Systemic Expression, Purification, and Initial Structural Characterization of Bacteriophage T4 Proteins Without Known Structure Homologs

Abstract

Bacteriophage T4 of Escherichia coli is one of the most studied phages. Research into it has led to numerous contributions to phage biology and biochemistry. Coding about 300 gene products, this double-stranded DNA virus is the best-understood model in phage study and modern genomics and proteomics. Ranging from viral RNA polymerase, commonly found in phages, to thymidylate synthase, whose mRNA requires eukaryotic-like self-splicing, its gene products provide a pool of fine examples for phage research. However, there are still up to 130 gene products that remain poorly characterized despite being one of the most-studied model phages. With the recent advancement of cryo-electron microscopy, we have a glimpse of the virion and the structural proteins that present in the final assembly. Unfortunately, proteins participating in other stages of phage development are absent. Here, we report our systemic analysis on 22 of these structurally uncharacterized proteins, of which none has a known homologous structure due to the low sequence homology to published structures and does not belong to the category of viral structural protein. Using NMR spectroscopy and cryo-EM, we provided a set of preliminary structural information for some of these proteins including NMR backbone assignment for Cef. Our findings pave the way for structural determination for the phage proteins, whose sequences are mainly conserved among phages. While this work provides the foundation for structural determinations of proteins like Gp57B, Cef, Y04L, and Mrh, other in vitro studies would also benefit from the high yield expression of these proteins.

Keywords: Cef; Mrh; NMR; T4; Y04L and Gp57B; bacteriophage; cryo-EM.

FIGURE 1

Multiple sequence alignment of selected T4 proteins. The multiple sequence alignments of Cef, ComCα, Mrh, and Pin of T4 showing the conserved regions of each viral protein with that of other phages. The purification and characterization of these proteins could provide a solid basis for future studies on these homologs of other phages. The red and yellow color is the visualization of “*,” which represents the fully conserved amino acid amongst all the sequences, and “:,” which represents amino acids with high similarities amongst the sequences (>0.5 in the Gonnet Pam 250 Matrix) used in the ClusterW alignment format.

FIGURE 2

Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) showing the expression and the purification of the viral proteins using N-terminal His-tag. (A) Cef, Pin, MRH, Y00H, Y00G, and DexA proteins were collected in the soluble fraction, evident by the presence of bands of correct molecular weight in the elusion fraction. (B) MotB, RpbA, SegF, Y01A, ComCα, and Valyl proteins were expressed into the inclusion body, evident by the absence of bands of correct molecular weight in the elusion fraction and the presence of the bands in the insoluble fraction. M, Marker; FT, Flow-through fraction; W, Wash fraction; E, elusion fraction; Ins, Resolublized cell debris containing insoluble protein.

FIGURE 3

SDS-PAGE showing the expression and the purification of viral proteins using solubilization tags and the cell-free expression system. (A) SUMO-Gp57B and SUMO-Y04L were expressed and collected in the soluble fraction, evident by the presence of bands of correct molecular weight in the elusion fraction (left). The native proteins were collected after SUMO cleavage, evident by the bands in the flow-through and wash fraction (right). M, Marker; FT, Flow-through fraction; W, Wash fraction; E, Elusion fraction; Ins, Resolublized cell debris containing insoluble protein. (B) Cef protein was expressed and collected in the soluble fraction, using the cell-free expression system, evident by the presence of a sharp band at the corresponding size in the elution fraction. M, Marker; FT, Flow-through fraction; W, Wash fraction; E, Elusion fraction.

FIGURE 4

Size exclusion chromatography of soluble viral proteins for assessment of multimeric state in solution. (A) Size exclusion chromatography of Pin, Y04L, Cef, and Gp57B demonstrated the viral proteins existed as a homogenous monomeric state in solution, shown by the absorption peak at the volume of the expected molecular weight. (B) Size exclusion chromatography of MRH and DexA demonstrated a homogenous multimeric state in solution, as the proteins eluted at a lower volume, indicating a larger molecular weight. The chromatographies of Y04L and Gp57B were performed using HiLoad 16/600 Superdex 200 pg while the experiments of Cef, MRH, Pin, and DexA were using HiLoad 16/600 Superdex 75 pg.

FIGURE 5

Characterization of the soluble phage proteins using NMR and Cryo-EM. (A) 1D ¹H-NMR spectra of Cef, Y04L, and Gp57B showed characteristics of folded structural features, such as the presence of the sharp peaks below 1.0 ppm as well as the dispersion of peaks between 6.0 and 11.0 ppm. The ¹H-NMR spectra of MRH and DexA showed the characteristics of a large multimeric protein, such as broad peak caused by the higher molecular weight, which was in agreement with the size exclusion chromatograms of MRH and DexA. (B) The 2D ¹H-¹⁵N HSQC spectra of Y04L showed crosspeaks that were dispersed, indicating a folded structure in solution. (C) The assigned 2D ¹H-¹⁵N HSQC spectra of Cef showed crosspeaks that were dispersed, indicating a folded structure in solution. The crosspeaks were assigned with the corresponding amino acid number and type. (D) The representative motion-corrected cryo-EM micrograph of DexA.