| Background:Hydrogels are a kind of polymers with three-dimensional network structure,which can significantly swell in water but do not dissolve.The raw materials for prepared hydrogels include natural materials and synthetic materials.Natural materials have good biocompatibility,but limited sources,low yield and different batches of materials have different properties.Although the composition and properties of synthetic materials are controllable,however,their functional groups are often single and their biocompatibility is poor.The recombinant proteins obtained by genetic engineering and protein engineering possess both the biocompatibility of natural materials and the controllability of synthetic materials.Elastin-like polypeptides?ELPs?are a class of protein molecules composed of the characteristic functional units of elastin by tandem repeat,which are usually prepared by genetic engineering.Similar to elastin,ELP not only has good mechanical properties,biocompatibility and biodegradability,but also has special reverse thermogel properties,so it has attracted extensive attention in recent years.Objective:A kind of elastin-like polypeptide was designed and its efficient expression in E.coli was established;hydrogels were prepared from the ELP by physical crosslinking,chemical crosslinking and enzymatic crosslinking.Methods:?1?The single sequence of ELP was amplified by overlap extension PCR?OE-PCR?,and the sequence was repeated to a certain extent by recursive directional ligation?RDL?.The prokaryotic expression vector pET28a-[ELP?]5-[ELP?]12-[ELP?]5 was constructed by genetic engineering.?2?The target protein [ELP?]5-[ELP?]12-[ELP?]5 was self-induced in E.coli ER2566 prokaryotic expression strain.The temperature responsive inverse transition cycling?ITC?of ELP was used to purify the target protein,and the purified target protein was quantified.?3?Physical,chemical and enzymatic crosslinking were used to explore the gel formation of ELP at different concentrations.Results:?1?Double digestion and sequencing results showed that the prokaryotic expression vector pET28a-[ELP?]5-[ELP?]12-[ELP?]5 was successfully constructed.?2?Solubility analysis showed that the target protein was soluble.After purification,the purity and yield of the target protein reached 92.6% and 80.46%,respectively.Quantitative results showed that the target protein of 1.05 g could be obtained from 1L of the induction solution.?3?Gelation results show: ELP concentration was 3%,the gelling temperature of 40 °C.ELP concentration was 12.5% and 15%,the gelling temperature of 37°C.The stability experiment showed that the gel network was completely degraded at 21 days when the sample concentration was 3%,and the gel network was completely degraded at 28 days when the sample concentration was 12.5% and 15% in the physical crosslinking experiment.Although the stability of the gel was improved with the increase of ELP concentration,the overall stability of physical crosslinking hydrogels was still poor.In the chemical crosslinking experiment,the remaining gel weight of 3% of the samples was 18.67% at 21 days,and the gel network still not completely degraded at 28 days.The remaining gel weights of 12.5% and 15% samples were 82% and 91.33%,respectively,at day 28.The stability of hydrogels formed by chemical crosslinking is much higher than that by physical crosslinking.In the enzyme cross-linking experiment,the remaining gel weight of 3% of the samples was 7.67% at 21 days,and the gel network basically degraded at 28 days.The remaining gel weights of 12.5% and 15% samples were 61.67% and 76.33%,respectively,at day 28.The stability of hydrogel formed by enzyme crosslinking is between physical crosslinking and chemical crosslinking.Conclusions: A kind of elastin-like peptides was designed and synthesized,and the protein hydrogels based on the peptide were prepared by different crosslinking methods. |
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