Human myocilin is a 55 kDa protein that is implicated in primary open angle glaucoma (POAG). Understanding the structure and folding of the native protein and the mutants that increase aggregation could lead to possible prevention of the condition. We report here the over expression and purification of the human myocilin in E. coli. The initial expression of recombinant myocilin in E. coli was found to be low. The problem of low yield was found to be due to multiple causes and was overcome using a suitable combination of vectors, tags, host background and expression protocols. The overexpressed human myocilin was purified by affinity column chromatography to yield about 8 mg of protein from 1 l of culture. The protein purity and folding were confirmed using electrophoresis, immunoblotting and fluorescence spectroscopy. Further biophysical characterization and crystallization trials using the recombinant human myocilin will pave the way for better understanding of the structure–aggregation relationship that is involved in causing POAG.