Synthesis of multiple copies of neurotrophic peptide and their high level expressions in E.coli

AIM: To construct and express containing multiple tandem copies of a peptide (neurotrophic peptide,NP),which was designed according to the NP sequence of prosaposin.METHODS: DNA sequence of peptide NP was synthesized by the preferred codons of E.coli.Two copies of NP fragments were produced by PCR and inserted into pUC18 vector.The fragments from pUC18-2NP by EcoT 14I were ligated into tandem multi-NP fragments through self-ligation,and subcloned into pET-EcoT vector,which has a unique EcoT14I cloning site allowing unidirectional insertion of a desired sequence.Multi-NP clones were screened by PCR-array.RESULTS: The constructs with different repeats of NP were obtained and expressed as fused-proteins at high level in E.coli BL21 (DE3).In order to get monomer peptide,each copy of peptide was interspersed by a unique site where the fused-protein could be cleaved by cyanogens bromide.CONCLUSION: Peptide NP could be highly expressed in E.coli.This work builds a solid foundation for further study on its bioactivity.