Refolding of TIMP-2 from Escherichia coli Inclusion Bodies

Abstract

E. coli is a convenient host in which to express recombinant proteins. The technology is available to most laboratories as it is relatively inexpensive and does not require extensive expertise. The major drawback of E. coli as an expression host is the inability of the organism to carry out many posttrans-lational modifications, including glycosylation and disulphide bond formation. High-level intracellular expression of many mammalian proteins in E. coli results in the formation of large insoluble aggregates, known as inclusion bodies (1). These dense bodies consist predominantly of the misfolded recombinant product, together with components of the transcription/translation machinery (i.e., RNA polymerase, ribosomal RNA, and plasmid DNA). The TIMPs are invariably insoluble when expressed in E. coli, their folding requirement for the formation of 6 disulphide bonds being incompatible with the reducing environment of the E. coli cell. Fortunately, active, correctly folded recombinant protein can often be recovered from insoluble inclusion bodies by a process of solubilization and in vitro refolding (2–3). Indeed, inclusion body formation has the advantage that the recombinant product often accumulates to high levels in the cell (up to 30% of total cell protein) and allows easy isolation of that protein, a relatively pure and stable form (inclusion bodies are typically >50% recombinant protein).