Modular broad-host-range expression vectors for single-protein and protein complex purification.
A set of modular broad-host-range expression vectors with various affinity tags (six-His-tag, FLAG-tag, Strep-tag II, T7-tag) was created. The complete nucleotide sequences of the vectors are known, and these small vectors can be mobilized by conjugation. They are useful in the purification of proteins and protein complexes from gram-negative bacterial species. The plasmids were easily customized for Thiocapsa roseopersicina, Rhodobacter capsulatus, and Methylococcus capsulatus by inserting an appropriate promoter. These examples demonstrate the versatility and flexibility of the vectors. The constructs harbor the T7 promoter for easy overproduction of the desired protein in an appropriate Escherichia coli host. The vectors were useful in purifying different proteins from T. roseopersicina. The FLAG-tag-Strep-tag II combination was utilized for isolation of the HynL-HypC2 protein complex involved in hydrogenase maturation. These tools should be useful for protein purification and for studying protein-protein interactions in a range of bacterial species.
Fodor
Barna D
BD
Institute of Biophysics, Biological Research Center, Hungarian Academy of Sciences, University of Szeged, H-6726 Szeged, Hungary.
Kovács
Akos T
AT
Csáki
Róbert
R
Hunyadi-Gulyás
Eva
E
Klement
Eva
E
Maróti
Gergely
G
Mészáros
Lívia S
LS
Medzihradszky
Katalin F
KF
Rákhely
Gábor
G
Kovács
Kornél L
KL
D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D003001:Cloning, Molecular; D003227:Conjugation, Genetic; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D005822:Genetic Vectors; D006639:Histidine / Q000235:genetics / Q000378:metabolism; D006864:Hydrogenase / Q000235:genetics; D020619:Methylococcus capsulatus / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011506:Proteins / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016354:Rhodobacter capsulatus / Q000235:genetics / Q000378:metabolism*; D019809:Streptavidin / Q000235:genetics / Q000378:metabolism; D020616:Thiocapsa roseopersicina / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D011506:Proteins; D011993:Recombinant Fusion Proteins; D006639:Histidine; D019809:Streptavidin; D006864:Hydrogenase