Expression And Purification Of HBRP And The Analysis Of Its Biological Function

The major protein of bovine seminal plasma namely BSP - Al, BSP - A2, BSP - A3 and BSP - 30kDa( coUectively called BSP protein ) represent a family of closely related proteins . They are secreted by the seminal vesicles . The protein were first purified on the basis of the stimulatory effect on the basal release of gonadotropins by rat anterior- pituitary cells in culture. Many scholars think they have not important physical roles because of their high working concentration . They have the same N - terminal amino acid and C - terminal amino acid, namely asparine and cysteine respectively. They form two largely the same hydrophobic domains . The cysteine polypeptide ring formed by them is called type - II construction domain . Now the type - fl construction domain is considered as the structural basis of multiple functions of BSP protein .The BSP proteins have many binding functions . Manjunath had pointed out that upon ejaculation , the BSP proteins bind to the spermatozoa surface and further studies have found the binding site are phospholipids of the sperm membrane . BSP proteins can bind to insulin - like growth factor - II (IGF - 2 ) , apolipoprotein A - I ( apo A- I ) , heparin, calmodulin ,fibrinogen and so on . BSP proteins can potentate sperm capacitation induced by heparin and HDL in vitro andBSP proteins can also stimulate cholesterol efflux from spermatozoa . Many reports suggested that the high cholesterol concentration of sperm membraneis a barricade of capacitation and in many mammals such as bull,ray and human . It had been corroborated there is cholesterol efflux while capacitation . These imply the BSP proteins have important functions in sperm lipid metabolism , sperm capacitation and acrosome reaction .The homologous proteins to BSP proteins have been found in the seminal plasma of boar and horse . These suggest the BSP proteins and its homologous proteins are ubiquitous in mammals and must have some physical significances . In our research , to find and study the important role of BSP proteinsand their related proteins in the development of zygotes and to find the new gene related to BSP , we cloned a cDNA gene sequence related to BSP proteins and make it express in eukaratic cells . The fusion protein acquired are purified are used to deduce its biological functions . We find the new cloned fusion protein has inhibitory effect on the activity of TPK. As we know TPK is the important key enzyme in cell growth and metabolism . So what we have done is of great significance to study further the biological functions of the new cloned protein related to BSP proteins .Materials and MethodsIn our study , first the E. coli BL21 transformed already are cultivated in smaller scale and then the plasmids DNA were extracted by the methods of alkaline lysis . The plasmids DNA extraction were processed 37 overnight by the restrictive endo - incisase Bam H I and Xho I . The incision products were used to check the integrality andvariability of the recombinant plasmids DNA by 1 % agarose gel elec-trophoresis . According to the result of restriction analysisof recombi-nants , the positive mono clones of recombinant BL21 were selected to be cultivated in larger scale in LB culture . Bonification and centrifu-gation were recommended for the lysis of collected cells and the supernatant and precipitation were collected respectively. As to the plenty of include bodies in the precipitation , denationalization, detergence, purification and dissolution, last regeneration were recommended to acquire great deal of expressed GST fusion proteins. After the GST fusion proteins were processed by factor Xa and were purified by Gluta-thione - sepharose 4B affinity chromatography, the recombinant protein HBRP with high purification would be acquired . During the process of separation and purification , the every result which can show the location of the aim protein was monitored by 10% SDS - PAGE . The GST fusion proteins acquired finally were examined and analyzed by the methods of Western - blottin...