Cloning DsRed2 And Prokaryotic Expression Of The Gene Encoding Monomer Red Flourescent Protein, And Its Bioinformatic Analysis

In this study, cloning and prokaryotic expression of DsRed2 gene encoding red fluorescent protein RFP2 from pDsRed2-1 were conducted, the prokaryotic expression vector pGEX4T-1-DsRed2 was constructed and the bioinformation of the protein was analysed. The results showed that[1] DNA sequence of target gene DsRed2 encoding a red fluorescent protein in pDsRed2-1 is 687bp, which involves a complete and functional open reading frame(ORF). Compared with the reference sequence Ref_DsRed2-DNA, the target DNA fragment had a single base mutation from the 537th C to T within its ORF, leading a change of the 179th codon TCC(UCC)to TCT(UCU). The identity between the target DNA and reference DNA was 99.85%.The single base mutation of the cloning target gene DsRed2 within its ORF is synonymous mutation, the encoding protein amino acid sequence remains the same and 100% identity between proteins encoded by both ORFs.[2] DsRed2 gene encoding protein RFP2 are composed of 225 amino acids; The protein belongs to the fluorescent protein family and the protein has no signal peptide, which does not involves in transshipment after synthesis.[3] RFP2 protein amino acid sequence contains five scanty water and five water affinity, the former are relatively small and comprised of the weak hydrophilic group, while the latter are big and comprised of the strong hydrophilic group; the number of hydrophilic amino acid is 2.2 times more than the number of the hydrophobic amino acid, the total value of the hydrophilic-hydrophobic property is-129.9; the hydrophobic core is small,which the amino acid sequence is folded.Hydrophobic amino acid are embedded by the hydrophilic amino acid exposing to the outside, most sequences constitute the hydrophilic surface and form the hydrophilic protein particles.[4] A recombinant prokaryotic expression vector pGEX4T1-DsRed2 was constructed by inserting the target gene DsRed2 into the vector pGEX-4T-1, and DsRed2 linked to 3’-end of the gene of GST tag, resulting in a fusion gene of GST-DsRed2 with a functional ORF which could be continuously read.[5] DsRed2 genes not only expressed in the strain of BL21 (DE3) but also express in the cloning strain DH5α. The quantity of target protein expressed in BL21(DE3) cells increased with an increase in induction time from 1 to 5h, but not obviously increased after 5h. In particular, the accumulation of the target proteins was sufficient by inducing 3h.[6] The RFP2 could dissolve in cells, but most of RFP2 appearing red color existed in hard-soluble inclusion bodies. RFP2 was monomeric proteins appearing red color under natural light without formation of polymers and special light source excitation.