A Novel “Prebinding” Strategy Dramatically Enhances Sortase

Article pubs.acs.org/bc

A Novel “Prebinding” Strategy Dramatically Enhances SortaseMediated Coupling of Proteins to Liposomes John R. Silvius* and Rania Leventis Department of Biochemistry, McGill University, 3655 Promenade Sir-William-Osler, Montréal, QC, Canada H3G 1A9 S Supporting Information *

ABSTRACT: We have examined quantitatively the efficiency and the kinetics of sortase A-mediated coupling of model substrate proteins (derived from green fluorescent protein and the SNAP variant of O-alkylguanine-DNA alkyltransferase) to large unilamellar liposomes incorporating low levels of oligopeptide-modified acceptor lipids. Under normal reaction conditions, even using high concentrations of S. aureus or S. pyogenes sortase A and optimal protein coupling substrates and acceptor lipids, protein−liposome coupling is slow, gives at best modest coupling yields, and is markedly limited by the hydrolytic activity of sortase. We demonstrate, however, that these limitations can be overcome under “prebinding” conditions that promote initial reversible association of sortase and the substrate protein with the liposome surface. Using oligohistidine-tagged sortase and substrate proteins and liposomes incorporating an acceptor lipid together with a Ni(II)-chelating lipid derivative, high coupling rates and yields can be obtained at low sortase concentrations, while virtually eliminating adverse effects of sortase hydrolytic activity on protein coupling. The prebinding approach described here can readily be adapted, and if necessary rendered virtually “traceless”, to accommodate diverse protein coupling substrates and end uses of the protein-modified liposomes.

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INTRODUCTION

S1‐LPXTG‐S2 + H 2N‐G‐Z

Protein coupling to liposomes (and other lipid surfaces) is an important process with a wide variety of applications, both to generate lipid−protein model membranes for in vitro studies and to create protein-bearing liposomes as in vivo delivery vehicles for therapeutic agents.1−4 While various methods are currently available to couple proteins to lipid membranes,5−7 most of these suffer from significant limitations. Traditional protein coupling chemistries are generally not site-specific, while newer approaches that afford site-specific coupling of proteins to surfaces typically require incorporation of noncanonical amino acids or other non-native chemical moieties, or the introduction of large functional domains, into the protein to be coupled.7−9 Sortase-mediated transpeptidation offers a potentially attractive means to couple proteins to liposomes in a manner that is site-specific, imposes minimal constraints on the nature of the protein to be coupled, and requires introduction of only a short coupling sequence into the substrate protein. Class A sortases, widely found in Gram-positive bacteria, physiologically are membrane-anchored proteins that catalyze covalent anchorage of proteins to peptidoglycan in the cell envelope,10−14 but soluble derivatives of these sortases can be utilized to prepare diverse bioconjugates via transpeptidation in vitro.15−18 Sortase A from S. aureus, the most widely used sortase, catalyzes the reversible transpeptidation reaction © XXXX American Chemical Society

↔ S1‐LPXTG‐Z + H 2N‐G‐S2

where S1-LPXTG-S2 is a substrate protein or peptide comprising arbitrary sequences S1 and S2 flanking the -LPXTG- sortase recognition motif, and the nucleophilic acceptor species H2N-G-Z can be any of a wide range of compounds incorporating an amide-linked glycine residue with a free amino group.19−23 This activity of sortase has been exploited to prepare a wide variety of protein−small molecule and protein−protein conjugates.15−18,21−32 While previous studies33−35 have used sortase to generate protein-coupled liposomes, no study to date has characterized quantitatively either the efficiency or the kinetics of this process. In this study we have examined in detail the sortasemediated coupling of proteins to liposomes incorporating oligoglycine-modified lipid acceptors. We demonstrate that sortase-mediated protein coupling to liposomes is normally a slow, relatively inefficient process that is adversely affected by the hydrolytic activity of sortase. We further show, however, that coupling can be greatly accelerated, and made much more efficient and robust, by using conditions that reversibly Received: February 15, 2017 Revised: March 27, 2017 Published: March 30, 2017 A

DOI: 10.1021/acs.bioconjchem.7b00087 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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liposomes (DOPC/POPG, labeled with a trace amount of RhoDOPE), in some cases including small proportions of other lipid components as indicated. Samples were withdrawn from coupling reaction mixtures (combining liposomes, sortase, and substrate protein) at various times, quenched with EDTA and assayed for protein−liposome coupling by gel filtration and fluorescence measurements as described under Experimental Procedures. In preliminary experiments, 18 to 24 h incubations of sortase and GFP-substrate proteins at 37 °C with liposomes incorporating acceptor lipids gave only low efficiencies of GFP-liposome coupling. Subsequent experiments showed that the time courses of these reactions were biphasic both at 37 °C and at 23 °C (Figure 1), with substantial initial coupling of GFP followed by a gradual decline in the level of coupled protein (Figure 1). Changing the concentration of sortase altered the rates of both phases of the reaction but not the peak extent of coupling (Figure 1B, inset). Peak coupling yields were slightly higher at 23 °C than at 37 °C. Biphasic time courses of liposomal coupling were also observed using S. pyogenes sortase A,36 or a mutated S. aureus sortase A variant with augmented, calcium- independent activity37 (Figure S1). No association of GFP with liposomes (100-

incubation conditions just noted, the level of liposome-coupled GFP initially rises to a maximum of ca. 40% but then falls to 60% of input GFPAA-His6 can be coupled to prebinding liposomes using low concentrations of His6SpySrt, while as described earlier (Figure S1), under “conventional” (non-prebinding) conditions, 6 h, suspended by vortexing in 150 mM NaCl/50 mM Tris, pH 7.5 and finally extruded through 0.1 μm polycarbonate filters to prepare large unilamellar liposomes. Liposomes incorporating nickel-free DOD-tri-NTA or NTA-PEG1500-DPPE were charged with Ni2+ by incubating with 5 mM NiSO4 (5 min, 23 °C) in the above buffer, then gelfiltered on Sephadex G-50 to remove unbound Ni2+. For coupling reactions, except where otherwise indicated liposomes (3.5 mM) were incubated with sortase and a protein coupling substrate (10 μM) in 150 mM NaCl, 50 mM Tris, 10 mM CaCl2, pH 7.5 at 23 °C. 150 μL aliquots were withdrawn from reaction mixtures at the indicated times, mixed with 50 μL 0.25 M EDTA, pH 7.5, and applied to columns of Sepharose 4B-CL (0.9 × 13 cm), which were eluted with 150 mM NaCl, 50 mM Tris, 2.5 mM EDTA, pH 7.5 to separate free from liposomecoupled proteins. For coupling reactions including His8- and His10-tagged proteins or liposomes containing DOD-tri-NTA· Ni, sample aliquots were incubated for 10 min after mixing with EDTA and before application to the Sepharose columns to allow complete dissociation of noncovalently bound protein from the vesicles. Protein and liposomal-marker (Rho-DOPE) fluorescence in collected fractions was measured using a PerkinElmer LS-50B luminescence spectrometer, using excitation/emission wavelengths of 490 nm/510 nm (slits 3 nm/5 nm) for GFP, 498 nm/516 nm (slits 5 nm/5 nm) for BG-CFlabeled SNAP, and 525 nm/596 nm (slits 5 nm/5 nm) for RhoDOPE. NiNTA-Agarose Binding Assays. Oligohistidine-tagged GFP or SNAP constructs (2.5 nmol) in 100 μL 150 mM NaCl, 50 mM Tris, pH 7.5 were mixed with 50 μL of a 50% suspension of NiNTA-agarose in the same buffer; the mixtures were gently rocked for 1 h at 23 °C with exclusion of light and then centrifuged (1 min at 18 000g). 100 μL of supernatant was recovered, and the pellet fraction was further washed by three times mixing with 100 μL of buffer, centrifuging, and recovering 100 μL of supernatant. Resin-bound protein was then eluted by mixing the pellet fraction four times with 100 μL of 150 mM NaCl, 50 mM Tris, 500 mM imidazole, pH 7.5, incubating for 5 min, centrifuging, and recovering 100 μL of supernatant. The pooled supernatant fractions from the wash and from the elution steps were diluted to 5 mL in 150 mM NaCl, 25 mM Tris, 2.5 mM EDTA, pH 7.5, and their fluorescence was measured as described above. From these data the percentages of input protein initially bound to NiNTA-agarose were determined, with small corrections for incomplete recovery of supernatant in the wash and elution steps.

product was subcloned into pET28a(+) via its NcoI and BamHI sites to give the plasmid pET28-His 6SNAPGK. Sequences encoding the amino acid sequences of GFP(His6)GG and GFP(His3)GGHis3, respectively, flanked by NcoI and HindIII sites, were prepared by two-stage PCR using pET28a-GFPGG-His6 as template and were subcloned into NcoI/HindIII-digested pET28a(+) to give the plasmids pET28a-GFP(His6)GG and pET28a-GFP(His3)GGHis3, respectively. Protein Preparations. Sortase-based constructs were expressed in E. coli BL21* cells using the autoinduction method of Studier;65 after incubating at 30 °C to OD660 nm = 0.6, cultures were shifted to 22 °C and further incubated for 18−20 h. Sortase constructs were purified as described by TonThat et al.,66 except that the NiNTA-agarose column was washed with 20 volumes of 5 mM imidazole in buffer C (150 mM NaCl, 50 mM Tris, 10% glycerol, pH 7.2) followed by 10 volumes of 20 mM imidazole in the same buffer; sortase was then eluted with 5 volumes of 500 mM imidazole in buffer C, concentrated and freed of imidazole on an Amicon filter, and stored as snap-frozen aliquots in buffer C at −80 °C. GFP derivatives were expressed in E. coli Rosetta 2 cells, cultured in Terrific Broth at 37 °C to OD660 nm = 0.6, supplemented with IPTG to 1 mM and further cultured for 18 h at 22 °C. GFP constructs were purified as described previously,24 but the NiNTA-agarose column was washed successively with 15 volumes each of 10 mM, 20 mM, and 30 mM imidazole in 300 mM NaCl, 100 mM sodium phosphate, 10 mM Tris, pH 8.0, before eluting with 5 volumes of 300 mM imidazole in the same buffer. The eluted protein was concentrated, and the buffer was exchanged for 150 mM NaCl, 20 mM Tris, 0.5 mM EDTA, pH 8.0, on an Amicon filter; the final protein stock was mixed with glycerol to 50% (v/v) and stored at −20 °C. His6SNAPGK was expressed in E. coli BL21* cells, culturing in LB medium to OD660 nm= 0.6 at 37 °C, adding IPTG (1 mM) and further culturing for 18 h at 22 °C. Cells pelleted from 700 mL of culture were resuspended and incubated for 1 h at 0 °C in 40 mL PBS (145 mM NaCl, 4 mM KCl, 15 mM phosphate, pH 7.4) supplemented with 10 mM imidazole, 4 mM βmercaptoethanol, 1 mM PMSF, 5 μg/mL each aprotinin and leupeptin, 2 μg/mL pepstatin, and 1 mg/mL lysozyme, then disrupted by probe sonication at 0 °C and centrifuged at 14 400g for 30 min. The supernatant was passed through a 0.22 μM Millipore filter and loaded at 4 °C onto a 4 mL column of NiNTA-agarose, which was washed successively with 10 volumes each of 10 mM, 20 mM, and 30 mM imidazole in PBS/4 mM β-mercaptoethanol; His6SNAPGK was then eluted with 4 volumes of 300 mM imidazole in the same buffer. The purified protein was concentrated, and the buffer was exchanged for 50 mM HEPES, 1 mM DTT, pH 7.2, on an Amicon filter; glycerol was added to 30% (v/v), and aliquots were snap-frozen and stored at −80 °C. His6SNAPGK was labeled at its active site by incubating with BG-FL (10 μM protein, 15 μM BG-FL in 50 mM HEPES, 1 mM DTT, pH 7.2) for 1 h at 23 °C with exclusion of light; uncoupled label was removed by gel filtration on BioGel P-6, and the labeled protein was concentrated and stored as described for the unlabeled form. Tev Protease Cleavage Reactions. Srt-His6 (14 mg) was incubated with His6-tagged Tev protease (0.28 mg), in 3.5 mL of 100 mM NaCl, 50 mM Tris, 1 mM DTT, pH 7.5 for 5 h at 23 °C. After exchanging the buffer for 150 mM NaCl, 50 mM Tris, 5 mM imidazole, 10% glycerol, pH 7.2 using an Amicon I

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.bioconjchem.7b00087. List of primers used for PCR reactions; Figure S1, time courses of protein coupling to nonprebinding liposomes mediated by S. pyogenes sortase A or calciumindependent S. aureus sortase A; Figure S2, lipid concentration dependence of sortase-mediated protein coupling to nonprebinding liposomes; Figure S3, time course of sortase-mediated coupling of His6SNAP-GK to prebinding liposomes; and 1H NMR spectral data (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel.: 1-(514)-398-7267. FAX: 1-(514)-398-7384. ORCID

John R. Silvius: 0000-0003-4050-509X Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by an Operating Grant (MOP115201) to JRS from the Canadian Institutes of Health Research.

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ABBREVIATIONS DOGS-NTA·Ni, 1,2-dioleoyl-sn-glycero-3-[(N-(5-amino-1carboxypentyl)iminodiacetic acid)succinyl] (nickel salt); DOPC, 1,2-dioleoyl-sn-glycero-3-phosphocholine; DPPE, 1,2dipalmitoyl-sn-glycero-3-phosphoethanolamine; GFP, green fluorescent protein; NiNTA-agarose, nickel(II)-nitrilotriacetic acid-substituted agarose; POPE-PEG2000-OMe, 1,2-dioleoylsn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]; POPG, 1-palmitoyl-2-oleoyl-sn-glycero-phospho-(1′-rac-glycerol); Rho-DOPE, 1,2-dioleoyl-sn-glycero-3phosphoethanolamine-N-(lissamine rhodamine B sulfonyl)

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DOI: 10.1021/acs.bioconjchem.7b00087 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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DOI: 10.1021/acs.bioconjchem.7b00087 Bioconjugate Chem. XXXX, XXX, XXX−XXX