Optimization Of Retinin Expression And Its Application In Nanocoating Materials

The discovery of antireflective coatings that mimic the eyes of the Drosophila insect provides a promising method to produce functional nanostructured coatings using biodegradable materials under mild conditions.However,the expression of the key retinin in the antireflective coating is currently low,leaving room for further optimization of the coating’s anti-reflection ability.To increase the anti-reflective properties of this artificial nanocoatings and reduce production costs,this study focused on host optimization,protein engineering modification of retinin expression,and optimization of artificial nanocoatings based on their anti-reflective ability.The study started with the key components of nanocoatings,retinin and corneal wax.The main findings of the paper are as follows:(1)Optimized expression of retinin by protein engineering.Using E.coli as the host,the expression of retinin was promoted by N-terminal sequence optimization,expression vector optimization,fusion protein and induction condition optimization.The results showed that the fusion protein strategy can promote the expression of retinin.On the basis of fusion enhanced green fluorescent protein(e GFP),the insertion of N-terminal short peptide and linker peptide can further increase the protein expression,and its fluorescence intensity is 4.8times that of single fusion e GFP.Based on the random mutation of SKIK short peptide to screen for high expression fusion protein,a short peptide mutant SKFM was screened and the fusion protein containing this short peptide was 50% more expressed than the fusion protein with SKIK short peptide;(2)Investigation of the optimal host for retinin expression.The expression of retinin was carried out in Pichia pastoris-GS115 with post-translational modification and high protein yield,Escherichia coli-Rosetta gami2 with fast growth rate and feasible operation,insect cell-Sf9 with similar source of retinin and suitable for secretory protein expression,and mammalian cell-Chinese hamster ovary(CHO)cells with high-density suspension growth and easy expression of difficult-to-express proteins,respectively,and the high expression potential of CHO cells for retinin was finally determined,and the purified retinin was 3.75 mg in 25 m L culture.(3)Investigation of the preparation of lanolin nanoemulsions.An effective method for producing lanolin nano-wax emulsions was established by ultrasonic emulsification technology.Nano-emulsions with a uniform particle size distribution and a stable system were obtained,with a particle size of 156.1 nm at 20% sonication power of ultrasonic crusher for 1 hour;(4)Optimization of nano-coating preparation and evaluation of antireflective Ability.Validation of the characterization and anti-reflectivity of specific retinin forming nanocoatings with waxes and to determine the effect of concentration ratio,p H and temperature in the nano-coating formation system on the characterization and anti-reflective properties of nano-coatings.The results showed that a single retinin was specific in the formation of antireflective ability of nanocoatings of proteins with wax emulsions.The most pronounced papillary structure of the nanocoatings with the best anti-reflective ability was observed at a specific concentration of retinin and a ratio of 16:4 with wax emulsion,a p H of7.0 in the nanocoatings formation system,and temperature of 30°C.The average transmittance of the coating in the wavelength range of 400-750 nm is 93.67%.This study aimed to investigate the potential of retinin in commonly expressed hosts and to investigate the effect of commonly used protein expression optimization methods in E.coli on retinin expression.Meanwhile,the composition of the nanocoatings and the optimization of the concentration,p H and temperature of the system components were determined to lay the foundation for the future application of artificial green degradable anti-reflective coatings.