Production of the MACPF Region of Complement C8α in Sf9 Insect Cells

Abstract

Composed of over 35 proteins, enzymes, and regulatory molecules, the complement system works in conjunction with the immune system to destroy and remove foreign substances (i.e. bacteria, virally infected particles, fungi, and parasites) from the body. The objective of this project is to produce a recombinant form of the putative membrane-binding region (MACPF) of the 64 kDa C8α subunit of the eighth component of human complement (C8) for structural analysis. C8 is an oligomeric protein made up of three non-identical subunits, C8α (64 kDa), C8β (64 kDa), and C8γ (22 kDa). The C8α and C8γ subunits are covalently bonded through a disulfide bond while C8β is non-covalently associated with C8α. C8α and C8β are homologous to one another and together with C6, C7, and C9 comprise the MAC protein family. The MACPF is considered to be self-folding, is ~ 43 kDa in size, and contains two disulfide bonds and no carbohydrates. The MACPF region has been cloned into the vector pIEx-4 and the sequence verified. The cloning experiments, the production and initial purification steps will be presented. This work was supported by the SC-INBRE NIH grant and Winthrop University.