Design and construction of hydrophobic ELP genes library

Abstract:

[Objective] To design and synthesis encoding hydrophobic ELP genes and construct ELP genes library. [Methods] The highest hydrophobic Ile(I) amino acid (hydrophobic parameters: 4.5) was chosen to replace the guest residues X of ELP pentapeptide repeat serial units building block (VPGXG)10 and the encoding block fragment was synthesized, flanking with a Dra Ⅲ and BglⅠ sites at upstream and downstream respectively. The Dra Ⅲ and Bgl Ⅰ were designed as a pair of isocaudamer for recursive directional clone to construct the ELP genes library. To confirm that the ELP library was constructed properly, the ELP[I]50 gene was randomly chosen to be expressed. The Tt of purified ELP[I]50 was detected via turbidity changing at OD350. [Results] The ELP genes library containing the ELP[I]n (n=10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120) was successfully constructed. The library was confirmed by expression of ELP[I]50 gene, protein purification by inverse transition cycling. The Tt of ELP[I]50 was 24.3 °C. [Conclusion] In order to increase the number of hydrophobic amino acids in ELP, the Ile(I) was chosen as a guest amino acid, the foundation for further screening ELP tag was builded up to get proper recombinant tag with smaller molecular weight, high levels expression, and low phase transition temperature.