Expression,purification And Characterization Of The Cuticular Protein OfCPT1 And OfCPT2 From Ostrinia Furnacalis

Asian corn borer(Ostrinia furnacalis)is the main pest that hurts crops including corn in China.Study on the cuticle of Asian corn borer will contribute to understand the formation of Asian corn borer cuticle and the way maintaining the cuticular physiological structure.Cuticular protein and chitin constitute the insect cuticle together as the two main components,therefore the discussion of the interaction between cuticular protein and chitin is significant in studying the formation mechanism of insect cuticle.CPT is an important cuticular protein family in insect cuticle,and absence of CPT may cause incomplete cuticular structure.Sequence analysis revealed that OfCPT1 and OfCPT2 belong to the cuticular protein Tweedle family and contain a conserved domain,which possibly has the ability to bind chitin.This study explored the physical and chemical properties of OfCPT1 and Of CPT2,and OfCPT1 and OfCPT2 interaction with chitin,we have obtained the following research results:1)Ofcpt1 and Ofcpt2 gene sequences were retrieved from the transcriptome data of O.furnacalis,and we searched the NCBI database to obtain protein sequences of OfCPT1 and OfCPT2.Through analyzing the protein sequence,we identified that OfCPT1 and OfCPT2 have signal peptide sequences at the N-terminus which are consist of 19 and 14 amino acids respectively.Sequences multiple alignments show that both OfCPT1 and Of CPT2 contain a conserved sequence of 100 amino acids,located in the middle of the protein sequence.Both proteins are hydrophilic proteins,and their isoelectric points are close.2)pET--28 a vector was used to express OfCPT1 in prokaryotic expression systems,protein was expressed as inclusion bodies.Purification by nickel-NTA chelate affinity chromatography with a yield of 50 mg/L.We also use pET--28 a vector to express OfCPT2 in prokaryotic expression systems,it was expressed as the soluble form,and purified by nickel-NTA chelate affinity chromatography with a yield of 5 mg/L.3)Chitin binding assay revealed that OfCPT1 is selective for the binding of different types of chitin.Of CPT1 has moderate binding ability to α-chitin,β-chitin,chitosan,colloid chitin and CNW,but strong binding ability to CNF.4)The chitosan binding experiment,circular dichroism spectrum showed that the structure of OfCPT1 has changed after binding chitosan solution.Protein secondary structure changed from α-helix(61.2%)to β-sheet(87.8%).OfCPT1 facilitates the binding to chitosan by changing its secondary structure.5)The mechanical properties of the protein-chitosan composite membrane were determined by the tensile method.The results show that OfCPT1 can effectively improve the elongation and toughness of the composite membrane at 1% protein concentration.6)Microscopic observation show that protein produces liquid-liquid phase separation in the presence of NaCl,size and density of droplets related to concentration of NaCl and pH.OfCPT1 contains an intrinsic disordered sequence at the N-terminus,which may cause the liquid-liquid phase separation phenomenon.This work revealed the specific chitin binding properties of OfCPT1 and changes in the OfCPT1 secondary structure after binding to chitosan,explored the modification of chitosan membrane caused by OfCPT1 protein,and analyzed the phenomenon of liquid-liquid phase separation of OfCPT1 and its potential impact on cuticular formation.These conclusions benefit our understanding of CPT family proteins,and lay a foundation for revealing the mechanism of insect cuticular formation.