New approach for identification of bacteriophage virion structural proteins.
The ability of the phage structural polypeptides to undergo post-translational modification makes the task of correlation of the primary nucleotide sequence data with the actual structural proteins of a virion extremely challenging. This study describes an alternative model approach based on two-stage chromatography for allocation of virion structural components and identification of their major polypeptides. Bacteriophage T4D, its amber mutant T4D23 (amH11) and its tail preparations were purified, concentrated and separated by ion exchange chromatograpgy based on fibrous DEAE-cellulose. The major tail fraction was then exposed to size-exclusion chromatography which enabled to separate tail components by size. This method proved itself as a highly efficient and gentle enough to save most of the biological material without changing the basic properties of the native phage. The result also shows that the accumulation of individual phage tails in the course of the amber mutant T4D23 (amH11) propagation on the permissive host Escherichia coli CR63 was resulted by changes in the conditions of reproduction. The ability of bacteriophages to form an excess of tails, capsids and other structures during reproduction on a non-traditional host provides an alternative way for obtaining highly concentrated preparations of virion components for further analysis of their major proteins and determination of the genes responsible for their synthesis.
Zabolotny Institute of Microbiology and Virology, National Academy of Sciences of Ukraine, 154 Acad Zabolotny St., Kyiv, MSP, D03680, Ukraine.
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Korol
Zabolotny Institute of Microbiology and Virology, National Academy of Sciences of Ukraine, 154 Acad Zabolotny St., Kyiv, MSP, D03680, Ukraine.
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Tovkach
D017122:Bacteriophage T4 / Q000737:chemistry* / Q000235:genetics; D002213:Capsid / Q000737:chemistry / Q000378:metabolism; D005591:Chemical Fractionation / Q000379:methods; D002852:Chromatography, Ion Exchange; D004926:Escherichia coli / Q000821:virology*; D058507:Host Specificity; D009154:Mutation; D015678:Viral Structural Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D014771:Virion / Q000737:chemistry* / Q000235:genetics; D014779:Virus Replication / Q000235:genetics*
D015678:Viral Structural Proteins