Optimized Expression and Purification for High-Activity Preparations of Algal [FeFe]-Hydrogenase

Iftach Yacoby, Lotta Tollstoy Tegler, Sergii Pochekailov, Shuguang Zhang, Paul W. King

Research output: Contribution to journalArticlepeer-review

32 Scopus Citations

Abstract

Background: 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. Principal Findings: 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. In combination, this process resulted in HydA1 purification yields of 5 mg L-1 of culture from E. coli with specific activities of 1000 U (U = 1 μmol hydrogen evolved mg-1 min-1). Significance: The [FeFe]-hydrogenases are highly efficient enzymes and their catalytic sites provide model structures for synthetic efforts to develop robust hydrogen activation catalysts. In order to characterize their structure-function properties in greater detail, and to use hydrogenases for biotechnological applications, reliable methods for rapid, high-yield expression and purification are required.

Original languageAmerican English
Article numbere35886
Number of pages8
JournalPLoS ONE
Volume7
Issue number4
DOIs
StatePublished - 2012

NREL Publication Number

  • NREL/JA-2700-52560

Keywords

  • hydrogenases
  • metallo-enzymes
  • purification
  • recombinant expression

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