Efficient affinity-tagging of M13 phage capsid protein IX for immobilization of protein III-displayed oligopeptide probes on abiotic platforms
We developed a genetic approach to efficiently add an affinity tag to every copy of protein IX (pIX) of M13 filamentous bacteriophage in a population. Affinity-tagged phages can be immobilized on a surface in a uniform monolayer in order to position the pIII–displayed peptides or proteins for optimal interaction with ligands. The tagging consists of two major steps. First, gene IX (gIX) of M13 phage is mutated in Escherichia coli via genetic recombineering with the gIX::aacCI insertion allele. Second, a plasmid that co-produces the affinity-tagged pIX and native pVIII is transformed into the strain carrying the defective M13 gIX. This genetic complementation allows the formation of infective phage particles that carry a full complement (five copies per virion) of the affinity-tagged pIX. To demonstrate the efficacy of our method, we tagged a M13 derivative phage, M13KE, with Strep-tag II. In order to tag pIX with Strep-tag II, the phage genes for pIX and pVIII were cloned and expressed from pASG-IBA4 which contains the E. coli OmpA signal sequence and Strep-Tag II under control of the tetracycline promoter/operator system. We achieved the maximum phage production of 3 × 1011 pfu/ml when Strep-Tag II-pIX-pVIII fusion was induced with 10 ng/ml of anhydrotetracycline. The complete process of affinity tagging a phage probe takes less than 5 days and can be utilized to tag any M13 or fd pIII-displayed oligopeptide probes to improve their performance.
KeywordsPhage-display M13 protein IX Affinity-tagging Phage immobilization
Zhou Tong, Laura Silo-Suh, Anwar Kalalah, and Paul Dawson performed the experiments. Bryan Chin and Sang-Jin Suh conceived and designed the study. Laura Silo-Suh and Sang-Jin Suh analyzed the data and supervised the project. Zhou Tong wrote the initial draft of the paper, and Laura Silo-Suh and Sang-Jin Suh revised the manuscript. All authors reviewed the manuscript.
This research was partially supported by the United States Department of Agriculture National Institute of Food and Agriculture grant USDA-2016-67,021-25,005 and Alabama Agricultural Experimental Station Hatch grant ALA0SUH to Suh and Chin. Zhou Tong was supported by the Chinese Scholarship Council fellowship. Anwar Kalalah was supported by a scholarship from the Kingdom of Saudi Arabia.
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Conflict of interest
The authors declare that they have no conflict of interest.
This article does not contain any studies with human participants or animals performed by any of the authors.
- Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K (eds)(1993) Curr Prot Mol Biol. Greene Publishing Associates, Inc. and John Wiley & Sons, Inc.Google Scholar
- Huang S, Li SQ, Yang H, Johnson ML, Wan J, Chen I, Petrenko VA, Barbaree JM, Chin BA (2008) Optimization of phage-based magnetoelastic biosensor performance. Sensor Trans J on Microsyst: Technol Appl 3:87–96Google Scholar
- Huang S, Yang H, Lakshmanan RS, Johnson ML, Wan J, Chen IH, Wikle HC 3rd, Petrenko VA, Barbaree JM, Chin BA (2009) Sequential detection of Salmonella typhimurium and Bacillus anthracis spores using magnetoelastic biosensors. Biosens Bioelectron 24(6):1730–1736. https://doi.org/10.1016/j.bios.2008.09.006 CrossRefPubMedGoogle Scholar