Cloning and Expression of C2 and V Domains of ALCAM Protein in E. coli
BL21 (DE3)

Introduction: ALCAM as a glycoprotein is a member of the immunoglobulin family and plays an important role in cell growth, survival and motility. This protein also contributes to the development of tumour invasion in colorectal and breast cancers and is typically considered as a cancer stem cell marker. ALCAM as a cell-surface antigen, with high expression in some cancers as colorectal and breast, has the potential to diagnose and treat these cancers. Objectives: As with other membrane proteins, ALCAM represents a potential target for therapy of colorectal and breast cancers, therefore, this study is aimed to clone and express C2 and V domains to be used in practical, diagnostic and remedial plans. Methods: In this study, the sequence of C2 and V domains were optimized for expression in prokaryotic host using online tools and cloned in pET-28a expression plasmids. E. coli BL21 (DE3) cells were transformed by pET-28a recombinant plasmids using heat shock method and the expression of recombinant V and C2 domains were examined by SDS-PAGE technique. Results: The synthetic genes of C2 and V domains were located between NcoI/BamHI and XhoI restriction sites and cloned into pBSK (+) vector. The presence of these genes in pET28a was determined by colony and confirmed by restriction digestion. Gens of C2 and V domains were expressed in E. coli BL21 DE3. The results of the SDSPAGE technique confirmed the expression of recombinant 23 and 29 kDa C2 and V domains in a bacterial expression system. Conclusions: The genes of C2 and V domains were expressed as the recombinant in E. coli. These recombinant fragments can be introduced as diagnostic and remedial candidates for screening and cancer-therapy in patients with colorectal and breast cancers.