Cloning and Expression of Chlamydia Trachomatis Inclusion Membrane Proteins

Abstract

Chlamydia trachomatis is an obligate intracellular human pathogen that resides inside host cells within a vacuole called an inclusion. To replicate & grow Chlamydia must usurp host cell proteins from within this vacuole. To do this, Chlamydia produce & secrete proteins, termed inclusion membrane proteins (Incs), that insert in the inclusion membrane with the N- and C- terminus facing the host cytosol. C. trachomatis is predicted to have 50 Incs, however few of these have known functions. Little knowledge about their function is gained via bioinformatics analysis as they lack similarity to any proteins outside of Chlamydia. This makes characterizing Incs or identifying possible functions difficult. The goal of this project is to clone & express the C-terminus of certain Incs which will be used to produce purified protein for future crystallography studies. This study focuses on the CT229-CT224 operon which is only found in human pathogens. Here, we present the cloning strategy of an Inc into the expression plasmid pET28a which will generate a C-terminus Inc fusion to a 6X His tag. To date CT226, CT227 & CT228 have been successfully cloned, verified by sequencing and transformed into BL21 for expression studies. Once the Inc proteins are produced they will be prepared for crystallography. By assessing the structures, insights may be gained as to possible functions based on similarity to other characterized proteins