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

• Published in: Scientific reports 2019-06, Vol.9 (1), p.9186
• Main Authors: Marino-Puertas, Laura, Del Amo-Maestro, Laura, Taulés, Marta, Gomis-Rüth, F Xavier, Goulas, Theodoros
• Format: Article
• Language: English
• Subjects: alpha-Macroglobulins - biosynthesis; alpha-Macroglobulins - metabolism; Animals; Bacterial Proteins - metabolism; Carrier Proteins - metabolism; Cell Line; Drosophila melanogaster; Humans; Protein Binding; Recombinant Proteins - biosynthesis; Recombinant Proteins - metabolism; Transforming Growth Factor beta2 - metabolism
• ISSN: 2045-2322

α -Macroglobulins (α Ms) regulate peptidases, hormones and cytokines. Mediated by peptidase cleavage, they transit between native, intact forms and activated, induced forms. α 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 M (hα M) at ~1.0 and ~0.4 mg per liter of cell culture, respectively. In both cases, hα M was mainly found in the induced form. Shorter hα 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 M to recombinant latent human transforming growth factor-β (pro-TGF-β ) and bacterial G-related α 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 M tetramers. The shorter recombinant hα M variants interacted after preincubation only. In contrast, pro-TGF-β did not interact, probably owing to hindrance by the N-terminal latency-associated protein of the cytokine.