Analysis of bacteriophage T4 coat proteins by two‐dimensional electrophoresis and computerized densitometry

Abstract

Analysis of bacteriophage T4 coat proteins by two-dimensional electrophoresis and computerized densitometryWe have designed treatment conditions which allow analysis of the coat proteins of bacteriophage T4D+ by high resolution two-dimensional electrophoresis. The best results were obtained when the mature phage particles were subjected to hypertonic lysis and DNase treatment before solubilization with sodium dodecyl sulfate (SDS). Samples containing 250 pg protein gave the optimum resolution, producing 45 spots as visualized by Coomassie Blue staining. Computerized densitometry measurements showed that these proteins range in relative abundance from less than 0.22 % to 11.8 % of the total. Bacteriophage T4 has a genome size large enough to accomodate about 170 genes, each consisting of 1000 base pairs [ll of which about 65 essential and 70 non-essential genes have been identified [21. However, electrophoretic analysis of SDS-solubilized T4 coat proteins has only resolved 21-25 bands in the protein pattern [3-51. Two-dimensional electrophoresis [61 has been successfully applied to various biological systems in detecting proteins which are not separable by one-dimensional techniques. Hence we have performed the separation of T4 coat proteins in a two-dimensional system. Our initial attempts to solubilize T4 coat protein by the lysis buffer technique of O'Farrell[6] or the SDS Figure I. SDS-polyacrylamide gel electrophoresis of DNase treated and control preparations. Lane [ I ] T4D+ treated with DNase as described in text. Lane [21 untreated control. Molecular weight standards were purchased from Pharmacia Fine Chemicals. Bacteriophage T4D+ and E. coli B strain WU 3610 were generously donated by Dr. R. K. Elespuru. E. coli B (leucine requiring) were grown to midlog phase in M-9 medium supplemented with 10 pg/ml leucine. Cultures were infected with T4D+ at a multiplicity of infeciton of 0.003 and incubated at 37OC for 4 h. Cell debris was removed by centrifugation at 1400 g for 15 min. The supematants were centrifuged at 27,000 g for 1 h. The resultant phage pellets were resuspended in 0.15 M NaCl and stored under chloroform until use. SDSpolyacrylamide gel electrophoresis was perfomed as described by Laemmli [3] in a 10 % slab gel, with a 30 pg protein load. Correspondence: Douglas M. Gersten, Abbreviation: SDS: Sodium dodecyl sulfate method of Ames and Nikaido [71 failed, resulting in poor entry of the proteins into the first dimension electrofocusing gel.The observation by OFarrellL61, that nucleic acids in the sample interfere with isoelectric focusing, suggested that treatment of the phage by hypertonic shock, followed by en-Table 1. Integrated density and percent abundance of each subunit. Subunit No.a) Integrated densityb) % Abundancec) 1 2 3 4 5 6 7 8

Structural proteins and DNA characteristics of 14 Listeria typing bacteriophages

“…The insolubility of some particular proteins could also be overcome by disrupting the phage particles (in the presence of surfactant and thiol reagent) by heating to 95 °C for 5 rain prior to addition of sample buffer. The use of SDS for treatment of phage proteins prior to IEF (Gersten et al, 1981) should, in general, be avoided. SDS may severely interfere with focusing and determination of true pI values, at least when conventional CA IEF is used.…”

“…However, little effort has been made to apply isoelectric focusing (IEF) and two-dimensional (2-D) electrophoresis to the study of phage proteins (Gersten et al, 1981 ;Lee & Stewart, 1985), presumably owing to the somewhat poor reproducibility of specific patterns. Methodological difficulties, such as the interference by phage DNA in the samples (O'Farrell, 1975), may have been another reason for the lack of such data in the literature.…”

Identification of some bacteriophage T4 prereplicative proteins on two-dimensional gel proteins

“…The high-resolution, two-dimensional electrophoretic methods of O'Farrell (16) and O'Farrell et al (18) have provided comprehensive profiles of cellular proteins from a variety of biological systems. Modifications of these methods have been used to analyze membrane proteins (2,21) and bacteriophage T4 coat proteins (8). Recently, another two-dimensional method has been used for the detection of labile protein phosphorylation modifications (3).…”