title: Gene selection and cloning approaches for co-expression and production of recombinant protein-protein complexes.
abstract: Multiprotein complexes play essential roles in all cells and X-ray crystallography can provide unparalleled insight into their structure and function. Many of these complexes are believed to be sufficiently stable for structural biology studies, but the production of protein-protein complexes using recombinant technologies is still labor-intensive. We have explored several strategies for the identification and cloning of heterodimers and heterotrimers that are compatible with the high-throughput (HTP) structural biology pipeline developed for single proteins. Two approaches are presented and compared which resulted in co-expression of paired genes from a single expression vector. Native operons encoding predicted interacting proteins were selected from a repertoire of genomes, and cloned directly to expression vector. In an alternative approach, Helicobacter pylori proteins predicted to interact strongly were cloned, each associated with translational control elements, then linked into an artificial operon. Proteins were then expressed and purified by standard HTP protocols, resulting to date in the structure determination of two H. pylori complexes.
authors.affiliation: Midwest Center for Structural Genomics, Biosciences Division, Argonne National Laboratory, 9700 S Cass Ave., Argonne, IL, 60439, USA. gbabnigg@anl.gov.
authors.forename: György
authors.initials: G
authors.lastname: Babnigg
authors.affiliation: Midwest Center for Structural Genomics, Biosciences Division, Argonne National Laboratory, 9700 S Cass Ave., Argonne, IL, 60439, USA.
authors.forename: Robert
authors.initials: R
authors.lastname: Jedrzejczak
authors.affiliation: Midwest Center for Structural Genomics, Biosciences Division, Argonne National Laboratory, 9700 S Cass Ave., Argonne, IL, 60439, USA.
authors.forename: Boguslaw
authors.initials: B
authors.lastname: Nocek
authors.affiliation: Midwest Center for Structural Genomics, Biosciences Division, Argonne National Laboratory, 9700 S Cass Ave., Argonne, IL, 60439, USA.
authors.forename: Adam
authors.initials: A
authors.lastname: Stein
authors.affiliation: Midwest Center for Structural Genomics, Biosciences Division, Argonne National Laboratory, 9700 S Cass Ave., Argonne, IL, 60439, USA.
authors.forename: William
authors.initials: W
authors.lastname: Eschenfeldt
authors.affiliation: Midwest Center for Structural Genomics, Biosciences Division, Argonne National Laboratory, 9700 S Cass Ave., Argonne, IL, 60439, USA.
authors.forename: Lucy
authors.initials: L
authors.lastname: Stols
authors.affiliation: Midwest Center for Structural Genomics, Biosciences Division, Argonne National Laboratory, 9700 S Cass Ave., Argonne, IL, 60439, USA.
authors.forename: Norman
authors.initials: N
authors.lastname: Marshall
authors.affiliation: Midwest Center for Structural Genomics, Biosciences Division, Argonne National Laboratory, 9700 S Cass Ave., Argonne, IL, 60439, USA.
authors.forename: Alicia
authors.initials: A
authors.lastname: Weger
authors.affiliation: Midwest Center for Structural Genomics, Biosciences Division, Argonne National Laboratory, 9700 S Cass Ave., Argonne, IL, 60439, USA.
authors.forename: Ruiying
authors.initials: R
authors.lastname: Wu
authors.affiliation: Midwest Center for Structural Genomics, Biosciences Division, Argonne National Laboratory, 9700 S Cass Ave., Argonne, IL, 60439, USA.
authors.forename: Mark
authors.initials: M
authors.lastname: Donnelly
authors.affiliation: Midwest Center for Structural Genomics, Biosciences Division, Argonne National Laboratory, 9700 S Cass Ave., Argonne, IL, 60439, USA. andrzejj@anl.gov.
authors.forename: Andrzej
authors.initials: A
authors.lastname: Joachimiak
mesh_terms: D003001:Cloning, Molecular / Q000379:methods*; D019295:Computational Biology / Q000379:methods; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D050939:Gene Fusion; D023061:Gene Order; D005822:Genetic Vectors; D008958:Models, Molecular; D046912:Multiprotein Complexes / Q000737:chemistry* / Q000378:metabolism; D009876:Operon; D011485:Protein Binding; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D012995:Solubility
chemical_list: D046912:Multiprotein Complexes; D011993:Recombinant Fusion Proteins
keywords: High-throughput structural biology; Ligation independent cloning; Multigene expression; Multiprotein complexes