Expression,Purification And Activity Identification Of Human Irisin Recombinant Protein

As a recently discovered muscle factor, human Irisin is an excretive polypeptide fragment whose precursor is fibronectin type III domain-containing protein 5(FNDC5). Irisin can transfer the signal of skeletal muscle and keep the relationship between skeletal muscle and the peripheral tissues. Irisin will certainly be a very promising active factor, and become the new targets for prevention and control of metabolic disease and its complications. Therefore, exploiting a method that can be used to mass produce and easily purify Irsin is very significant. In the current study, according to various characteristics of the expression system, the P.pastoris expression system and E.coli expression system would be chosen to express recombinant human Irisin protein. Moreover, the recombinant protein would be purified and its activity would be identified. The main results of this study were as follows:(1)The human FNDC5 cDNA was obtained from total RNA of muscle tissue, meanwhile, the human Irisin gene was amplified by the specific primers.(2)The human Irisin gene was inserted into E.coli expression vector pET-30 a to form pET-30a-Irisin. And the recombinant plasmid was transformed to E.coli Rosetta(DE3) pLysS to form the Rosetta- pET-30a-Irisin strain.(3)The E.coli recombination strain Rosetta- pET-30a-Irisin could realize the dissoluble expression of human Irisin induced with IPTG. The 14 KDa recombinant protein was purified by Ni-sepharose, and the recovery rate was about 2mg/L.(4)The human Irisin gene with his-tag was inserted into pPIC9 K to construct the P.pastoris recombinant expression vector p PIC9K-Irisin-his. The recombinant plasmid lineared by Sac I was transformed into P.pastoris GS115 competent cell, and the GS115-pPIC9K-Irisin-his recombination strain of P.pastoris with Mut+ phenotype was screened out.(5)The GS115-pPIC9K-Irisin-his recombinant strain of P.pastoris could realize the secretory expression of human Irisin induced by methanol, and the optimized induction time was 96 h. The recovery rate of recombinant protein purified by Ni-sepharose was more than 20mg/L, and the recombinant protein generated four strips of various sizes between 14-29 k Da by shearing and modification of glycosylation(6)The level of UCP1 and elovl3 mRNA in C2C12 cells were increased after treated with the purified recombinant human Irisin protein from P.pastoris, certifing that the recombinant Irisin had biological activity. However, the recombinant human Irisin protein purified from E.coli expression system hasn’t biological activity.It was the first time that human FNDC5/Irisin gene was expressed in P.pastoris expression system and E.coli expression system, and obtained high purity recombinant protein by nickel column. Due to modification of glycosylation, the recombinant protein from P.pastoris produced different size of strips with biological activities. The protein from recombinant E.coli without modification was shown single band and had no biological activities. The establishment of the two kinds of recombinant expression systems laid a foundation for furture study in structure, function and application of human Irisin protein.