2024-03-29T06:19:29Zhttp://digital.csic.es/dspace-oai/requestoai:digital.csic.es:10261/2017132021-12-28T16:07:18Zcom_10261_41com_10261_1col_10261_294
2020-02-24T12:13:05Z
urn:hdl:10261/201713
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
Amo-Maestro, Laura del
Taulés, Marta
Gomis-Rüth, F. Xavier
Goulas, Theodoros
Ministerio de Economía y Competitividad (España)
Generalitat de Catalunya
Fundació La Marató de TV3
Ministerio de Ciencia, Innovación y Universidades (España)
Biochemistry
Biological techniques
© The Author(s) 2019.
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 pure human α2M (hα2M) at ~1.0 and ~0.4 mg per liter of cell culture, respectively. In both cases, hα2M was mainly found in the induced form. Shorter hα2M 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 hα2M to recombinant latent human transforming growth factor-β2 (pro-TGF-β2) and bacterial G-related α2M 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 hα2M tetramers. The shorter recombinant hα2M variants interacted after preincubation only. In contrast, pro-TGF-β2 did not interact, probably owing to hindrance by the N-terminal latency-associated protein of the cytokine.
2020-02-24T12:13:05Z
2020-02-24T12:13:05Z
2019
2020-02-24T12:13:05Z
artículo
Scientific Reports 9: 9186 (2019)
http://hdl.handle.net/10261/201713
10.1038/s41598-019-45712-z
2045-2322
http://dx.doi.org/10.13039/501100002809
http://dx.doi.org/10.13039/501100003329
http://dx.doi.org/10.13039/100008666
31235767
Publisher's version
http://dx.doi.org/10.1038/s41598-019-45712-z
Sí
info:eu-repo/grantAgreement/MINECO/Plan Estatal de Investigación Científica y Técnica y de Innovación 2013-2016/BFU2015-64487-R
info:eu-repo/grantAgreement/MINECO/Plan Estatal de Investigación Científica y Técnica y de Innovación 2013-2016/MDM-2014-0435-03
http://creativecommons.org/licenses/by/4.0/
openAccess
Springer Nature