Detection and optimization of microbial expression systems for extracellular production and purification of Ca2+-responsive phase-changing annexin fusions

• Published in: Protein expression and purification Vol. 226; p. 106617
• Main Authors: Li, Jinjing, Wu, Baokang, Ji, Yiting, Zhang, Shuncheng, Ge, Yuanyuan, Fan, Jun
• Format: Journal Article
• Language: English
• Published: Elsevier Inc 01-02-2025
• Subjects: Escherichia coli; Extracellular expression systems; Tag removal; Tandem affinity tag; Two-step purification; Tandem affinity tag; Two-step purification; Extracellular expression systems; Escherichia coli; Tag removal
• ISSN: 1046-5928; 1096-0279; 1096-0279
• DOI: 10.1016/j.pep.2024.106617

Previously, we identified the human annexin A1 as a purification tag for column-free purification with gentler calcium-responsive precipitation. In this work, we used the annexin A1 tagged green fluorescent protein constructs for detecting extracellular production in Escherichia coli, Bacillus subtilis, and Pichia pastoris, and identified that the leaderless fusion protein was transported extracellularly in E. coli with supply of additives including Triton X-100. The coexpressed enzymes, culture compositions, and induction conditions in E. coli extracellular expression systems were optimized. With coexpression of phospholipase C from Bacillus cereus and addition of 0.2 % Triton X-100 after induction for 60 h at 28 °C, the annexin A1 tagged green fluorescent protein and 5-aminolevulinate dehydratase from E. coli were overexpressed and purified from lysogeny broth by precipitation with 20 mM Ca2+ and redissolution with 25 mM EDTA with the acceptable protein purities and recoveries. The silica binding peptide was fused to the annexin A1 tagged fluorescent protein fusion for successive affinity precipitation and purification. With incubation of the specific protease, the released tag-free protein displayed higher purity via on-resin cleavage than that through cleavage of the free fusion protein. The tandem tag is applicable for two-step purification of small or large amounts of other fusion proteins in the culture and recovery of tag-free proteins at low cost.
•The extracellular expression of the hanA1- EmGFP constructs in E. coli, B. subtilis, and P. pastoris was detected, only the fusion protein was secreted into the culture from E. coli cells.•Different E. coli extracellular expression systems were optimized for improving the protein yield and purification efficiency.•Two hanA1 tagged proteins were purified by Ca2+ precipitation and EDTA redissolution.•The silica binding peptide was fused to the hanA1-EmGFP for successive purification and on-resin cleavage.