Expression profiling of proteins in L-threonine biosynthetic pathway of Escherichia coli by using antibody microarray.
We demonstrate the use of an antibody (Ab) microarray for a comparative expression profiling of proteins in an L-threonine biosynthetic pathway of Escherichia coli between a parental strain (W3110) and L-threonine overproducing mutant (TF5015). On the basis of a global comparative transcriptome analysis between the two strains, 28 analytical target proteins were selected and subjected to a production of polyclonal Abs against them. An Ab microarray was constructed by spotting a set of produced antibodies on a glass slide, and was employed for a comparative expression profiling of the proteins between the two strains by a two-color fluorescence assay method. The performance of the Ab microarray was evaluated with respect to cross-reactivity of the antibodies, dye-labeling efficiency, and the nature of antigenic proteins. Of these, the cross-reactivity of the used antibodies was found to mainly cause the deviation of the observed expression ratios from the expected ones. To offset the deviations, correction factors were derived from a statistical analysis and introduced. As a result, ten proteins were categorized to be up-regulated, while one was down-regulated in TF5015. Expression profiling of proteins using the Ab microarray was further verified by comparison with Western blotting and 2-DE.
Han
Min-Kyu
MK
Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Guseong-dong, Daejeon, Korea.
Hong
Mi-Young
MY
Lee
Dohoon
D
Lee
Dong-Eun
DE
Noh
Geon Youp
GY
Lee
Jin-Ho
JH
Kim
Sung-Ho
SH
Kim
Hak-Sung
HS
D000907:Antibodies, Bacterial / Q000145:classification / Q000235:genetics / Q000276:immunology*; D015153:Blotting, Western; D004926:Escherichia coli / Q000235:genetics / Q000276:immunology*; D029968:Escherichia coli Proteins / Q000276:immunology*; D020869:Gene Expression Profiling; D015964:Gene Expression Regulation, Bacterial; D007123:Immunoelectrophoresis, Two-Dimensional; D040081:Protein Array Analysis / Q000379:methods* / Q000592:standards; D013050:Spectrometry, Fluorescence / Q000379:methods; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013194:Staining and Labeling / Q000379:methods; D013912:Threonine / Q000378:metabolism*
D000907:Antibodies, Bacterial; D029968:Escherichia coli Proteins; D013912:Threonine