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Nuclear factor kappa B (NF-kappaB) is a ubiquitous, inducible transcription factor that regulates the initiation and progression of immune and inflammatory stress responses. NF-kappaB activation depends on phosphorylation and degradation of its inhibitor protein, IkappaB, initiated by an IkappaB kinase (IKK) complex. This IKK complex includes a catalytic heterodimer composed of IkappaB kinase 1 (IKK1) and IkappaB kinase 2 (IKK2) as well as a regulatory adaptor subunit, NF-kappaB essential modulator. To better understand the role of IKKs in NF-kappaB activation, we have cloned, expressed, purified, and characterized the physiological isoform, the rhIKK1/rhIKK2 heterodimer. We compared its kinetic properties with those of the homodimers rhIKK1 and rhIKK2 and a constitutively active rhIKK2 (S177E, S181E) mutant. We demonstrate activation of these recombinantly expressed IKKs by phosphorylation during expression in a baculoviral system. The K(m) values for ATP and IkappaBalpha peptide for the rhIKK1/rhIKK2 heterodimer are 0.63 and 0.60 micrometer, respectively, which are comparable to those of the IKK2 homodimer. However, the purified rhIKK1/rhIKK2 heterodimer exhibits the highest catalytic efficiency (k(cat)/K(m)) of 47.50 h(-1) micrometer(-1) using an IkappaBalpha peptide substrate compared with any of the other IKK isoforms, including rhIKK2 (17.44 h(-1) micrometer(-1)), its mutant rhIKK2 (S177E, S181E, 1.18 h(-1) micrometer(-1)), or rhIKK1 (0.02 h(-1) micrometer(-1)). Kinetic analysis also indicates that, although both products of the kinase reaction, ADP and a phosphorylated IkappaBalpha peptide, exhibited competitive inhibitory kinetics, only ADP with the low K(i) of 0.77 micrometer may play a physiological role in regulation of the enzyme activity.

Human tissue factor pathway inhibitor (TFPI) was expressed in E. coli as a non-glycosylated protein with an additional alanine attached to the aminoterminus of the wild type molecule. High-level expression was obtained with pMON6875, a plasmid containing a tac promoter, Gene 10 leader from bacteriophage T7, methionine-alanine-TFPI coding sequence, and the p22 transcriptional terminator. In this system, TFPI accounted for about 5-10% of the total cell protein. The inclusion bodies containing. TFPI were sulfitolyzed, purified by anion-exchange chromatography, refolded through a disulfide interchange reaction, and further fractionated by Mono S cation exchange chromatography. The Mono S resin resolved a peak of highly active TFPI from relatively inactive and possibly misfolded molecules. The E. coli TFPI was shown to be about two-fold more active, on a molar basis, than full-length human SK hepatoma TFPI in a tissue factor-induced clotting assay in human plasma.