Accurate disulfide formation in Escherichia coli: Overexpression and characterization of the first domain (HF6478) of the multiple Kazal-type inhibitor LEKTI

The human hemofiltrate peptide HF6478, a putative serine proteinase inhibit or, which is part of the precursor protein LEKTI, was cloned, overexpressed , and purified. HF6478 contains two disulfide bridges with 1-4, 2-3 connect ivity, sharing partial homology to Kazal-type domains and other serine prot einase inhibitors. It was expressed as thioredoxin (Trx) fusion protein, an d disulfide formation occurred in the oxidative cytoplasm of Escherichia co li Origami (DE3) strain which carries a trxB(-)/gor522(-) double mutation. The soluble fusion protein was purified using metal-chelating affinity chro matography, Cleavage of the Trx fusion protein with factor Xa and subsequen t purification yielded the final product in amounts sufficient for structur al studies. Characterization of recombinant HF6478 was done by amino acid s equencing, mass spectrometry, capillary zone electrophoresis, and CD spectr oscopy. Taking the blood filtrate peptide HF6478 as example, we present a s trategy which should facilitate the expression of different extracellular p roteins in the E. coli cytoplasm. (C) 2001 Academic Press.