TY - JOUR
T1 - Isotopic labeling of c-type multiheme cytochromes overexpressed in E. coli
AU - Fernandes, Ana P.
AU - Couto, Isabel
AU - Morgado, Leonor
AU - Londer, Yuri Y.
AU - Salgueiro, Carlos A.
N1 - info:eu-repo/grantAgreement/FCT/POCI/60060/PT#
info:eu-repo/grantAgreement/FCT/3599-PPCDT/74498/PT#
program under contract no.DE-AC02-06CH11357.
Sem PDF conforme despacho.
PY - 2008/5/1
Y1 - 2008/5/1
N2 - Progresses made in bacterial genome sequencing show a remarkable profusion of multiheme c-type cytochromes in many bacteria, highlighting the importance of these proteins in different cellular events. However, the characterization of multiheme cytochromes has been significantly retarded by the numerous experimental challenges encountered by researchers who attempt to overexpress these proteins, especially if isotopic labeling is required. Here we describe a methodology for isotopic labeling of multiheme cytochromes c overexpressed in Escherichia coli, using the triheme cytochrome PpcA from Geobacter sulfurreducens as a model protein. By combining different strategies previously described and using E. coli cells containing the gene coding for PpcA and the cytochrome c maturation gene cluster, an experimental labeling methodology was developed that is based on two major aspects: (i) use of a two-step culture growth procedure, where cell growth in rich media was followed by transfer to minimal media containing 15N-labeled ammonium chloride, and (ii) incorporation of the heme precursor delta-aminolevulinic acid in minimal culture media. The yields of labeled protein obtained were comparable to those obtained for expression of PpcA in rich media. Proper protein folding and labeling were confirmed by UV-visible and NMR spectroscopy. To our knowledge, this is the first report of a recombinant multiheme cytochrome labeling and it represents a major breakthrough for functional and structural studies of multiheme cytochromes.
AB - Progresses made in bacterial genome sequencing show a remarkable profusion of multiheme c-type cytochromes in many bacteria, highlighting the importance of these proteins in different cellular events. However, the characterization of multiheme cytochromes has been significantly retarded by the numerous experimental challenges encountered by researchers who attempt to overexpress these proteins, especially if isotopic labeling is required. Here we describe a methodology for isotopic labeling of multiheme cytochromes c overexpressed in Escherichia coli, using the triheme cytochrome PpcA from Geobacter sulfurreducens as a model protein. By combining different strategies previously described and using E. coli cells containing the gene coding for PpcA and the cytochrome c maturation gene cluster, an experimental labeling methodology was developed that is based on two major aspects: (i) use of a two-step culture growth procedure, where cell growth in rich media was followed by transfer to minimal media containing 15N-labeled ammonium chloride, and (ii) incorporation of the heme precursor delta-aminolevulinic acid in minimal culture media. The yields of labeled protein obtained were comparable to those obtained for expression of PpcA in rich media. Proper protein folding and labeling were confirmed by UV-visible and NMR spectroscopy. To our knowledge, this is the first report of a recombinant multiheme cytochrome labeling and it represents a major breakthrough for functional and structural studies of multiheme cytochromes.
KW - Escherichia coli
KW - Geobacter sulfurreducens
KW - Heterologous expression
KW - Isotopic labeling
KW - Multiheme cytochromes
KW - PpcA
UR - http://www.scopus.com/inward/record.url?scp=40849087788&partnerID=8YFLogxK
U2 - 10.1016/j.pep.2008.02.001
DO - 10.1016/j.pep.2008.02.001
M3 - Article
C2 - 18343156
AN - SCOPUS:40849087788
SN - 1046-5928
VL - 59
SP - 182
EP - 188
JO - Protein Expression And Purification
JF - Protein Expression And Purification
IS - 1
ER -