| Summary: | Recombinant Protein G (rPG), an engineered form of streptococcal protein G with a theoretical molecular weight of 22.26 kDa was successfully cloned and expressed in E.coli BL 21(DE3) cells. The albumin binding domain was removed during the gene synthesis to avoid unspecific binding. This recombinant form of protein G contains only the IgG binding domains along with the 6X histidine tag at the N terminal. The removal of non-specific domains maximizes the specificity of IgG binding through the Fc region. The recombinant protein G was purified through heat treatment and using immobilized metal affinity chromatography (IMAC). On an SDS-PAGE gel there was only a single band of the purified preparation which migrated at 32 KDa, however when analyzed by mass spectrometry it was ~22.4 kDa, this phenomenon of retarded migration on SDS-PAGE has been known from previous studies. The production of recombinant protein has also been optimized. The effects of expression temperature, inducer type, inducer concentration and media composition have been investigated. The expression was done at 10 liter scale using the best expression conditions, and the protein was purified to homogeneity, dialyzed and lyophilized. The pure protein was immobilized on a POROS AL (Self Pack® POROS® 20 AL, Applied Biosystems, USA). The immobilized protein IgG binding has been monitored using a VersAFlo system. This system allowed real time monitoring of IgG binding characteristics. === <p></p><p> </p>
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