The ease of genetic manipulation， low cost， rapid growth and number of previous studies have made Escherichia coli one of the most widely used microorganism species for producing recombinant proteins. In this post-genomic era， challenges remain to rapidly express and purify large numbers of proteins for academic and commercial purposes in a high-throughput manner. In this review， we describe several state-of-the-art approaches that are suitable for the cloning， expression and purification， conducted in parallel， of numerous molecules， and we discuss recent progress related to soluble protein expression， mRNA folding， fusion tags， post-translational modification and production of membrane proteins. Moreover， we address the ongoing efforts to overcome various challenges faced in protein expression in E. coli， which could lead to an improvement of the current system from trial and error to a predictable and rational design.