title: Optimized expression and purification for high-activity preparations of algal [FeFe]-hydrogenase.
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.
  
  
  RESULTS
  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)).
  
  
  CONCLUSIONS
  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.
authors.affiliation: Center for Biomedical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States of America. iftachy@post.tau.ac.il
authors.forename: Iftach
authors.initials: I
authors.lastname: Yacoby
authors.forename: Lotta Tollstoy
authors.initials: LT
authors.lastname: Tegler
authors.forename: Sergii
authors.initials: S
authors.lastname: Pochekailov
authors.forename: Shuguang
authors.initials: S
authors.lastname: Zhang
authors.forename: Paul W
authors.initials: PW
authors.lastname: King
mesh_terms: D016825:Chlamydomonas reinhardtii / Q000201:enzymology; D010450:Endopeptidases / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D015951:Factor Xa / Q000378:metabolism; D005288:Ferredoxins / Q000235:genetics / Q000378:metabolism; D006864:Hydrogenase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D007506:Iron-Sulfur Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D005288:Ferredoxins; D007506:Iron-Sulfur Proteins; D011993:Recombinant Fusion Proteins; C118830:iron hydrogenase; D006864:Hydrogenase; D010450:Endopeptidases; C099488:TEV protease; D015951:Factor Xa