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Published on: December 1, 2020
A bipartite bacteriophage T4 SOC and HOC randomized peptide display library: detection and analysis of phage T4
Naglis Malys1, Dau-Yin Chang, Richard G Baumann
1Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, MD 21201-1503, USA.
Abstract:
HOC and SOC are dispensable T4 capsid proteins that can be used for phage display of multiple copies of peptides and proteins. A bipartite phage T4 peptide library was created by displaying on tetra-alanine linker peptides five randomized amino acids from the carboxyl-terminus of SOC and five randomized amino acids from the amino terminus of HOC. The bipartite library was biopanned against the phage T4 terminase large subunit gp17 to identify T4 gene products that may interact with the terminase. The sequences of selected phages displayed matches to those T4 gene products previously known by genetic and biochemical criteria to interact with gp17: gp20 (portal protein), gp32 (single-stranded DNA binding protein), gp16 (terminase small subunit), and gp17 (self). In addition, matches were found to gp55 (T4 late sigma factor), gp45 (sliding clamp), gp44 (clamp loader), gp2 (DNA end protein), and gp23 (major capsid protein). Abundant amino acid sequence matches were found to aa region 118-134 of gp55. Immunoprecipitation and affinity column chromatography demonstrated direct binding of gp17 and gp55; moreover, gp17 bound specifically to a column-coupled peptide corresponding to gp55 residues 111-136. Measurements of gene 17 and other mRNA levels in mutant-infected bacteria did not support a role of gp17-gp55 interaction in regulation of terminase or other late gene transcription. However, whereas DNA concatemers that accumulate in prohead and terminase defective phage T4 infections could be packaged in vitro to approximately 10% wild-type efficiency, 55am33am defective concatemeric DNA was packaged at least 100-fold less efficiently. Moreover, gp55 residues 111-136 peptide specifically blocked DNA packaging in vitro. These results suggest that the T4 terminase interaction with T4 late sigma factor gp55 plays a role in DNA packaging in vivo. The gp55 interaction may function to load the terminase onto DNA for packaging.
