Single-step conjugation of bioactive peptides to proteins via a self-contained succinimidyl bis-arylhydrazone - PubMed (original) (raw)

. 2009 Oct 21;20(10):1950-7.

doi: 10.1021/bc9002794. Epub 2009 Sep 29.

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Single-step conjugation of bioactive peptides to proteins via a self-contained succinimidyl bis-arylhydrazone

Joy A Phillips et al. Bioconjug Chem. 2009.

Abstract

This paper describes a method for a single-step, site-specific conjugation of bioactive peptides to proteins that exploits the monitoring advantages provided by the unique UV signature absorbance of a bis-arylhydrazone. The utility of this method is demonstrated by the conjugation of a decapeptide molecular adjuvant, YSFKDMP(MeL)aR (EP67), to two test proteins, ovalbumin (OVA) and bovine serum albumin (BSA), and to proteins expressed on intact influenza virons and fungal arthroconidia (spores) of Coccidioides. Conjugation is accomplished with a version of EP67 in which its N-terminus is modified with succinimidyl-4-benzoylhydrazino-nicotinamide (S4BHyNic) (peptide 7), thus enabling conjugation to these large entities via formation of amide bonds with surface-exposed amino groups. The presence of the strongly absorbing bis-arylhydrazone S4BHyNic (ε(354 nm) = 29 000 L mol(-1) cm(-1)) allows for determination of EP67-to-protein molar substitution ratios (MSR), which are in good agreement with the MSRs determined by amino acid analysis. Conjugation to OVA does not compromise the ability of EP67 to engage C5a receptor bearing antigen presenting cells (APC) as measured by the EP67-mediated release of interleukin-6 (IL-6) from APCs. Mice immunized with the resulting OVA-EP67 vaccine conjugate produce high serum titers of OVA-specific IgG antibodies relative to OVA alone. Also, the conjugation of EP67 does not affect the surface integrity of influenza virons or the biological viability of Coccidioides spores. This method of conjugating bioactive peptides to proteins and other large biological entities may represent a convenient and effective way of generating various bioconjugates for use in mechanistic studies or novel therapeutic entities such as EP67-containing vaccines.

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Figures

Figure 1

Figure 1

Panel A. Absorbance spectra of 4.3 mg OVA and OVA reacted in the presence of a 20-fold molar excess of 7 in 1.0 ml of phosphate buffer pH 8.0 for 2 hours at room temperature. Spectra were generated from 20 μl of the solutions (after dialysis) diluted to 1.0 ml with water. Panel B. Absorbance spectra of 4.0 mg BSA and BSA reacted in the presence of a 20-fold molar excess of 7 in 1.0 ml of phosphate buffer pH 8.0 for 2 hours at room temperature. Spectra were generated from 20 μl of the solutions (after dialysis) diluted to 1.0 ml with water.

Figure 2

Figure 2

Absorbance spectra of intact, UV-inactivated influenza virons equivalent to 1.7 mg of protein in the presence of an approximate 20-fold molar excess of 7 in 1.5 ml phosphate buffer pH 8.0 for 3 hours at room temperature. The spectrum was generated from 50 μl of the reaction solution (after dialysis) diluted to 1.0 ml with water.

Figure 3

Figure 3

Scanning electron micrograph of intact, UV-inactivated influenza virons before (Panel A) and after (Panel B) conjugation with 7.

Figure 4

Figure 4

Absorbance spectra of arthroconidia (107 viable cells) incubated with 7(1000 μg/ml) in 0.1 M phosphate buffer pH 8.0 for 2 hours at room temperature (closed circles) and arthroconidia (107 viable cells) only (open circles).

Figure 5

Figure 5

Arthroconidia of C. posadasii (isolate C735) grown on GYE plate cultures for 4 weeks at 30° C, harvested in PBS (ca. 107 cells), incubated for 2 hours with and without 1000 μg/ml of 7 at room temperature, and assessed for viability.

Figure 6

Figure 6

Arthroconidia of C. posadasii (isolate C735) grown on GYE plate cultures for 4 weeks at 30° C. Panel A: Cells (ca. 107) incubated with 7 (1000 μg/ml) for 2 hours at room temperature in 0.1 M phosphate buffer pH 8.0, washed with PBS, fixed with 1% paraformaldehyde, washed with PBS, and blocked with 10% FBS overnight at 4°C. Panel B: Cells from Panel A incubated with rabbit anti-EP67 followed by incubation with FITC-labeled goat anti-rabbit IgG and examined by fluorescence microscopy. Control cells (Panels C and D) were not incubated with 7, but were otherwise prepared and examined as in Panels A and B.

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