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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
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 heterogen...
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Published in: | Scientific reports 2019-06, Vol.9 (1), p.1-12, Article 9186 |
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Main Authors: | , , , , |
Format: | Article |
Language: | English |
Subjects: | |
Citations: | Items that this one cites Items that cite this one |
Online Access: | Get full text |
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Summary: | 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. |
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ISSN: | 2045-2322 2045-2322 |
DOI: | 10.1038/s41598-019-45712-z |