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Recombinant production of human ?2-macroglobulin variants and interaction studies with recombinant G-related ?2-macroglobulin binding protein and latent transforming growth factor-?2.

ABSTRACT: ?2-Macroglobulins (?2Ms) regulate peptidases, hormones and cytokines. Mediated by peptidase cleavage, they transit between native, intact forms and activated, induced forms. ?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 ?2M (h?2M) at ~1.0 and ~0.4?mg per liter of cell culture, respectively. In both cases, h?2M was mainly found in the induced form. Shorter h?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 h?2M to recombinant latent human transforming growth factor-?2 (pro-TGF-?2) and bacterial G-related ?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 h?2M tetramers. The shorter recombinant h?2M variants interacted after preincubation only. In contrast, pro-TGF-?2 did not interact, probably owing to hindrance by the N-terminal latency-associated protein of the cytokine.

SUBMITTER: Marino-Puertas L

PROVIDER: S-EPMC6591361 | biostudies-literature | 2019 Jun

REPOSITORIES: biostudies-literature

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

Marino-Puertas Laura L Del Amo-Maestro Laura L Taulés Marta M Gomis-Rüth F Xavier FX Goulas Theodoros T

Scientific reports 20190624 1

α2-Macroglobulins (α2Ms) regulate peptidases, hormones and cytokines. Mediated by peptidase cleavage, they transit between native, intact forms and activated, induced forms. α2Ms 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 pu ...[more]

PMID: 31235767

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Dataset Information

Recombinant production of human ?2-macroglobulin variants and interaction studies with recombinant G-related ?2-macroglobulin binding protein and latent transforming growth factor-?2.

Publications

Recombinant production of human α<sub>2</sub>-macroglobulin variants and interaction studies with recombinant G-related α<sub>2</sub>-macroglobulin binding protein and latent transforming growth factor-β<sub>2</sub>.

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