Purification of phage display-modified bacteriophage T4 by affinity chromatography

<p>Abstract</p> <p>Background</p> <p>Affinity chromatography is one of the most efficient protein purification strategies. This technique comprises a one-step procedure with a purification level in the order of several thousand-fold, adaptable for various proteins, differentiated in their size, shape, charge, and other properties. The aim of this work was to verify the possibility of applying affinity chromatography in bacteriophage purification, with the perspective of therapeutic purposes. T4 is a large, icosahedral phage that may serve as an efficient display platform for foreign peptides or proteins. Here we propose a new method of T4 phage purification by affinity chromatography after its modification with affinity tags (GST and Histag) by <it>in vivo </it>phage display. As any permanent introduction of extraneous DNA into a phage genome is strongly unfavourable for medical purposes, integration of foreign motifs with the phage genome was not applied. The phage was propagated in bacteria expressing fusions of the phage protein Hoc with affinity tags from bacterial plasmids, independently from the phage expression system.</p> <p>Results</p> <p>Elution profiles of phages modified with the specific affinity motifs (compared to non-specific phages) document their binding to the affinity resins and effective elution with standard competitive agents. Non-specific binding was also observed, but was 10²-10⁵times weaker than the specific one. GST-modified bacteriophages were also effectively released from glutathione Sepharose by proteolytic cleavage. The possibility of proteolytic release was designed at the stage of expression vector construction. Decrease in LPS content in phage preparations was dependent on the washing intensity; intensive washing resulted in preparations of 11-40 EU/ml.</p> <p>Conclusions</p> <p>Affinity tags can be successfully incorporated into the T4 phage capsid by the <it>in vivo </it>phage display technique and they strongly elevate bacteriophage affinity to a specific resin. Affinity chromatography can be considered as a new phage purification method, appropriate for further investigations and development.</p

Recombinant expression and purification of T4 phage Hoc, Soc, gp23, gp24 proteins in native conformations with stability studies.

Understanding the biological activity of bacteriophage particles is essential for rational design of bacteriophages with defined pharmacokinetic parameters and to identify the mechanisms of immunobiological activities demonstrated for some bacteriophages. This work requires highly purified preparations of the individual phage structural proteins, possessing native conformation that is essential for their reactivity, and free of incompatible biologically active substances such as bacterial lipopolysaccharide (LPS). In this study we describe expression in E. coli and purification of four proteins forming the surface of the bacteriophage T4 head: gp23, gp24, gphoc and gpsoc. We optimized protein expression using a set of chaperones for effective production of soluble proteins in their native conformations. The assistance of chaperones was critical for production of soluble gp23 (chaperone gp31 of T4 phage) and of gpsoc (chaperone TF of E. coli). Phage head proteins were purified in native conditions by affinity chromatography and size-exclusion chromatography. Two-step LPS removal allowed immunological purity grade with the average endotoxin activity less than 1 unit per ml of protein preparation. The secondary structure and stability of the proteins were studied using circular dichroism (CD) spectrometry, which confirmed that highly purified proteins preserve their native conformations. In increasing concentration of a denaturant (guanidine hydrochloride), protein stability was proved to increase as follows: gpsoc, gp23, gphoc. The denaturation profile of gp24 protein showed independent domain unfolding with the most stable larger domain. The native purified recombinant phage proteins obtained in this work were shown to be suitable for immunological experiments in vivo and in vitro

Size distribution in purified preparations of non-denatured T4 head proteins.

<p>Dynamic Light Scattering patterns of the native gphoc (green), gp24 (blue), gp23 (red), and gpsoc (black).</p

CD spectra and unfolding transitions of the four major T4 capsid proteins.

<p>(A) (B) CD spectrum and representative set of chemical denaturation spectra (based on gp23 unfolding induced by increasing concentrations of GdmCl) monitored by changes in ellipticity. (C) Normalized chemical denaturation curves of the gphoc (dark green), gp24 (blue), gp23 (red), and gpsoc (light green); the wave length: 220 nm. The data were analysed assuming a two-state reversible equilibrium transition (data summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038902#pone-0038902-t003" target="_blank">Table 3</a>).</p

SDS-PAGE of purified proteins of T4 phage head.

<p>(A) gp23, (B) gp24, (C) gphoc and (D) gpsoc. Proteins are marked with dashes. M- molecular weight marker.</p

SDS-PAGE of expressed recombinant GST-tagged proteins of T4 phage head.

<p>(A) gp23, (B) gp24, (C) gphoc and (D) gpsoc. Overexpressed proteins are marked with dashes. M- molecular weight marker. 1- insoluble fraction of the culture before induction (control). 2- insoluble fraction of the culture after induction (expression). 3- soluble fraction of the culture before induction (control). 4- soluble fraction of the culture after induction (expression).</p

Endotoxin activity in the phage head protein preparations.

<p>Mean, maximum and minimum values of six independent preparations were presented (LPS units per 10 microgram of protein preparation).</p

Normalized chemical denaturation curves of the gp24.

<p>Domain A (green) and domain B (blue).</p

Relative yields of soluble recombinant proteins after co-expression with selected chaperones.

*<p>ND- not determined.</p><p>Relative yields of soluble recombinant proteins after co-expression with selected chaperones, estimated as a fraction of the total amount of the proteins. Parameters representing combinations used in further work were underlined.</p