HIGH-LEVEL PRODUCTION OF UNIFORMLY N-15-ENRICHED AND C-13-ENRICHED FUSION PROTEINS IN ESCHERICHIA-COLI

An approach to produce C-13- and N-15-enriched proteins is described. The concept is based on intracellular production of the recombinant pr oteins in Escherichia coil as fusions to an IgG-binding domain, Z, der ived from staphylococcal protein A. The production method provides yie lds of 40-200 mg/l of isotope-enriched fusion proteins in defined mini mal media. In addition, the Z fusion partner facilitates the first pur ification step by IgG affinity chromatography. The production system i s applied to isotope enrichment of human insulin-like growth factor II (IGF-II), bovine pancreatic trypsin inhibitor (BPTI), and Z itself. H igh levels of protein production are achieved in shaker flasks using t otally defined minimal medium supplemented with C-13(6)-glucose and (( NH4)-N-15)(2)SO4 as the only carbon and nitrogen sources. Growth condi tions were optimized to obtain high protein production levels and high levels of isotope incorporation, while minimizing C-13(6)-glucose usa ge. Incorporation levels of C-13 and/or N-15 isotopes in purified IGF- II, BPTI, and Z were confirmed using mass spectrometry and NMR spectro scopy. More than 99% of total isotope enrichment was obtained using a defined isotope-enriched minimal medium. The optimized systems provide reliable, high-level production of isotope-enriched fusion proteins. They can be used to produce 20-40 mg/l of properly folded Z and BPTI p roteins. The production system of recombinant BPTI is state-of-the-art and provides the highest known yield of native refolded BPTI.