Molecular cloning of BIR3 domain from XIAP in pET28a vector

The inhibitor of apoptosis protein (IAP) family is a functionally and structurally related group of proteins that serve as endogenous inhibitors of programmed cell death, or apoptosis. In addition, some family members are regulators of another form of programmed cell death termed necroptosis. Apoptosis is one type of programmed cell death which includes external and internal pathways. Some of the effector proteins that play rolls in this procedure are Caspases. These cysteine proteases were inhibited by IAPs. This family of protein has 8 members and the common point between them is similar domains name BIRs. XIAP is of the important members of this family and inhibits Caspase-9 and Caspase-3, 7. XIAP are additionally thought to contribute to cancer cell invasion and metastasis through their ability to drive NF-κB mediated expression of genes involved in cell motility, migration, and invasion. Caspase-9 inhibition is done by BIR3 domain of XIAP and Caspase-3 by BIR2. In this research, we cloned BIR3 domain of XIAP in a pET28a vector. PCR product was digested by EcoRI and HindIII and ligated into the EcoRI/HindIII digested pET-28a vector. The ligation mixture was transformed in the cloning host E. coli DH5α and screened by antibiotic selection. Positive colonies were selected by colony PCR and screened by double digestion of isolated plasmid and then sequenced to check the inserted DNA.

Keywords:

Apoptosis , XIAP , BIR , Cloning.