Intact cell matrix-assisted laser desorption/ionization mass spectrometry as a tool to screen drugs in vivo for regulation of protein expression.
Here we demonstrate for the first time the application of intact cell matrix-assisted laser desorption/ionization mass spectrometry (ICM-MS) to study the regulation of protein expression. This technique can be extended to screen the drugs that inhibit protein synthesis in various diseases. We have used Escherichia coli cells expressing a recombinant glutathione-S-transferase (GST) gene under an arabinose-inducible promoter as a model system. Using ICM-MS analysis, we have detected a 28 kDa peak corresponding to the production of recombinant GST under the arabinose-induced condition. Furthermore, recombinant GST protein was purified by a single-step affinity purification using a glutathione Sepharose 4B affinity column from arabinose-induced E. coli cells. The purified GST protein was found to be a 28 kDa protein by MALDI analysis suggesting the arabinose-induced protein is indeed GST. The regulation of protein expression was studied using glucose as an alternative metabolite. The glucose-mediated regulation of the ara-operon was followed using the ICM-MS technique. All the results obtained from ICM-MS data were validated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The present technique can be extended for in vivo screening of drugs and it holds tremendous potential to discover novel drugs against specific protein expressions in different diseases.
Kulkarni
Mahesh J
MJ
Division of Organic Chemistry Synthesis, National Chemical Laboratory, Pune 411 008, India. mj.kulkarni@ncl.res.in
Vinod
V P
VP
Umasankar
P K
PK
Patole
Milind S
MS
Rao
Mala
M
D001426:Bacterial Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects*; D011500:Protein Synthesis Inhibitors / Q000032:analysis / Q000494:pharmacology; D011994:Recombinant Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000295:instrumentation / Q000379:methods*
D001426:Bacterial Proteins; D011500:Protein Synthesis Inhibitors; D011994:Recombinant Proteins