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

α 2 -Macroglobulins (α 2 Ms) regulate peptidases, hormones and cytokines. Mediated by peptidase cleavage, they transit between native, intact forms and activated, induced forms. α 2 Ms 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 α 2 M (hα 2 M) at ~1.0 and ~0.4 mg per liter of cell culture, respectively. In both cases, hα 2 M was mainly found in the induced form. Shorter hα 2 M variants encompassing N-/C-terminal parts were also expressed and yielded pure material at ~1.6/~1.3 and ~3.2/~4.6 mg per liter of insect or mammalian cell culture, respectively. We then analyzed the binding of recombinant and authentic hα 2 M to recombinant latent human transforming growth factor-β 2 (pro-TGF-β 2 ) and bacterial G-related α 2 M 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 hα 2 M tetramers. The shorter recombinant hα 2 M variants interacted after preincubation only. In contrast, pro-TGF-β 2 did not interact, probably owing to hindrance by the N-terminal latency-associated protein of the cytokine.