Recombinant production of human α(2)-macroglobulin variants and interaction studies with recombinant G-related α(2)-macroglobulin binding protein and latent transforming growth factor-β(2)

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.