Sepharose affinity chromatography, ultrafiltration, and Sephacryl S-100 chromatography to obtain high-purity recombinant Lys-C. Finally, the activity of recombinant Lys-C was measured by chromogenic reaction and digestion of insulin aspart precursors. The recombinant Lys-C fermentation yield was determined to be 2.4 g/L, and the final yield reached 48 mg/L after renaturation and purification. The enzymatic activity of Lys-C was improved by 4.8-fold (13.8 U/L) in the presence of 80 mg/L of pre-N-pro. The specific enzyme activity of Lys-C achieved 10.2 U/mg after multi-step purification, and the digestion efficiency of Lys-C for insulin precursors reached 93.5%. In this study, recombinant Lys-C with high yield and good activity was obtained allowing for accurate digestion of the insulin precursors, which provides an insight into Lys-C recombinant expression and potential industrial application.