Protein Over Expression

Materials and methods Overexpression of proteins. An overnight culture (15 mL) of transformed E. coli BL21 expressing GST (glutathione S-transferase) -peptide, GST-EBNA 1 (Epstein-Barr nuclear antigen-1) peptide and 6 × His-USP 7 peptide requires 45 mL of LB Amp + Km + medium was assigned to GST and 6 × His-USP 7 cultures and LB Amp + medium was assigned to GST EBNA 1 cultures. Three flasks, each with a bacterial culture, were placed in a shaking incubator (250 rpm) for 1 hour at 37 ° C. to obtain optimal cell growth.

Integrins are a type of membrane bound alpha / beta heterodimeric protein that is frequently overexpressed in cancer cells by 50-100 fold. They mediate cell survival, proliferation and tumor invasion. In addition, integrins recognize tripeptide binding sites on several extracellular matrix (ECM) proteins (eg, fibronectin) and this integrin-fibronectin interaction is important for metastasis and also ing. Resistance to many chemotherapies Current cancer treatments are inappropriate and nonspecific, and the determination of therapeutic strategies that specifically inhibit cancer progression remains the main focus of cancer biology research. Toxin drugs have been the focus of research in the past decade, especially snake venom is a useful source of some protein drugs and other new protein drugs. In addition, Zazakanin has no effect on cell adhesion to Matrigel or fibrinogen, indicating that Zazakanin does not bind to α v β 1 integrin.

The cell-free protein expression method synthesizes proteins without using viable cells. Cell-free protein expression systems are used in protein analysis and graduate processes. It transcribes DNA into RNA and further translates the RNA into protein by an in vitro solution consisting of a selective cell lysate type. They are not used in situations where high yield protein synthesis is required and are preferred when small amounts of recombinant protein are required. Various cell lysates, E. coli, wheat germ are routinely used, and recently mammalian cell lysates are believed to be the best choice for protein synthesis. Commercialization of cell-free protein systems, increased research activities in the pharmaceutical field, and the demand for biologics are important factors in promoting the growth of cell-free protein expression markets.

Mammalian expression systems can be used to generate proteins temporarily or by stable cell lines in which the expression construct is integrated into the host genome. Stable cell lines can be used in multiple experiments, but transient production can produce large amounts of protein in 1 to 2 weeks. These transient high yield mammalian expression systems utilize suspension culture and can yield yields per liter. In addition, these proteins have more natural folding and post-translational modifications such as glycosylation compared to other expression systems. In the following examples, recombinant proteins were expressed using three different mammalian expression systems.