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Rapid analysis of protein interactions: On‐chip micropurification of recombinant protein expressed in Esherichia coli
Author(s) -
Natsume Tohru,
Taoka Masato,
Manki Hiroshi,
Kume Shouen,
Isobe Toshiaki,
Mikoshiba Katsuhiko
Publication year - 2002
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/1615-9861(200209)2:9<1247::aid-prot1247>3.0.co;2-v
Subject(s) - recombinant dna , protein chip , flag tag , computational biology , protein expression , chemistry , biology , microbiology and biotechnology , biochemistry , gene , fusion protein , bioinformatics
We describe a rapid analysis of interactions between antibodies and a recombinant protein present in total cell lysates. Using a surface plasmon resonance biosensor, a low concentration of glutathione‐S‐transferase (GST) fused protein expressed in small scale Esherichia coli culture was purified on an anti‐GST antibody immobilized sensor chip. The ‘on‐chip purification’ was verified using matrix‐assisted laser desorption/ionization‐time of flight mass spectrometry by measuring the molecular masses of recombinant proteins purified on the sensor chip. The specific binding of monoclonal antibodies for the on‐chip micropurified recombinant proteins can then be monitored, thus enabling kinetic analysis and epitope mapping of the bound antibodies. This approach reduced time, resources and sample consumption by avoiding conventional steps related to concentration and purification.