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Cloning, expression and purification of the luminal domain of spinach photosystem 1 subunit PsaF functional in binding to plastocyanin and with a disulfide bridge required for folding
Institutionen för kemi, Göteborgs Universitet, Department of Chemistry, University of Gothenburg.
Högskolan i Halmstad, Sektionen för ekonomi och teknik (SET), Bio- och miljösystemforskning (BLESS), Växtcellbiologi: Energiomvandling i växtceller.ORCID-id: 0000-0002-0494-3992
Institutionen för kemi, Göteborgs Universitet, Department of Chemistry, University of Gothenburg.
2011 (engelsk)Inngår i: Protein Expression and Purification, ISSN 1046-5928, E-ISSN 1096-0279, Vol. 78, nr 2, s. 156-166Artikkel i tidsskrift (Fagfellevurdert) Published
Abstract [en]

The photosystem 1 subunit PsaF is involved in the docking of the electron-donor proteins plastocyanin and cytochrome c6 in eukaryotic photosynthetic organisms. Here we report the expression, purification and basic characterization of the luminal domain of spinach PsaF, encompassing amino-acid residues 1-79. The recombinant protein was expressed in Escherichia coli BL21 (DE3) using a pET32 Xa/LIC thioredoxin fusion system. The thioredoxin fusion protein contained a His6 tag and was removed and separated from PsaF through proteolytic digestion by factor Xa followed by immobilized metal affinity chromatography. Further purification with size-exclusion chromatography resulted in a final yield of approximately 6 mg PsaF from one liter growth medium. The correct identity after the factor Xa treatment of PsaF was verified by FT-ICR mass spectrometry which also showed that the purified protein contains an intact disulfide bridge between Cys residues 6 and 38. Secondary structure and folding was further explored using far-UV CD spectroscopy indicating a α-helical content in agreement with the 3.3 Å-resolution crystal structure of photosystem I Ref. [5] and a helix-coil transition temperature of 29 °C. Thermofluorescence studies showed that the disulfide bridge is necessary to keep the overall fold of the protein and that hydrophobic regions become exposed at 50-65 °C depending on the ionic strength. The described expression and purification procedure can be used for isotopic labeling of the protein and 15N-HSQC NMR studies indicated a slow or intermediate exchange between different conformations of the prepared protein and that it belongs to the molten-globule structural family. Finally, by using a carboxyl- and amine-reactive zero-length crosslinker, we have shown that the recombinant protein binds to plastocyanin by a specific, native-like, electrostatic interaction, hence, confirming its functionality.

sted, utgiver, år, opplag, sider
San Diego: Academic Press, 2011. Vol. 78, nr 2, s. 156-166
Emneord [en]
NMR, Photosynthesis, Plastocyanin, Protein–protein interaction, PsaF, Thioredoxin
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Identifikatorer
URN: urn:nbn:se:hh:diva-15882DOI: 10.1016/j.pep.2011.02.007ISI: 000292020400007PubMedID: 21354471Scopus ID: 2-s2.0-79958155132OAI: oai:DiVA.org:hh-15882DiVA, id: diva2:433517
Tilgjengelig fra: 2011-08-10 Laget: 2011-08-10 Sist oppdatert: 2019-04-04bibliografisk kontrollert

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