Modular Assembly And Functional Characterization Of Artificial LEA3 Protein

Late Embryogenesis Abundant group 3 proteins are highly hydrophilic proteins that play an important role in responding to diverse stresses in numerous organisms.Most LEA3 proteins and their hydrophilic domains(HD)have random coils as their main secondary structure in aqueous solutions,which transform into ordered ? helices to function under abiotic stress conditions.Unlike most LEA3 proteins,DosH derived from Dinococcus radiodurans is a naturally ordered LEA3 protein,and previous studies have found that the N-terminal domain(position 1-103)of DosH protein is the key region for its folding into an ordered secondary structure.Synthetic biology provides the possibility for artificial assembling ordered LEA3 proteins or their analogues.In this study,we used the N-terminal domain of DosH protein as module A(named DS)and the hydrophilic domains of LEA3 protein from different sources as module B,and artificially assembled four non-natural LEA3 proteins.The relationship between the order and stress resistance of the modularly assembled LEA3 proteins were clarified by circular dichroism and the construction of recombinant Escherichia coli.The main results obtained are as follows:1.Based on the 11 amino acid motif specific to LEA3 protein,we identified 6 motifs(position 41-162),24 motifs(position 224-701)and 27(position 86-661)motifs from Brassica napus(plant),Caenorhabditis elegans(animal)and Yarrowia lipolytica(fungus),respectively,and the hydrophobic domain containing the motifs was named BnHD,CeHD and YlHD,respectively.The N-terminal DS domain of DosH protein was reassembled with BnHD,CeHD,YlHD and its own hydrophilic domain(DrHD)to form new hydrophilic proteins,named DS+BnHD,DS+CeHD,DS+YlHD and DS+DrHD,respectively.2.Bioinformatics analysis showed that the order of the modularly assembled LEA3 proteins DS+DrHD,DS+ BnHD,DS+CeHD and DS+YlHD was 31.5%,23.3%,18.6% and23.9%,respectively,which were higher than the order of its natural LEA3 protein and hydrophilic domain.The results of circular dichroism showed that the order of DS+DrHD,DS+BnHD,DS+CeHD and DS+YlHD was 82.3%,74.5%,75.4% and 83.3% in normal phosphate buffer,respectively,and the ? helix content was 56.1%,53.7%,49.1% and 64.6%,respectively,which indicating that the modularly assembled LEA3 proteins were ordered with high ? helices.3.The phenotype analysis of the heterologous expression of the modularly assembled LEA3 proteins in E.coli showed that all the strains containing the ordered LEA3 proteins had better resistance under high salt,oxidation and drought stress compared to the BL21-DosH strain containing the natural LEA3 protein,and the resistance ability was:BL21-DS+YlHD > BL21-DS+DrHD > BL21-DS+BnHD > BL21-DS+CeHD >BL21-DosH.4.In vivo enzyme activity assays showed that after treatment with 1.5 M Na Cl for 4 h or 20 m M H2O2 for 10 min,the strains BL21-DS+DrHD,BL21-DS+BnHD,BL21-DS+CeHD and BL21-DS+YlHD had higher catalase,superoxide dismutase activity and lower malondialdehy production than strains containing hydrophilic domain,indicating that the modularly assembled ordered LEA3 protein plays an important role in protecting enzyme activity and reducting oxidative damage of E.coli.cell membraneTaken together,we used the N-terminal domain of DosH protein as module A and the hydrophilic domains of LEA3 protein from different sources as module B,and artificially assembled four non-natural hydrophilic proteins.Circular dichroism showed that the four hydrophilic proteins were highly ordered.Ordered artificial LEA3 protein might protect Escherichia coli against stress by protecting enzyme activity and oxidative cell membrane damage in vivo.Based on these results,the N-terminal domain of DosH protein can be applied in synthetic biology due to it can change the order of hydrophilic domains and thus increase stress resistance.