Expression profiling of proteins in L-threonine biosynthetic pathway of Escherichia coli by using antibody microarray

• Published in: Proteomics (Weinheim) 2006-11, Vol.6 (22), p.5929-5940
• Main Authors: Han, Min-Kyu, Hong, Mi-Young, Lee, Dohoon, Lee, Dong-Eun, Noh, Geon Youp, Lee, Jin-Ho, Kim, Sung-Ho, Kim, Hak-Sung
• Format: Article
• Language: English
• Subjects: Analytical, structural and metabolic biochemistry; Antibodies, Bacterial - classification; Antibodies, Bacterial - genetics; Antibodies, Bacterial - immunology; Antibody microarrays; Biological and medical sciences; Blotting, Western; Cross-reactivity; Escherichia coli - genetics; Escherichia coli - immunology; Escherichia coli Proteins - immunology; Expression profiling; Fundamental and applied biological sciences. Psychology; Gene Expression Profiling; Gene Expression Regulation, Bacterial; Immunoelectrophoresis, Two-Dimensional; L-threonine biosynthesis; Miscellaneous; Protein Array Analysis - methods; Protein Array Analysis - standards; Proteins; Spectrometry, Fluorescence - methods; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Staining and Labeling - methods; Threonine - metabolism
• ISSN: 1615-9853; 1615-9861
• DOI: 10.1002/pmic.200600324

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.