Gene Cloning And Prokaryotic Expression Of Human Mammaglobin

Human mammaglobin (hMAM), a 93 amino acid globin(MW=10.5 kDa), is identified to be a member of the uteroglobin (UG) superfamily. Although its physiological function is not clear, all members of this family, small, secretory, rarely glycosylated dimeric proteins, are proven to be mainly expressed in mucosal tissues, and share similar structures and functions, e.g, regulating the inflammation, binding to steroids and inhibiting immune response, etc. Studies have shown that the expression of hMAM is restricted to the adult mammary gland and mammary gland tumor, so the protein can be used as a specific breast tumor marker. In this way, the application of hMAM includes early detection and/or relapse monitoring of breast cancer, identification of tumor source, and monitoring of metastatic breast tumors in peripheral blood, lymph node and bone marrow. At the same time, hMAM shows therapeutic promise because of its tissue specificity. The results are as follows:1. hMAM cDNA was amplified through RT-PCR from breast cancer tissue and breast cancer cell line MDA-MB453, and two subtypes of hMAM cDNA, a wild type and its isoform, were found. The isoform hMAM cDNA consisting of 270 bp, was different from the wild type one of 279 bp due to nine continuous base pair missing. Therefore the translational product of the isoform was seen to be lack of three continuous amino acid (-79~-81, VFM) compared with that of the wild type. The homology of the wild type and the isoform are testified to be 96% in both DNA and amino acid sequences. Whereafter the prokaryotic expression vectors pGEX-KG/hMAM and pGEX-KG/hMAM (Isoform) were constructed respectively.2. The E.coli. BL21 transformed with the recombinant vectors were cultured and induced with IPTG to express the corresponding fusion protein and optimal conditions of induction were achieved. SDS-PAGE analysis showed that an about 36 kDa recombinant fusion protein were expressed and the proportion of the fusion protein was about 30% of the total protein of induced bacteria. And the fusion protein formed inclusion bodies in prokaryotic expression system.3. The inclusion body was isolated from the Escherichia coli. BL21 and dissolved in denaturing agent. The crude product was firstly purified by reverse phase chromatography, and then by anion exchange chromatography after renaturation. With a yield of 3.5%, one gram of wet bacteria could produce 11 mg purified GST fusion proteins with a purity of 96.5% and 95.0% respectively, and a concentration of 0.53 mg/ml.4. Wild type and isoform of hMAM showed no significant difference in expressing property, renaturaturing condition and chromatography characterhMAM cDNA and its isoform were cloned successfully and the fusion protein GST-/iMAM were expressed in Escherichia coli BL21. We have obtained purified GST-&MAM through our modified purification procedure, paving the way for studying the structure, function of two subtypes of hMAM and their application in immunological diagnosis for breast cancer.