Separation of flavivirus membrane and capsid proteins by multistep high-performance liquid chromatography optimized by immunological monitoring.
Complete separation of the three structural proteins, E, C and M, of an enveloped virus (tick-borne encephalitis virus) was achieved by means of a two-step high-performance liquid chromatography (HPLC) technique in less than 1 h. The hydrophobically associated membrane proteins E and M were successfully separated by high-performance gel permeation chromatography (TSK-3000 SW column) in the presence of sodium dodecyl sulphate (SDS), whereas the separation of M and C as well as desalting and removal of SDS was achieved by subsequent reversed-phase chromatography on a C3 column. With regard to further characterization by peptide mapping, analysis of the amino acid composition and aminoterminal sequencing, the second step was performed with volatile buffer systems. Quality control of the separation was achieved by a combination of HPLC with a highly sensitive dot immunoassay by the use of polyclonal as well as monoclonal antibodies. This method proved extremely sensitive and revealed strong tailing effects and cross-contaminations of peaks well-separated in reversed-phase chromatography, which were neither apparent in the absorbance curve at 214 nm nor in the analysis by SDS-polyacrylamide gel electrophoresis. By visualization of the peak-tailing effect, the chromatographic conditions could be modified in order to achieve an optimum separation of proteins.
Winkler
G
G
Heinz
F X
FX
Guirakhoo
F
F
Kunz
C
C
D000911:Antibodies, Monoclonal; D002213:Capsid / Q000032:analysis*; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D004669:Encephalitis Viruses, Tick-Borne / Q000032:analysis; D005416:Flavivirus / Q000032:analysis*; D006023:Glycoproteins / Q000032:analysis; D007118:Immunoassay; D008565:Membrane Proteins / Q000302:isolation & purification*; D014764:Viral Proteins / Q000302:isolation & purification*
D000911:Antibodies, Monoclonal; D006023:Glycoproteins; D008565:Membrane Proteins; D014764:Viral Proteins