Oriented attachment of VNAR proteins, via site-selective modification, on PLGA–PEG nanoparticles enhances nanoconjugate performance

Conjugation of Variable New Antigen Receptors (VNARs) to PLGA–PEG nanoparticles in a site-selective manner provides superior nanoparticle–protein constructs


SDS-PAGE
Non-reducing glycine-SDS-PAGE at 18% acrylamide were performed following standard lab procedures. A 6% stacking gel was used and a broad-range MW marker (10-250 kDa, Prestained PageRuler Plus Protein Standards, ThermoScientific) was co-run to estimate protein weights. Samples (5 μL at 33 μM) were mixed with loading buffer (2 μL, composition for 5 × SDS: 1 g SDS, 3 mL glycerol, 6 mL 0.5 M Tris buffer pH = 6.8, 2 mg bromophenol blue in 10 mL), heated at 75 °C for 5 mins, and centrifuged at 14000 rcf for 1 min. Samples were subsequently loaded into the wells in a volume of 5 μL. All gels were run at 80 V for 15 mins, then 150 V until complete. Gels were stained using a Coomasie stain.

UV-Vis spectroscopy
UV-Vis spectroscopy was used to determine protein concentrations, using a Varian Cary 100 Bio UV-Visible spectrophotometer operating at 21 °C. Sample buffer was used as blank for baseline correction. Extinction coefficient of V NAR E4 is ε 280 = 24075 M -1 cm -1 .

Determination of Protein Masses by Liquid Chromatography Mass Spectrometry (LC-MS)
LC-MS was performed on protein samples using a Waters Acquity uPLC connected to Waters Acquity Single Quad Detector (SQD). Column: Hypersil Gold C4, 1.9 μm, 2.1 × 50 mm. Wavelength: Nitrogen was used as the nebulizer and desolvation gas at a total flow of 600 L/h. Total mass spectra for protein samples were reconstructed from the ion series using the MaxEnt1 algorithm preinstalled on MassLynx software.

Characterisation of nanoformulations
Nanoparticle size, polydispersity index and zeta potential were analysed using a NanoBrook Omni (Brookhaven Instruments Corp), after resuspension at 200 µg polymer/mL in PBS. A Micro BCA protein assay kit (Thermo Scientific) was used to quantify V NAR loading on each nanoformulation as previously described. 1

Modified enzyme-linked immunosorbent assay (modified ELISA)
Binding of NP to human DLL4 Fc chimera protein (Sino Biological) was evaluated as previously described. 1 For targeting specificity studies, NP were pre-incubated with DLL4 Fc chimera (10 µg/mL) in free format for 30 mins at room temperature, prior to addition to microtiter plates containing the same protein in immobilised format. Alternatively, targeting specificity was assessed through simultaneous addition of NP and a competing anti-DLL4 monoclonal antibody (0-40 µg/mL; R&D systems) to antigen-coated plates.

E4 clone data V NAR Clone E4 ACA version design and synthesis
The Alanine Cysteine Alanine (ACA) insertion in the C-terminal region of V NAR E4 clone was designed in silico using DNASTAR® software. Flanking the V NAR gene sequence NcoI and EcoRI restriction endonuclease sites as well as a poly-histidine (6×histidine) tag were incorporated to facilitate cloning into a prokaryotic expression and IMAC protein purification and immunodetection respectively ( Figure S1). The in-house designed gene ( Figure S1) was synthesised by PCR amplification of the V NAR E4 DNA template using oligonucleotides #249_Forward and #256_Reverse using 2×Phusion High-Fidelity PCR Master Mix (New England Biolabs). Figure S1. A detailed in-silico sequence of clone E4 highlighting the pelB leader sequence, restriction sites, ACA insertion, and poly histidine tag.

Cloning V NAR E4 ACA into pIMS147 and E. coli TG1 Transformation
This was achieved by digestion of the pIMS147 and E4 ACA amplicon with NcoI and EcoRI restriction endonucleases. Digested plasmid and insert DNA were ligated using T4 ligase enzyme (New England Biolabs). Resultant ligated mixture was transformed into electrocompetent E. coli TG1 and plated out on selective growth media 2×TY-AG agar plates. After overnight growth, colonies were selected and grown overnight in 2×TY-AG broth. Plasmid DNA was prepared from overnight cultures using a Qiagen Miniprep DNA kit and samples sent for sequencing. A clone with confirmed correct sequence was selected and used for protein expression.

Soluble V NAR Protein Expression and Purification
1 L of 2×TY-AG growth media was inoculated with a 5 mL overnight culture of V NAR E4. The inoculated culture was grown overnight at 37 °C, 250 rpm, followed by centrifugation at 6000 x g for 10 mins. The resulting cell paste was resuspended in 1 L of TB-Amp media, and grown at 30 °C, 250 rpm for 1 h. Protein expression induction was initiated by adding IPTG to the media at a final concentration of 1 mM and grown for a further 4 h at 30 °C, 250 rpm.
Cells were centrifuged at 6000 × g for 30 mins, and supernatant discarded. The cell pellet was resuspended in 100 mL of osmotic shock buffer (20 % w/v sucrose, 100 mM Tris HCl pH 7.6, 5 mM EDTA). Osmotic shock sample was placed on a rolling platform mixer at 50 rpm, for 15 mins at room temp. Finally, 100 mL of 5 mM MgCl 2 was added to the sample and mixing continued for a further 15 mins.
Sample was centrifuged at 15000 × g for 30 mins, 4 °C and supernatant was collected. A 1/10 volume of 10×PBS was added to the resulting supernatant.
IMAC purification of expressed his-tagged protein was performed by addition of 3 mL of Ni 2+ charged IMAC resin to the supernatant then the resultant suspension mixed for 1 h on a roller mixing platform at 50 rpm. Post batch binding, the IMAC resin was collected in a column and unbound proteins passed through the column under gravity. Nickel resin was washed with 50 mL of 1×PBS containing 10 mM Imidazole. Bound protein was eluted with 10 mL of 250 mM Imidazole, pH 8.
The batch IMAC purified protein was subject to a second round of purification using Anion Exchange chromatography. The eluted sample was buffer exchanged to 30 mM Tris HCl, pH 9.5, and loaded onto the AKTA Prime Chromatography system with a gradient elution using 20 column volumes of 1 M NaCl 30 mM Tris HCl pH 9.5. Analysis of protein samples across the eluted peaks by SDS PAGE confirmed fractions contained E4 ACA protein. Fractions were then polled, and buffer exchanged to 1×PBS, pH 7.4. Protein concentration was determined using the A280 spectrophotometer.

3.
Terrific Broth (TB)-Amp: In 900 L de-ionised water, dissolve 12 g and 24 g of Tryptone and Yeast extract respectively. Add 4 mL of glycerol, then autoclave. To autoclaved media, add 100 mL of sterile Phosphate salts (In 500 mL de-ionised water, dissolve 11.55 g and 62.7 g of
Data expressed as mean ± SEM.