Soluble Protein Expression in Bacteria

Abstract

Abstract This article summarizes methods for increasing the fraction of recombinant proteins expressed in a soluble form in the cytoplasmic fraction of bacteria. Many recombinant proteins, when expressed at very high levels in bacteria, form insoluble aggregates called inclusion bodies (IBs). Growth conditions can be manipulated to keep protein in a soluble form, including lowering the temperature during induction, choice of host strain and expression vector, or coculturing of proteins that assist in folding. Higher yields can be obtained by mutating the protein or fusing it with various linkers (which can also improve purification by enhancing column chromatography interactions). Eliminating protease or RNase recognition sites can stabilize the protein or mRNA, as can mutations that improve recognition by cofactors that assist in folding. Methodology to simultaneously produce many different recombinant proteins in a soluble form has been developed as part of the structural genomics initiative; these can also be used to for scanning mutagenesis studies. High density bacterial cultivation can be used to produce gram per liter quantities of recombinant proteins at the industrial scale. However, maintaining high levels of soluble protein requires optimizing all aspects of growth, ranging from how the inoculum is stored, to medium details and bacterial harvesting and lysis.