Processing of the tail lysozyme (gp5) of bacteriophage T4

Abstract

The processing site of gp5 has been determined to be between residues Val-390 and His-391, instead of Ser-351 and Ala-352 as previously reported (H. Kanamaru, N. C. Gassner, N. Ye, S. Takeda, and F. Arisaka, J. Bacteriol. 181:2739-2744). Moreover, the maturation of gp5 is abolished by null mutations in other hub genes, indicating that cleavage requires the interactions of several baseplate proteins.

FIG. 1.

SDS-PAGE analysis of gene products from tube baseplates. The polyacrylamide concentration was 12.5%. Tube baseplates were treated with 6 M urea at 37°C for 15 min (lane A), with SDS buffer at 70°C for 3 min (lane B), or with SDS buffer at 90°C for 3 min (lane C). Lane D, protein molecular weight standards.

FIG. 2.

Recombinant gene 5-expressed protein products detected by Western blot analysis. After electrophoresis on SDS-12.5% PAGE, proteins were transferred to PVDF membranes. Lane A, Prestained protein molecular weight standards; lane B, gp5 separated from tube baseplates; lane C, sample from induced E. coli BL21(DE3) cells containing recombinant gene 5 with the codon for C-terminal Ser-360; lane D, sample from uninduced E. coli BL21(DE3) cells containing recombinant gene 5 with the codon for C-terminal Ser-360; lane E, sample from induced E. coli BL21(DE3) cells containing recombinant gene 5 with the codon for C-terminal Val-380; lane F, sample from uninduced E. coli BL21(DE3) cells containing recombinant gene 5 with the codon for C-terminal Val-380; lane G, sample from induced E. coli BL21(DE3) cells containing recombinant gene 5 with the codon for C-terminal Val-390; lane H, sample from uninduced E. coli BL21(DE3) cells containing recombinant gene 5 with the codon for C-terminal Val-390.

FIG. 3.

Western blot analysis of processed gp5 from lysates. After electrophoresis on SDS-8% PAGE, proteins were transferred to PVDF membranes. Lane A, 25am N67 lysate; lane B, 26am N131 lysate; lane C, 27am N120 lysate; lane D, 28am A452 lysate; lane E, 29am B7 lysate; lane F, tube baseplates; lane G, 5am B256 lysate; lane H, 48am NO22X lysate; lane I, 51am S29 lysate.

FIG. 4.

Western blot analysis of processed gp5 from lysates. After electrophoresis on SDS-8% PAGE, proteins were transferred to PVDF membranes. Lane A, prestained protein molecular mass standards (Bio-Rad Laboratories); lane B, 25am N67 lysate; lane C, 26am N131 lysate; lane D, 28am A452 lysate; lane E, 29am B7 lysate; lane F, 5am N135 lysate; lane G, 23am H11 lysate (tails); lane H, uninfected E. coli BE (sup⁰); lane I, 51am S29 lysate.

FIG. 5.

Similar regions of the amino acid sequence between gp5 (′) and gp23 ("). The ratio of identical amino acid pairs in whole gp5/gp23 (1′/46" to 390′/435") is 8.2%, much higher than 5.0% in two common proteins. When the number of conservative changes is included, the total similarity ratio of amino acid pairs between gp5 and gp23 reaches 30.0%. In the main region from 155′/200" to 362′/407" (208 amino acid pairs, the region indicated by arrows), the ratio of identical amino acid pairs in gp5 and gp23 is 11.5% and the total similarity ratio including conservative changes is 35.1%. Colons indicate identical amino acids. Periods indicate conservative changes.

FIG. 6.

Western blot analysis of gp26. The tube baseplate proteins were transferred from SDS-12.5% PAGE gels to PVDF membranes. Lane A, gp26 band detected with antiserum against the C-terminal peptide of gp26; lane B, gp5 band detected with antiserum against gp5; lane C, gp26 and gp5 bands detected with a mixture of the two antisera.