Identification of the binding of sceptrin to MreB via a bidirectional affinity protocol.
A bidirectional affinity system was used to screen for marine natural products that bound to Escherichia coli proteins. A system was developed and applied to isolate the natural product sceptrin from an Agelas conifera extract and its affinity partner MreB from E. coli lysate. The use of a dual immunoaffinity fluorescent (IAF) tag permitted this process to co-immunoprecipitate the bacterial equivalent of actin, MreB, from E. coli lysate. MreB was subsequently validated as a target for sceptrin using a resistance mapping approach. The combination of these studies suggests that natural products and their protein targets can be isolated in concert using a melody of forward and reverse affinity matrices. While the structure of sceptrin was elucidated by NMR analysis, the bulk of effort was conducted without knowing the structure of the natural product, thereby elevating a key bottleneck in the development of high-throughput methods for natural product discovery.
Rodríguez
Abimael D
AD
Department of Chemistry, University of Puerto Rico, P.O. Box 23346, UPR Station, San Juan, Puerto Rico 00931-3346, USA.
Lear
Martin J
MJ
La Clair
James J
JJ
D000345:Affinity Labels / Q000737:chemistry; D047230:Agelas / Q000737:chemistry; D000818:Animals; D001483:Base Sequence; D048168:Escherichia coli K12 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry; D047468:Immunoprecipitation; D008969:Molecular Sequence Data; D020125:Mutation, Missense; D017354:Point Mutation; D011758:Pyrroles / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism
D000345:Affinity Labels; D029968:Escherichia coli Proteins; D005456:Fluorescent Dyes; D011758:Pyrroles; C078473:MreB protein, E coli; C077210:sceptrin