Ligation Independent Cloning of Polycistronic, Genetically Modified, Humab4d5-8 F (Ab') 2, in Bacterial Plasmid



Farahmand L^{1, 2} ; Majidzadeha K^{2, 3} ; Sepehrizadeh Z¹ ; Mofid MR⁴ ; Esmaeili R² ; Yazdi MT¹ Show Affiliations
Source: Avicenna Journal of Medical Biotechnology Published:2012

Abstract

In recent years, recombinant monoclonal antibodies and their derivatives have emerged as important targeted therapy agents. Monoclonal antibodies are extremely difficult to produce. So, the cost of production is very high and many people cannot afford these drugs. In this regard, choosing inexpensive and easy ways to manipulate production systems such as bacterial hosts to reduce the cost of manufacturing these critical components are considered as vital step for developmental issues in recombinant expression systems. We, therefore, attempted to generate a polycistronic construct of anti HER-2 F(ab')2 fragment antibody for insertion in an expression bacterial plasmid. Also some modifications were made in the hinge region to express antibody F(ab')2 fragment in its authentic form preventing from multiple varieties of disulfide bond formation. Finally, synthesized construct was cloned in pET-32 Ek/LIC vector without using restriction enzyme digestion or ligation reactions. The results of this study showed that modified F(ab')2 fragment was simply and successfully inserted in Escherichia coli (E.coli) using the Ligation Independent Cloning technology. © 2012, Avicenna Journal of Medical Biotechnology. All rights reserved.