Optimized expression and purification for high-activity preparations of algal [FeFe]-hydrogenase.

PloS One
Iftach YacobyPaul W King

Abstract

Recombinant expression and purification of metallo-enzymes, including hydrogenases, at high-yields is challenging due to complex, and enzyme specific, post-translational maturation processes. Low fidelities of maturation result in preparations containing a significant fraction of inactive, apo-protein that are not suitable for biophysical or crystallographic studies. We describe the construction, overexpression and high-yield purification of a fusion protein consisting of the algal [2Fe2S]-ferredoxin PetF (Fd) and [FeFe]-hydrogenase HydA1. The maturation of Fd-HydA1 was optimized through improvements in culture conditions and media components used for expression. We also demonstrated that fusion of Fd to the N-terminus of HydA1, in comparison to the C-terminus, led to increased expression levels that were 4-fold higher. Together, these improvements led to enhanced HydA1 activity and improved yield after purification. The strong binding-affinity of Fd for DEAE allowed for two-step purification by ion exchange and StrepTactin affinity chromatography. In addition, the incorporation of a TEV protease site in the Fd-HydA1 linker allowed for the proteolytic removal of Fd after DEAE step, and purification of HydA1 alone by StrepTactin...Continue Reading

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Citations

Oct 7, 2014·Journal of the American Chemical Society·David W MulderPaul W King
Sep 13, 2015·Protein Science : a Publication of the Protein Society·Simone MorraFrancesca Valetti
Apr 9, 2015·Biotechnology and Applied Biochemistry·Simone MorraFrancesca Valetti
May 16, 2020·The Journal of Biological Chemistry·Jacob H ArtzPaul W King
Oct 9, 2013·The Journal of Biological Chemistry·Erin A PedenAlexandra Dubini
Aug 23, 2020·International Journal of Molecular Sciences·Qin FanMatthias Gimpel
Apr 13, 2021·ACS Nano·Oren Ben-ZviLihi Adler-Abramovich
Apr 13, 2013·Journal of the American Chemical Society·David W MulderPaul W King
Jan 13, 2015·Journal of the American Chemical Society·Kevin D SwansonJohn W Peters

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Methods Mentioned

BETA
affinity purification
ion exchange chromatography

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