The Feasibility Explore Of Elastin-like Polypeptide As Protein Marker

As a standard of protein molecular weight, Protein marker is widely used inprotein electrophoresis, its weight ranges from6KDa to200KDa. According to theweight of the protein, it can be divided into low molecular weight protein marker andhigh molecular weight protein marker；Protein marker can also be divided into theprestained type and non-prestained type if needed dyeing treatment afterelectrophoresis. The preliminary findings of the market price show that the marketprice of non-prestained protein marker is10times than that of the prestained onehaving the same molecular weight range.Statistics reveal that the componant of marker is mostly constituted by lots ofnatural proteins enduring heated denaturation well, through investigating thecomposition of the commercially available protein marker such as Takara company,Fermentas company and Shanghai Biological Engineering company. The purificationmethod almost uses the conventional chromatography. But in order to obtain highpurity of the target protein, the purification should combine several other traditionalpurification methods. Moreover, the purification process is relatively complicated,time consuming, expensive, and the chromatographic materials waste seriously.Therefore, it has an important significance to use of a single protein specie and novelpurification method for reducing manufacturing cost and simplifying themanufacturing process.Elastic polypeptide (Elastin-like polypeptides, ELPs) is a class of artificialprotein which is temperature sensitive and has a characteristic of reversible phasetransition. Because ELPs can aggregate together when the temperature of theenvironment was changed, we are able to use this property to separate and purify thetarget protein from other proteins. Owing to this particular performance, ELPs has ahuge potential applications in the field of medicine, biology (protein purification),and materials science. But few studies were found for protein marker.In this article, we have successfully constructed six cloning vector pUC19-ELP [KV8F] and six expression vector pET-22b(+)-ELP [KV8F] through RDL (recursivedirectional ligation) method. We extended the ELP [KV8F] from20repetitions to140repetitions step by step, then introduced into E. coli, after induction by adding IPTG,different lengths of the elastin-like polypeptides with different molecular weight wereobtained by ITC process. The SDS polyacrylamide gel electrophoresis revealed thatthe visible strip distributed well with high purity and hardly observed other proteinbands. Moreover, the purity of each strip almost reached98%analysed using thesoftware of BandScan.The purity was consistent with the requirements of the proteinmarker. The result provided the basis for futher study of the elastin-like polypeptideas the protein marker. Under laboratory conditions, the average production of ELPswas about120mg/L, and marker products only required about21μg for onceelectrophoresis. Therfore, the ELPs marker only costs2.465￥,which is used for100times. However, marker from Fermentas company costs180￥. In summary,elastin-like polypeptide marker will have a widely applications and broad prospectsbecause of its low cost.