The Soluble Expression Of Recombinant Human Epididymal Protease Eppin

Family planning is the key in keeping the economic development in our country, therefore the contraceptive research is given great attention.In recent years, the human epididymal protease inhibitor(Eppin) has been thought as a male contraceptive target. Eppin expresses three m RNAs encoding two isoforms of cysteine-rich protein containing both Kunitz-type and WAP-type which locates at testis and epididymis. In 2004, Professor O’Rand immuned male monkeys with recombinant Eppin protein, which caused seven out of nine males(78%) infertility with high title antibodies in several months. However when antibodies dropped, five(72%) males restore fertility. In the process of ejaculation, Eppin combines with Semenogelin(SEMG1), and regulates the prostate specific antigen(PSA) hydrolysis SEMG1. This combination delays the process of hydrolysis to protect the sperm from dying in female vaginal environment resulting in liquefaction of the semen coagulum and the progress release of motile spermatozoa.When the antibodies specific to SEMG1 bind with Eppin, the PSA cannot hydrolyze antibodies and result infertility. We parse the three-dimensional structure of Eppin by the protein crystallization to explain the molecular mechanism how Eppin regulates the movement of sperm. The rsearch on the three-dimensional structure of Eppin protein will screen the potential contraceptive vaccine by explaining the molecular mechanism of the conformation change. This topic is proposed to express adequate soluble Eppin recombinant protein to prepare for protein crystallization.Firstly, We predicted the amino acid sequence, secondary structure and disulfide bond pairs of Eppin. Secondly, we have try to express and purify the soluble Eppin protein in eukaryotic and prokaryotic expression systems. We try to express Eppin protein in the eukaryotic insect Bac-to-bac baculovirus expression system and Drosophila expression system. In the meantime it was designed that various combination of prokaryotic expression vector and host as well as the methods to release protein and the best induced temperature and concentration of inducer. Then with the help of maltose binding protein MBP label and the expression strain Rossetta2-Origami B(DE3), the first soluble Eppin protein was obtained. For getting large quantity and high purity of soluble protein, we modified the plasmid by adding the TEV restriction sites and changing the six histidine sequence from the C-terminal to the N-terminal amino acids. The protein purification methods include Ni-NTA 、Amylose column and Molecular sieve purification system. In the meantime,The experimental results are as follows: ①The N-terminal amino acids of Eppin has a flexible loop area and compared with the C-terminal are more disorder. The Eppin is predicted to form 7 disulfide bond, respectively located between 33aa-65aa、40aa-60aa、48aa-61aa、54aa-69aa、77aa-127aa、86aa-110aa、102aa-123 aa. ②In Bac-to-Bac baculovirus system, Eppin become inclusion bodies in the cells. And it doesn’t express in Drosophila expression system. ③ The recombinant plasmids includes pET22b-Eppin 、p ET40b-Eppin、p ET28a-Eppin、pET32m-Eppin、p ET15a-Eppin have been successfully builded. However the Eppin protein neither become inclusions or unexpressed. ④The recombinant plasmid p MALc-2x-Eppin firstly express soluble recombinant protein MBP-Eppin,and without the label the Eppin is still soluble with antigenicity. The mass spectrometry confirmed it as Eppin protein. ⑤The soluble Eppin protein is in polymer form with MBP. And the six histidines for Ni column purification are unexposed on the space. Far more researches on the protein crystallization may focus on the C-terminal amino acids of Eppin protein.The soluble Eppin protein provides important experimental base for the determination of the Eppin’s three-dimensional structure and screening the potential contraceptive vaccine.