Expression And Purification Of The Genetically Encoded Synthetic Human Elastin-like Polypeptide

With the increasing extensive application and the valuable usefulness of the elastin-like polypeptides (ELPs), in vivo high throughout expression of ELPs molecules has always been a crucial problem. Herein we constructed a synthetic gene of the most structurally regular hexapeptide VAPGVG from the human tropoelastin and an unusual highly distribution Ser residues in its hydrophilic domain, exon 26A, with precisely specified molecular weight and sequence, which corresponds to [(VAPGVG)3S]32. Applying the method of orthogonal experimental design, by addition of gelatin and changing the osmotic pressure of the culture medium and induction time, finally we determined the optimal expression condition was 0.2M NaCl,0.5% gelatin in TB culture medium and after 5-6h induction, the expression level of the HELP was improved by 30%-40% as high as the total proteins of the E.coli bacteria. Then we exploited the thermal property of the ELPs, applying the inverse transition cycling to purify the target protein. Under the condition of transition temperature (49℃), the rate of purity can reach up to 80%. To improve the rate of purity and recovery furthermore, we made use of the traditional His-tag affinity chromatography, finally abtained the high purity human elastin-like polypeptide, which is up to 97%. The binding buffer should be 20mM sodium phosphate,0.5M NaCl,40mM imidazole, pH 7.4, and the elution buffer should be 20mM sodium phosphate,0.5M NaCl,500mM imidazole, pH 7.4, respectively.