Milk-Derived Bioactive Peptides Expressed In E.coli BL21 Using Genetic Engineering Technology

Bioactive peptides (Bioactive peptides, BPs) are multifunctional compound which come from protein, having 25 natural amino acids in different composition and arrangement, consisting of two peptides , complex linear, annular structure different peptides. Milk protein is an important source of bioactive peptides, milk-derived bioactive peptides mainly refer to the micro molecule peptide sections which are soluble in water and easier digested and absorbed by human body than the original protein and come from whey protease and casein hydrolyzed by hydrolytic enzymes .The molecular weight of them are between protein and amino acids, they are acid and heat resistance, low permeability, and the largest and most widely used demand of health food material. Now scientists have isolated dozens of active peptides from different milk protein digestion products, scientific research personnel produced these milk-derived bioactive small peptides by biological fermentation, chemical synthesis and gene engineering methods . In these research topics, the preparation methods and active protection technology have become an international hotspot. Our experiment was designed to focus on the target gene of 4 milk-derived immunostimulating peptides, they are Gly-Leu-Phe, Tyr-Gly-Gly, Leu-Leu-Tyr, Phe-Phe-Ser-Asp-Lys. Then the prokaryotic expression vector pTYB11 with target gene was constructed and transduced into E.coli BL21 by recombinant DNA technique to successfully build the E.coli BL21 express recombinant protein system. This research mainly divided into the following content: prokaryotic expression of milk–derived bioactive peptide and intein mediated self-splicing purification of target peptides. Choose four milk-derived immunoactive peptides which have been reported by science research , according to the E.coli preference anticodon table design purpose gene sequences and primer, constructing a purpose gene expression plasmid and recombinant express bacterial strain by polymerase chain reaction (PCR) amplification. After the induction of IPTG ,the recombinant proteins were purificated by Chitin affinity column, and analysised by Western Blotting through the antibody affinity Tag to identify the immunogenicity validated. Results show that target protein has high purity and immunogenicity, thus to provide candidate protein for further research the bioactivitiy of bioactive peptides.