Molecular Design,Preparation And Characterization Of Novel Elastin-like Peptides

Recombinant elastin like peptides(ELPs)are polypeptide polymers synthesized by genetic engineering.They are generally composed of VPGXG pentapeptide repeat units.They have reversible phase transition,good biocompatibility,degradability and self-assembly characteristics,and thus have great application potential in the field of new biomedical materials.The phase transition temperature and thermal behavior of ELPs have a great impact on their functions and applications,but the understanding of the molecular mechanism of their thermal behavior is still very limited.It is still a major challenge to design ELPs polypeptide nanomaterials with suitable thermal behavior for specific application scenarios.Based on gene recombination technology,material characterization and molecular dynamics simulation,this paper adopts(VPGIG)n,which is containing the most hydrophobic ile as the guest amino acid,as the starting material to explore the influence of ELP amino acid composition on its thermal behavior.On this basis,two new ELP molecules are successfully designed and created,which enhance our understanding and enrich ELPs molecular library.The research contents and main results are as follows:1.Bioinformatics analysis of ELPs.The hydrophilicity,relative molecular weight,isoelectric point and three-dimensional structure of ELPs in this study were analyzed and predicted by using the common analysis tools of bioinformatics.The results showed that all ELPs studied were hydrophobic proteins.The three-dimensional structure prediction results showed that such proteins were mainly barrel structures composed of internal hydrophobic stacking force and external hydrogen bond network.2.Cloning and recombinant expression of ELPs.The ELP genes were obtained by PCR and homologous recombination with p ET-28 a vector to construct p ET28a-ELPs,which was expressed in Escherichia coli BL21.Among them,(VPGIG)30,(VPGIG)60,(VPGIG)90 and(VPTGIG)25 were soluble proteins.They were isolated and purified by ITC method,and the average yield was 12 mg/L;(VPTIG)30 was expressed as inclusion body.After renaturation and purification,the average yield was 15 mg/L.SDS-PAGE of all ELPs can get a single band,indicating that a relatively pure ELPs protein obtained,and the purity can reach 95%.3.Thermal behavior of starting material ELPs.The effects of concentration and chain length on the phase transition temperature of(VPGIG)n(n= 30,60,90)were studied by dynamic light scattering.The results showed that the chain length and concentration were inversely proportional to the Tt value of ELPs,ranged from 13 to27 ℃,showing reversible phase transition manner.The secondary structure of ELPs was analyzed by circular dichroism spectrometer(CD).Upon heating,the phase transition of ELPs occurred,and the secondary structure changed from random coil to b-sheet;Upon cooling,(VPGIG)n can recover to its original random coil structure.4.Thermal behavior of a novel ELP(VPTIG)30.The substitution of gly3 by thr slightly increases the low critical transition temperature(LCST),and its thermal behavior changes to large thermal hysteresis;The secondary structure of(VPTIG)30was analyzed by circular dichroism spectrometer(CD).Upon heating,the secondary structure of(VPTIG)30 changed from random coil to b-sheet;However,upon cooling,the negative peak intensity of(VPTIG)30 did not recover and remained in an orderly structure state.Small molecule probe 1,6-hexanediol was added to(VPTIG)30.With the increase of 1,6-hexanediol concentration,the turbidity of ELP decreased until it was completely dissolved,indicating that the main force to maintain the phase transition structure of ELPs is hydrophobicity.5.Molecular dynamics simulation of ELPs.The distribution of local hydration layer around the peptide at different temperatures was quantified by radial distribution function(RDF),and the number of hydrogen bonds between the peptide and water molecules was calculated.It was found that the phase transition of ELPs with the increase of temperature may be due to the destruction of hydration layer.The secondary structure after heating aggregation was analyzed by Ramachandran diagram.It is found that(VPTIG)30 and(VPGIG)30 contain both random coil and βStructure below the Tt,and forming a new hydrophobic ordered structure above the Tt value.In order to explain the difference of thermal hysteresis behavior between(VPTIG)30 and(VPGIG)30,the inter and intra chain dynamics of ELPs in soluble and aggregated structures were further simulated.The results showed that the number of intra chain hydrogen bonds of(VPTIG)30 was higher than that of(VPGIG)30,indicating that the intra chain force was stronger and may enhance the maintenance of surface hydrophobicity.The Heatmap results of hierarchical clustering show that the hydrogen bond alternation patterns of the two types of ELPs have similar internal laws in different temperature ranges,but the hydrogen bond and hydrophobic interaction in the(VPTIG)30 enhance the interaction force in the internal space of the peptide,so that the whole peptide structure is self-locking.Upon coolling,the re-exposure of the hydrophilic part is prevented,and the aggregation state of ELPs is maintained;Therefore,it is speculated that the main reason for the large thermal hysteresis of(VPTIG)30 is that the introduction of thr promotes the hydrogen bond and hydrophobic interaction,enhances the intramolecular and interchain interactions of ELP,and forms intermolecular self-locking.Therefore,when the temperature decreases,it still maintains the molecular aggregation state and cannot return to the solution state.6.Design and characterization of a novel elastin like protein(VPTGIG)25.Based on the above understanding of the motif and thermal behavior properties of ELPs,a new ELP molecule(VPTGIG)25 was rationally designed.Through the characterization of its thermal behavior and properties,it was found that the Tt value of(VPTGIG)25was significantly increased to 37 ℃,showing reversible phase transition manner,suitable for the application in the field of biomedicine.In conclusion,this study systematically studied the molecular basis of motif and thermal behavior of ELPs,designed two new and diversified ELPs molecules,enriched the ELPs molecular library and expanded its application scenarios.These in-depth explorations are of great value for the design of diversified ELPs materials with special thermal behavior requirements for the fields of medicine and biotechnology.