Surface Display of Domain Ⅲ of Japanese Encephalitis Virus E Protein on Salmonella Typhimurium by Using an Ice Nucleation Protein

A bacterial cell surface display technique based on an ice nucleation protein has been employed for the development of live vaccine against viral infection. Due to its ubiquitous ability to invade host cells, Salmonella typhimurium might be a good candidate for displaying viral antigens. We demonstrated the surface display of domain III of Japanese encephalitis virus E protein and the enhanced green fluorescent protein on S. typhimurium BRD509 using the ice nucleation protein. The effects of the motif in the ice nucleation protein on the effective display of integral protein were also investigated. The results showed that display motifs in the protein can target integral foreign protein on the surface of S. typhimurium BRD509. Moreover, recombinant strains with surface displayed viral proteins retained their invasiveness, suggesting that the recombinant S. typhimurium can be used as live vaccine vector for eliciting complete immunogenicity. The data may yield better understanding of the mechanism by which ice nucleation protein displays foreign proteins in the Salmonella strain.

Surface display of carbonic anhydrase on Escherichia coli for CO_(2) capture and mineralization

Mineralization catalyzed by carbonic anhydrase(CA)is one of the most promising technologies for capturing CO_(2).In this work,Escherichia coli BL21(DE3)was used as the host,and the N-terminus of ice nucleation protein(INPN)was used as the carrier protein.Different fusion patterns and vectors were used to construct CA surface display systems forα-carbonic anhydrase(HPCA)from Helicobacter pylori 26695 andα-carbonic anhydrase(SazCA)from Sulfurihydrogenibium azorense.The surface display system in which HPCA was fused with INPN via a flexible linker and intermediate repeat sequences showed higher whole-cell enzyme activity,while the enzyme activity of the SazCA expression system was significantly higher than that of the HPCA expression system.The pET22b vector with the signal peptide PelB was more suitable for the cell surface display of SazCA.Cell frac-tionation and western-blot analysis indicated that SazCA and INPN were successfully anchored on the cell’s outer membrane as a fusion protein.The enzyme activity of the surface display strain E-22b-I RL S(11.43 U⋅mL^(−1) OD 600−1)was significantly higher than that of the intracellular expression strain E-22b-S(8.355 U⋅mL^(−1) OD 600−1)under optimized induction conditions.Compared with free SazCA,E-22b-I RL S had higher thermal and pH stability.The long-term stability of SazCA was also significantly improved by surface display.When the engineered strain and free enzyme were used for CO_(2) mineralization,the amount of CaCO_(3) deposition catalyzed by the strain E-22b-I RL S on the surface(241 mg)was similar to that of the free SazCA and was significantly higher than the intracellular expression strain E-22b-S(173 mg).These results demonstrate that the SazCA surface display strain can serve as a whole-cell biocatalyst for CO_(2) capture and mineralization.

Combinatorial cell surface display system in Escherichia coli for noninvasive colorectal cancer detection

Navigation and recognition of disease lesions remain challenging during colorectal cancer diagnosis and treatment,given that the precision and capacity of using specific surface antigens as recognition sites are relatively lacking.Moreover,the low penetration rate of noninvasive detection methods has delayed disease diagnosis in developing countries.We developed a bacterial device targeting the Thomsen-Friedenreich antigen,which is abundant on lesion tissue,and produced vesicles that serve as markers for ultrasonic detection.The device uses a bacterial cell surface display system and acoustic reporter gene to function as a screening device for colorectal cancer detection.The diagnostic efficiency of this device was determined by flow cytometry,immunohistochemistry,microfluidic chip-based assay,and ultrasonic examinations on both cellular and tissue scales.In all scales and experiments,our device showed great feasibility upon differentiating disease lesion and normal tissue,while foundation of the usage of vesicle reporter system as noninvasive method has also been laid.The application of this device provides insight into the practicability and prospect of bacterial detecting agents in the field of diagnostics.All animal studies were approved by the Institutional Animal Care and Use Committee of Shanghai Jiao Tong University,China(approval No.201801015)on February 23,2018.