title: Cell-free synthesis of the H-cluster: a model for the in vitro assembly of metalloprotein metal centers.
abstract: Many organometallic cofactors are highly complex and require multiple accessory proteins for both their assembly and transfer to a target protein. A cell-free system in which the biosynthetic pathway for a prosthetic group has been fully or even partially reconstructed enables investigations of the reaction sequence as well as the cofactor itself. As a model for the in vitro assembly of protein-bound metal centers, we describe a procedure for the cell-free synthesis of the H-cluster in the context of producing purified and active [FeFe] hydrogenase samples for spectroscopic studies. In general terms, this in vitro system is a combination of non-purified accessory proteins, exogenous substrates, and purified hydrogenase apoprotein. We also describe methods for making the required components used in the cell-free system. Specifically, these procedures include anaerobic expression of heterologous metalloproteins in Escherichia coli, anaerobic cell lysate production, and anaerobic metalloprotein purification using Strep-Tactin(®) chromatography.
authors.affiliation: Department of Chemical Engineering, Stanford University, 381 North South Mall, Stanford, CA, 94305-5025, USA, kuchenreuther@gmail.com.
authors.forename: Jon M
authors.initials: JM
authors.lastname: Kuchenreuther
authors.forename: Stacey A
authors.initials: SA
authors.lastname: Shiigi
authors.forename: James R
authors.initials: JR
authors.lastname: Swartz
mesh_terms: D000693:Anaerobiosis; D000818:Animals; D001671:Biochemistry / Q000379:methods*; D002457:Cell Extracts; D002474:Cell-Free System; D002645:Chickens; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D006864:Hydrogenase / Q000378:metabolism*; D008667:Metalloproteins / Q000378:metabolism*; D008670:Metals / Q000378:metabolism*
chemical_list: D002457:Cell Extracts; D008667:Metalloproteins; D008670:Metals; D006864:Hydrogenase