Evaluation of the growth medium of a new ice-nucleating-active Pseudomonas: A response surface methodology approach

Aims@#Attention to ice nucleation proteins has increased for more than two decades. Ice nucleation proteins have been utilized for artificial snow-making known as Snowmax™, cryopreservation of tissues and cells, and cloud condensation nuclei. There is a direct relationship between bacterial growth and ice nucleation activity. Therefore, the optimization of the culture medium seems necessary.@*Methodology and results@#The effect of different carbon and nitrogen sources on the growth of a new native Pseudomonas sp. IRL.INP1 was evaluated by using fractional factorial design, the path of the steepest ascent experiment and central composite design. Ice nucleation activity, biomass and whole-cell protein were identified afterward. The model predicted by response surface methodology indicated that the maximum bacterial growth was observed when sucrose, ammonium sulfate [(NH4)2SO4] and manganese (II) (Mn2+) were utilized at 12.46 g/L, 321.97 mg/L and 938.09 µM, respectively. Also, 1.10 g/L biomass and 0.85 µg/µL whole-cell proteins were gained, and the isolate showed ice nucleation activity 31 sec sooner after optimization.@*Conclusion, significance and impact of study@#Ice nucleation proteins are growth-dependent and the growth condition optimization leads to higher bacterial cells growth. Therefore, best bacterial growth was obtained when proper carbon and nitrogen sources were used, and ice nucleation activity was observed in shorter time. This is the first study concerning ice nucleation activity optimization using different carbon and nitrogen sources.

Optimization of the whole-cell catalytic activity of recombinant Escherichia coli cells with surface-immobilized organophosphorus hydrolase.

Previous studies have verified the feasibility of using Escherichia coli systems that display organophosphorous hydrolase (OPH) on the cell surface as whole-cell catalysts. However, the inefficient display of the enzyme on cell surfaces remains unaddressed. In the present study, multiple optimization experiments on full-length and truncated ice nucleation protein anchors, E. coli host cells, culture media, and culture conditions were performed to optimize whole-cell OPH enzymatic activity. The results show that apart from the dramatic effect of isopropyl-b-d-thiogalactoside concentration and culture temperature, the coordination between the anchor protein, culture media, and host cells is essential for highly efficient OPH display. Under optimal conditions, namely, culturing in M9 medium, 20 °C induction temperature, 0.1 mmol l-1 IPTG, and 100 μmol l-1 Co2+, the engineered E. coli strain MB109-406 that expresses the fusion enzyme InaK-N-OPH exhibited a whole-cell OPH activity of 0.62 U mg-1 ?cell d.wt. This result is much higher than that of several currently available OPH-displaying systems, which shows the potential of the current system for further large-scale industrial or environmental applications.

Cloning, protein expression and display of synthetic multi-epitope mycobacterial antigens on Salmonella typhi Ty21a cell surface.

Expressing proteins of interest as fusion to proteins of bacterial envelope is a powerful technique for biotechnological and medical applications. The synthetic gene (VacII) encoding for T-cell epitopes of selected genes of Mycobacterium tuberculosis namely, ESAT6, MTP40, 38 kDa, and MPT64 was fused with N- terminus of Pseudomonas syringae ice nucleation protein (INP) outer membrane protein. The fused genes were cloned into a bacterial expression vector pKK223-3. The recombinant protein was purified by Ni-NAT column. VacII gene was displayed on the cell surface of Salmonella typhi Ty21a using N-terminal region of ice nucleation proteins (INP) as an anchoring motif. Glycine method confirmed that VacII was anchored on the cell surface. Western blot analysis further identified the synthesis of INP derivatives containing the N-terminal domain INP- VacII fusion protein of the expected size (52 kDa).

Surface Display of Domain Ⅲ of Japanese Encephalitis Virus E Protein on Salmonella Typhimurium by Using an Ice Nucleation Protein / 中国病毒学·英文版

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.