A preliminary study of expression of human neonatal Fc-receptor (FcRn) in Escherichia coli

Abstract

IntroductionHuman neonatal Fc-receptor is a potential novel antibody binding protein for development of immunoassay. Previous studies had shown that this human protein has high affinity to Ig G either in vivo or in vitro. However, none of the studies had attempted exploring the utility of human FcRn in antibody immobilisation strategy. In this study, we are in the direction towards development of a novel antibody binding protein by using human FcRn as an immobilisation platform. The three-dimensional structure of the protein was analysed. This protein was successful expressed in bacteria E. coli BL21 (DE3) by using expression vector pET-28b. The pET-28b vector contains poly-histidine tagged which allows detection and purification of expressed proteins. Objectives(1) To study the three dimensional structure of human FcRn, (2) To clone the human FcRn into pET-28b expression vector, (3) To express human FcRn in bacteria E. coli BL21 (DE3). MethodsHuman FcRn cDNA was ligated into pET-28b vector through EcoRI restriction site. The successful clone was transformed into E. coli BL21 (DE3) for expression. Expression in bacteria was induced by IPTG. Induction was conducted under 30°C and 37°C. Expression products were analysed by performing SDS-Page and Western Blot. The 3D structure of human FcRn was studied by using ViewerLite program. Results & DiscussionHuman FcRN was expressed under two sets of temperature, 30°C and 37°C, in 1 hour, 2 hours, 3 hours, and 6 hours. The induction for 3 hours showed highest amount of expressed products, under the above mentioned temperature. The amount of protein expressed in 6 hours of induction showed almost same thickness of band in SDS-PAGE compare to the induction under 3 hours period. ConclusionExpression of human FcRn can be conducted using bacteria expression system, instead of mammalian cell expression system which requires longer time and complicated process. Further study will be conducted to determine the antibody-binding activity and stability of the expressed protein.