Bacteriophage T4 capsid packaging and unpackaging of DNA and proteins.
Bacteriophage T4 has proven itself readily amenable to phage-based DNA and protein packaging, expression, and display systems due to its physical resiliency and genomic flexibility. As a large dsDNA phage with dispensable internal proteins and dispensable outer capsid proteins it can be adapted to package both DNA and proteins of interest within the capsid and to display peptides and proteins externally on the capsid. A single 170 kb linear DNA, or single or multiple copies of shorter linear DNAs, of any sequence can be packaged by the large terminase subunit in vitro into protein-containing proheads and give full or partially full capsids. The prohead receptacles for DNA packaging can also display peptides or full-length proteins from capsid display proteins HOC and SOC. Our laboratory has also developed a protein expression, packaging, and processing (PEPP) system which we have found to have advantages over mammalian and bacterial cell systems, including high yield, increased stability, and simplified downstream processing. Proteins that we have produced by the phage PEPP platform include human HIV-1 protease, micrococcal endonuclease from Staphylococcus aureus, restriction endonuclease EcoRI, luciferase, human granulocyte colony stimulating factor (GCSF), green fluorescent protein (GFP), and the 99 amino acid C-terminus of amyloid precursor protein (APP). Difficult to produce proteins that are toxic in mammalian protein expression systems are easily produced, packaged, and processed with the PEPP platform. APP is one example of such a highly refractory protein that has been produced successfully. The methods below describe the procedures for in vitro packaging of proheads with DNA and for producing recombinant T4 phage that carry a gene of interest in the phage genome and produce and internally package the corresponding protein of interest.
Institute for Genome Sciences, University of Maryland School of Medicine, Baltimore, MD, USA.
Julienne M
JM
Mullaney
Lindsay W
LW
Black
D017122:Bacteriophage T4 / Q000235:genetics* / Q000378:metabolism* / Q000502:physiology; D002213:Capsid / Q000378:metabolism*; D042003:DNA Packaging; D004274:DNA, Recombinant; D005818:Genetic Engineering / Q000379:methods; D005822:Genetic Vectors / Q000235:genetics; D010948:Viral Plaque Assay / Q000379:methods; D019065:Virus Assembly
D004274:DNA, Recombinant