A novel approach for separating bacteriophages from other bacteriophages using affinity chromatography and phage display

Abstract

Practical applications of bacteriophages in medicine and biotechnology induce a great need for technologies of phage purification. None of the popular methods offer solutions for separation of a phage from another similar phage. We used affinity chromatography combined with competitive phage display (i) to purify T4 bacteriophage from bacterial debris and (ii) to separate T4 from other contaminating bacteriophages. In 'competitive phage display' bacterial cells produced both wild types of the proteins (expression from the phage genome) and the protein fusions with affinity tags (expression from the expression vectors). Fusion proteins were competitively incorporated into the phage capsid. It allowed effective separation of T4 from a contaminating phage on standard affinity resins.

Figure 1. Expression of recombinant GST- or His-tagged proteins Hoc and Soc.

M – molecular weight marker.

Figure 2. Modifications of bacteriophage T4 capsid with affinity tags before affinity chromatography.

Schema of recombinant protein types. Information on relevant panels in Figure 3 presenting results of purification of all types is given in the bottom of each chart.

Figure 3. Efficacy of T4 phage binding to standard affinity chromatography resins.

Presented according to: type of T4 capsid protein presenting affinity tags (Hoc protein or Soc protein), type of the affinity tags presented (GST or His-tag), and to localization of the affinity tags in the protein (N- or C-terminal). Phages were modified competitively, i.e. without genetic manipulation of the phage genome. Phage titres [pfu/ml] in wash fraction and in three subsequent elution fractions are presented (each fraction was eluted by 500 mM of imidazole or 20 mM of glutathione). W – wash fraction, E1 to E3 – successive elution fractions. (A): Hoc protein of T4 was modified with the affinity tags and then the phage was isolated by GST-affinity chromatography. (B): Hoc protein of T4 was modified with the affinity tags and then the phage was isolated by Ni²⁺-affinity chromatography. (C): Soc protein of T4 was modified with the affinity tags and then the phage was isolated by GST-affinity chromatography. (D): Hoc protein of T4 was modified with the affinity tags and then the phage was isolated by Ni²⁺-affinity chromatography.

Figure 4. Separation of T4 bacteriophage modified with GST-Hoc by competitive phage display from contaminating phages: φ9, TuIb, and 76.

Bacteriophages were mixed 1:1, incubated with glutathione Sepharose, washed and eluted. Phage titre in subsequent fractions is presented: W – wash fraction, E1 to E3 – elution fractions.

Figure 5. The new method for bacteriophage purification: competitive phage display combined with affinity chromatography.