Recombinant production of human α2-macroglobulin variants and interaction studies with recombinant G-related α2-macroglobulin binding protein and latent transforming growth factor-β2

alpha(2)-Macroglobulins (alpha Ms-2) regulate peptidases, hormones and cytokines. Mediated by peptidase cleavage, they transit between native, intact forms and activated, induced forms. alpha Ms-2 have been studied over decades using authentic material from primary sources, which was limited by sample heterogeneity and contaminants. Here, we developed high-yield expression systems based on transient transfection in Drosophila Schneider 2 and human Expi293F cells, which produced pure human alpha M-2 (h alpha M-2) at similar to 1.0 and similar to 0.4 mg per liter of cell culture, respectively. In both cases, h alpha M-2 was mainly found in the induced form. Shorter h alpha M-2 variants encompassing N-/C-terminal parts were also expressed and yielded pure material at similar to 1.6/similar to 1.3 and similar to 3.2/similar to 4.6 mg per liter of insect or mammalian cell culture, respectively. We then analyzed the binding of recombinant and authentic h alpha M-2 to recombinant latent human transforming growth factor-beta(2) (pro-TGF-beta(2)) and bacterial G-related alpha M-2 binding protein (GRAB) by surface plasmon resonance, multiple-angle laser light scattering, size-exclusion chromatography, fluorogenic labelling, gel electrophoresis and Western-blot analysis. Two GRAB molecules formed stable complexes of high affinity with native and induced authentic h alpha M-2 tetramers. The shorter recombinant h alpha M-2 variants interacted after preincubation only. In contrast, pro-TGF-beta(2) did not interact, probably owing to hindrance by the N-terminal latency-associated protein of the cytokine.