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

Abstract

α ₂-Macroglobulins (α ₂Ms) regulate peptidases, hormones and cytokines. Mediated by peptidase cleavage, they transit between native, intact forms and activated, induced forms. α ₂Ms 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 ₂M (hα ₂M) at ~1.0 and ~0.4 mg per liter of cell culture, respectively. In both cases, hα ₂M was mainly found in the induced form. Shorter hα ₂M 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 ₂M to recombinant latent human transforming growth factor-β ₂ (pro-TGF-β ₂) and bacterial G-related α ₂M 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 ₂M tetramers. The shorter recombinant hα ₂M 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.