Research On The Application Of PhyA/PhyB And Its Homologous Phase Separation Proteins

Many anticancer drugs have defects such as poor water solubility,low plasma concentration,high toxicity profiles,and lack of selectivity.Therefore drug carrier provides a good idea to solve the problem in improving drug insolubility,realizing accurate drug release,reducing drug toxicity profiles,increasing plasma concentration and so on.Because of its clean,efficient,non-toxic and other characteristics,photo-controlled drug carrier is one of the hot spots in the field of biological medicine.By stimulating the photo-responsive groups,the instability of the system can be destroyed,thus realized the release of drugs accurately.Phase separation is a new development direction in the field of life science.With the rapid development of life science,phase separation materials have great application prospects in biological medicine materials,especially in targeted drug transportation.Phase separation is often mediated by interactions between domains of low complexity.By domain analysis,we found that phytochrome-specific,PHY,and c GMP phosphodiesterase/Adenylyl cyclase/Fhl A,GAF domain are homologous genes with low complex domains,which may lead to phase separation of the homologous proteins.This project will construct p ET30-phy A（1-591）and p ET30-All2699g_1-2plasmids to explore whether the recombinant proteins expressed can occur phase separation.It is committed to developing a light-controlled drug carrier with low toxicity,easy controllability and clean characteristics on the base of this research.When the concentration of PhyA（1-591）-PCB protein was 0.1060 mg/m L,the phase separation rate reached the maximum 8.070%within 5 minutes under 650 nm monochromatic light treatment.After 5 minutes,the phase separation rate slowed down gradually,indicating that the degree of solid-liquid separation was limited.So the drug could be released at a uniform and slow speed within a certain time range based on this.At the same time,after being treated with different p H solutions for 3 h,the protein can maintain the stability of 78.73%-84.67%,and there is little difference between various p H solutions treatment.However,in consideration of the low phase separation rate of this protein,a low drug release rate may occur.Besides,with high extraction difficulty and low stability easily affected by temperature,it is not suitable for developing into drug carriers.When the concentration of GAF_1-2-PCB protein was 0.0876 mg/m L,the liquid-liquid phase separation could occur under 650 nm monochromatic light irradiation,and the phase separation rate could reach 12.85%within 5 min.When the concentration of GAF_1-2-PCB protein was 0.1000 mg/m L,protein and ice crystal coagulant were prepared into drug capsules in a ratio of 25:1.Under the stimulation of 650 nm monochromatic light,phase separation of protein occurred,the internal structure of the capsule was broken,and the system was unstable,leading to the release of drug.As a gelatum agent,the drug capsules formed by ice crystal forming agent are relatively stable.The gelatum formed is colorless and transparent,thus it will not block the action of monochromatic light on the carrier,for which it is suitable for the development of photo-controlled drug carriers.Besides,vitamin C and vitamin E as the representatives of water-soluble and oil-soluble medicine,the encapsulation rates are 89.98%and 99.99%.According to the monochromatic light treatment time of 5-25 min,the vitamin C release rate reached25.16%-30.39%,the vitamin E release rate reached 11.47%-80.08%.At room temperature for 4 h,the stability of the capsule was 89.88%.