Amine modification of filamentous virions (phage particles) is widely used in phage display technology to couple small groups such as biotin or fluorescent dyes to the major coat protein pVIII. We have developed a generalized kinetic model for protein amine modification and applied it to the modification of pVIII with biotin and the near-infrared fluorophor Alexa Fluor 680. Empirically optimized kinetic parameters for the two modification reactions allow the modification level to be predicted for a wide range of virions and modifying reagent concentrations. Virions with 0.03 biotins per pVIII subunit have 50% of the maximal binding capacity for a streptavidin conjugate.

Introduction

Filamentous phages of the Ff class (derivatives of natural strains fd, f1 and M13) are the most common vectors in phage display technology (1). A foreign peptide or protein domain is fused genetically to one of the phage coat proteins: in most cases, either to 1 to 5 copies of the minor coat protein pIII, or to 1 to ∼150 copies of the major coat protein pVIII. The foreign protein or domain is thereby displayed on the outer surface of the virion, where it is accessible to antibodies, receptors, or other solutes in the medium. Random peptide libraries (RPLs)—large populations of virions collectively displaying millions or billions of random peptides—are a rich source of high-affinity peptide ligands for a great diversity of target biomolecules. Such ligands can be specifically affinity-selected from the RPL by using the target biomolecule as an immobilized selector. Analogously, specific peptide ligands—for target cells such as cancer cells, or for defined tissues in vivo such as tumors—can be affinity-selected using intact cells (2) or tissues in living animals (3) as selectors.

As shown in Figure 1, the filamentous virion has thousands of surface-exposed e-amino groups—one on each subunit of the major coat protein pVIII (4,5)—to which small chemical groups may be coupled without impairing structural integrity or infectivity (6). The α-amino groups on the pVIII subunits, though mostly buried in the structural model in Figure 1 and unreactive with acetimidate at pH 10.0 (6), may be reactive with amine-reactive reagents other than acetimidate. The ability to modify surface amines can be exploited in many research contexts. For instance, virions bearing tumor-avid peptides can be lightly modified with biotin and used to image tumors in vivo by a two-step “pretargeting” regimen (7). In the first step, tumor-bearing mice are injected with the biotinylated virions. In the second step—initiated only after non-tumor-bound virions have been allowed to clear—the mice are injected with ¹¹¹In-labeled streptavidin. The labeled streptavidin binds tightly to the tumor-targeted biotinylated virions, allowing the tumor to be imaged by single-photon emission computed tomography. Similarly, virions bearing tumor-avid peptides and lightly modified with near-infrared (NIR) fluorophors can be used to image tumors optically in vivo (8).

Figure 1. (Click to enlarge)

Success in such experiments requires that the exposed amines be modified to a sufficient level for the purpose at hand while avoiding the ill effects of over-modification, including detrimentally changing the virion's physical properties or sterically hindering specific binding by its displayed peptides. The effect of modification level is well illustrated with biotinylated virions. Figure 2 shows a saturation curve for a streptavidin conjugate binding to virions biotinylated to various levels. According to that curve, virions reach half their maximum binding capacity at a modification level of about 0.03 biotins per pVIII subunit. That number would be an appropriate target modification level for almost all applications.

Figure 2. Saturation curve for the binding of a streptavidin conjugate to biotinylated virions. (Click to enlarge)

It is desirable to be able to achieve a specific target modification level without a laborious series of pilot modifications. In this article, building on previous work (9), we develop a generalized kinetic model for protein amine modification that uses the results of test reactions at a few protein and reagent concentrations to calculate the expected results of similar modification reactions over a vast continuum of other protein and reagent concentrations. Modifications of filamentous phage pVIII with biotin and the NIR fluorophor Alexa Fluor 680 (AF680) serve as examples, but the model is applicable to any protein and amine-modifying reagent.

Materials and methods

Two N-hydroxysuccinimide (NHS) esters were chosen as amine-reactive reagents: the biotinylating reagent NHS-PEO₄-biotin (Catalog no. 21329; Pierce Chemical Co., Rockford, IL, USA), and the NIR fluorophor labeling reagent sulfo-NHS-AF680 (Catalog no. A20008; Invitrogen, Carlsbad, CA, USA). The pre-weighed reagents were dissolved just before use in dimethyl sulfoxide (DMSO) to a range of concentrations (up to 17.4 mM sulfo-NHS-AF680; up to 34 mM NHS-PEO₄-biotin).

1.33 × 10¹³ fd virions (equivalent to 59.7 nmol pVIII subunits) in calcium- and magnesium-free buffer (CMF; 136.9 mM NaCl, 2.68 mM KCl, 8.1 mM Na₂HPO₄, 1.47 mM KH₂PO₄, pH adjusted to 7.2 with HCl or NaOH) were diluted with additional CMF to total volumes ranging 108–639 µL. Then 1/9 volume of 1 M NaH₂PO₄ (pH adjusted to 7.00 with NaOH) and up to 1/50 volume of reagent (either NHS-PEO₄-biotin or sulfo-NHS-AF680) in DMSO were added. The vessels were vortexed immediately. Reactions were allowed to continue overnight (∼12–18 h) at room temperature in the dark; in view of the high reactivity of NHS esters, this was deemed sufficient time for the reactions to go to completion. Meanwhile, in order to determine the actual concentration of sulfo-NHS-AF680 reagent, 2 µL of the same fresh reagent stock solution used for the phage reactions (nominally 17.4 mM reagent in DMSO) were mixed with 120 µL of 10.67 mM lysine in 0.1 M NaH2PO4, pH adjusted to 7.0 with NaOH. After overnight reaction in the dark, the reaction mixture was diluted 160-fold in CMF and its dye content measured spectrophotometrically, assuming a molar extinction coefficient of 184,000 at 678 nm (as reported by the supplier); reactive dye was assumed to constitute 95% of total dye, in accordance with the reagent's certificate of analysis. The sulfo-NHS-AF680 concentrations reported here were based on this determination.