Construction of a Signal Peptide Library in Escherichia coli and Its Application in Guiding Protein Secretion Expression

Expression of extracellular proteins in Escherichia coli is crucial for boosting recombinant protein production, but single signal peptide use is restricted by its targeting and efficiency limitations. This study integrated endogenous E. coli signal peptides, exogenous microbial signal peptides, and atypical signal peptides to build a library of 30 signal peptides for protein secretion in E. coli . Using maltose-binding protein (MBP) of E. coli and Cellobiose 2-epimerase (CE) of Caldicellulosiruptor bescii as target proteins, the library was fused with their genes via T4 DNA ligase. After extracellular protein concentration measurement and SDS - PAGE analysis, high - efficiency secretion signal peptides were identified. Results showed that signal peptide LTIIb increased MBP secretion 3.39-fold compared to its original peptide, and signal peptides LacZ and N20 enabled CE secretion. Considering CE's high economic value, fermentation conditions for high-yielding were optimized. Under optimized conditions (adding 0.5 mM IPTG and 25 mM glycine to 2×YT medium after 2.5 h culture, followed by 24 h fermentation at 37℃ and 200 rpm), extracellular CE yield increased 4.15 fold. This signal peptide library overcomes traditional single target protein screening limits, allows multi - target - protein compatibility screening, and offers a generalized tool for industrial - scale recombinant protein production and purification. The study confirms the library's feasibility and diversity, providing a basis for new bioagent development.