Analysis of recombinant protein expression by MALDI-TOF mass spectrometry of bacterial colonies.
E. coli expressing soluble recombinant HIV antigens were analyzed directly by MALDI-TOF mass spectrometry (MS) from bacterial colonies picked from agar plates. An HIV envelope (ENV) antigen construct, penvA, was expressed in E. coli by transformation of the plasmid pPL/penvA-M. The plasmid was co-transformed into E. coli DH5 alpha cells with an equal quantity of the plasmid pKRR826, the parent vector without the penvA insert, and plated at medium density on L-agar plus ampicillin plates. A total of 24 colonies from four agar plates (six colonies per plate) were picked and transferred into 50% acetonitrile--0.1% trifluoroacetic acid aliquots for analysis by MALDI-TOF MS. The MS analysis detected 10 of 24 colonies expressing the recombinant protein; one colony expressed a mutant penvA protein; eleven of 24 colonies showed ions only from E. coli; and two of 24 colonies showed no detectable proteins. When E. coli transformed only with plasmid pPL/penvA-M were examined, all (10 of 10) colonies showed the penv insert by the MALDI-TOF MS method. The method is fast (less than 1.5 h for 24 colonies) and allows identification of colonies expressing intact or mutant proteins directly from culture plates without sample purification.
Winkler
M A
MA
Dept. 90T, Abbott Laboratories, Abbott Park, IL 60064, USA. martin.winkler@abbott.com
Hickman
R K
RK
Golden
A
A
Aboleneen
H
H
D001431:Bacteriological Techniques; D003001:Cloning, Molecular / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics*; D015967:Gene Expression Regulation, Viral; D015686:Gene Products, env / Q000032:analysis / Q000235:genetics; D015497:HIV-1 / Q000235:genetics; D016296:Mutagenesis / Q000235:genetics; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000032:analysis* / Q000235:genetics*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D014170:Transformation, Genetic
D015686:Gene Products, env; D011994:Recombinant Proteins