DataSheet_1_Immunogenic epitope scanning in bacteriolytic enzymes Pal and Cpl-1 and engineering Pal to escape antibody responses.docx

Bacteriolytic enzymes are promising antibacterial agents, but they can cause a typical immune response in vivo. In this study, we used a targeted modification method for two antibacterial endolysins, Pal and Cpl-1. We identified the key immunogenic amino acids, and designed and tested new, bacteriolytic variants with altered immunogenicity. One new variant of Pal (257-259 MKS → TFG) demonstrated decreased immunogenicity while a similar mutant (257-259 MKS → TFK) demonstrated increased immunogenicity. A third variant (280-282 DKP → GGA) demonstrated significantly increased antibacterial activity and it was not cross-neutralized by antibodies induced by the wild-type enzyme. We propose this variant as a new engineered endolysin with increased antibacterial activity that is capable of escaping cross-neutralization by antibodies induced by wild-type Pal. We show that efficient antibacterial enzymes that avoid cross-neutralization by IgG can be developed by epitope scanning, in silico design, and substitutions of identified key amino acids with a high rate of success. Importantly, this universal approach can be applied to many proteins beyond endolysins and has the potential for design of numerous biological drugs.</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

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

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

CD spectra of recombinant T4 major head proteins.

<p>Comparison of CD spectra acquired in the range of 190–265 nm for recombinant gphoc (dark green), gp24 (blue), gp23 (red), and gpsoc (light green). Analysis of CD spectra showed that produced proteins are folded and contain secondary structures as predicted based on their primary structure (unpublished data).</p

The sequences of PCR primers used for bacteriophage gene cloning.

<p>F: Forward Primer; R: Reverse primer.</p

Normalized chemical denaturation curves of the gp24.

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

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