key: cord-0686214-gxn2yx2d
authors: Rahmati-Yamchi, Mohammad; Babaahmadi, Mohammad Karimi; Mohammadi, Seyed Abolghasem; Makvandi, Manoochehr; Mamoueie, Morteza; Wood, David
title: Column-free purification and coating of SpyCatcher protein on ELISA wells generates universal solid support for capturing of SpyTag-fusion protein from the non-purified condition
date: 2020-04-29
journal: Protein Expr Purif
DOI: 10.1016/j.pep.2020.105650
sha: 68e3ed1d39cfee66085edd1e85370f57457bebff
doc_id: 686214
cord_uid: gxn2yx2d

• Spy Tag-Protein covalent interaction is rapid and specific method for protein immobilization. • By utilization of Non-Chromatographic method and SpyCatcher protein it is possible rapidly develop a universal solid support for SpyTag protein purification. • This method is highly simple and applicable to other proteins.

A variety of display technologies, such as Phage or mRNA display, are used for selection of the new binders against wide range of antigens [1] [2] [3] . In Phage display, a DNA library encoding millions of variants of a specific antibody is cloned in fusion to a phage coat protein such that each phage expresses a slightly different specific binder on its surface. The DNA encoding each binder is then contained within each phage, allowing a direct linkage between the DNA sequence and functionality of the binding protein to the target antigen. The selection relies on an affinity enrichment process known as Biopanning [3, 4] . The Biopanning process includes three parts: binding of phage to the target antigen, washing out the non-binding phages, and then elution of binding phage. The target antigens used for Biopanning are typically highly purified recombinant proteins that are immobilized on either beads or solid supports such as ELISA multi-well plates. Solid-support immobilization methods require less target protein and facilitate the selection multiple targets simultaneously. However, in both methods, the quality and yield of the recombinant protein have an essential role in the generation of antibodies [5] . For every new Biopanning selection process ~ 1 mg of purified recombinant protein is required (typically 1-100µg/ml for each well of a microtiter plate). In many laboratory applications, the purification of new target antigen proteins can present a serious bottleneck to developing new binders, since each new target can require an entirely new expression and purification method to be developed.

To circumvent purification steps, several methods have been established. Lim et al have developed a methods denoted as Yin-Yang panning [6] . This method was developed for affinity selection of a specific protein in crude lysate without purification. This procedure was done by saturation of nonbinder antibodies in non-expressed bacterial lysate, with blocking agents in ELISA wells, followed by selection of specific binders from expressed lysates in wells. This method was used for development of MERS-CoV nucleoprotein specific antibody. Although this method is very cost effective, it must be optimized for every target protein. Fierle et al. have used bacterial superglue for specific capture of antigen from crude lysate [7] . This method is based on covalent peptide-protein interaction of SpyTag (SpyT) and SpyCatcher (SpyC) from Streptococcus pyogenes [8] . In practice, the SpyTag is chemically synthesized and conjugated onto beads, while the corresponding SpyCatcher protein is expressed as a fusion partner with several cancer antigens expressed in a recombinant eukaryotic host system. The SpyCatcher fusion protein was specifically captured onto the bead-based solid phase and then used specific-antibody selection. This interaction is efficient and highly specific. However, it is relatively expensive due to chemical synthesis and conjugation of SpyTag.

The aforementioned works have inspired us to develop a very simple and cost-effective method for specific capture of antigen from crude lysate. To achieve this purpose, we have designed an indirect sandwich-like ELISA by non-chromatographic purification of faster variant SpyCatcher002 [9] protein and coating it on an ELISA plate to capture a SpyT002 fusion protein (in this work GFP) from crude lysate. We show that this coated SpyC002 protein has the protential to be used as universal platform for immobilization SpyT002 fusion proteins.

All chemicals were purchased from Sigma Aldrich (St. Louis, MO, USA) unless otherwise stated. All cloning enzymes were purchased from Thermo Fisher Scientific. All oligonucleotides were synthesized by Macrogen (Seoul, South Korea) and synthetic DNA constructs were synthesized by Generay Biotech (Hongkong). All DNA extraction kits such as plasmid, Gel extraction and PCR clean up were purchased from Dena Zist Asia (Mashhad, Iran). For cloning and expression of recombinant proteins TOP10 and BL21(DE3) (RIL) were used respectively. Antibodies were purchased from BioLegend.

Primer sequences used for plasmid construction in this study are available in Table 1 . First, the sequence encoding the RTX tag (BRT 17 ) [10] was synthesized by Generay and then cloned with NcoI and EcoRI into pET32a to generate pET32-RTX. SpyC002 (Addgene ID:102827) and GFP were amplified and cloned by EcoRI and HindIII into pET32-RTX to make pET32-RTX-SpyC002 (Also referred as SpyC) and pET32-RTX-GFP respectively. The SpyT002 encoding plasmid was constructed according following steps: First, SpyT002-MBP was amplified from the pMAL-p5X vector and cloned into pET28(a) to make pET28-SpyT002-MBP. In parallel, RTX tag was cloned into a pMAL-p5X by NcoI and EcoRI (pMALp5X-RTX) and then RTX from pMALp5X-RTX subcloned into pET28-SpyT002MBP by SacI and EcoRI (for retrieve pMAL linker) to make pET28-SpyT002-MBP-RTX. Finally, the GFP DNA sequence was amplified and cloned by EcoRI and HindIII to generate pET28-SpyT002MBP-RTX-GFP (also referred as SpyC).

All protein expression experiments were performed in the E. coli strain BL21(RIL). Cells harboring pET32-based plasmids (pET32-RTX-GFP and pET32-RTX-SpyC002) were cultured in Luria Broth (LB) media supplemented with 100 µg/mL ampicillin and 30 µg/mL chloramphenicol, while pET28based plasmids (pET28-SpyT002-MBP-RTX-GFP) were cultured in LB media supplemented with 50 µg/mL kanamycin and 30 µg/mL chloramphenicol. The expression cells were cultured in 5 mL LB overnight at 37 °C. The cultures were diluted 1:100 (v/v) into 200 ml LB media supplemented with the appropriate antibiotics. The cells were then grown at 37 °C until OD600 reached to 0.6-0.8, at which point 0.5 mM (final concentration) isopropyl β-D-1-thiogalactopyranoside (IPTG) was added for protein expression induction at 18 °C for 20 h.

Cells were harvested by centrifugation at 5000 xg for 10 min at 4 °C. The cell pellets were resuspended in Low Salt Buffer (40 mM Tris-HCL, 200 mM NaCl at pH 8.5). Low Salt Buffer was used for further steps, which include washing and dissolving steps. In each case, the cell pellet was resuspended in 1/20 of original culture volume. The resuspended cultures were then sonicated for 10 cycles of 30 s sonication at a setting of 4-5 W, with 30 s on ice. The resulting lysate was then clarified by centrifugation at 18,000 rpm for 30 min at 4 °C. The supernatant was recovered for purification of the target proteins.

This step was done according to the original Fan et al. procedures [10, 11] . Briefly, calcium chloride was added to the cleared lysate of pET32-RTX-GFP, pET32-RTX-SpyC002 and pET28-SpyT002MBP-RTX-GFP to a final concentration of 25 mM (diluted from a 2 M stock solution). The sample was then mixed by inverting, and then incubated at room temperature for 15 min. The sample was then centrifuged at 16,000 g for 5 min and the supernatant was discarded. The remaining pellet was resuspended in Low Salt Buffer (40 mM Tris-Hcl, 200 mM NaCl at pH 8.5) by a short sonication. The sample was then centrifuged again at 16,000 g for 5 min and supernatant was discarded. the pellet was washed according previous 

The RTX (BRT 17 ) tag sequence (GGAGNDTLY) 9 is derived from the consensus block V RTX domain from the adenylate cyclase toxin (Cya A) of B. pertussis. It reversibly aggregates and becomes insoluble in the presence of low concentrations of calcium (25mM) and remains insoluble until the calcium is removed by EDTA or other strong chelating agent. At this calcium concentration most bacterial proteins remain soluble, making it possible to isolate RTX-fusion proteins from whole lysates by simple centrifugation. By washing the RTX fusion pellet several times, it is possible to achieve pure a protein. Addition of metal chelators such as EDTA/EGTA in equivalent concentration reverts the RTX tag to soluble form. The RTX-based purification is illustrated in Figure 1 . Addition of a solubility enhancer (in this work Trx) enhanced the production yield. For pET32-RTX-SpyC002, ~6.5 mg of pure protein was purified from 200ml of shake-flask culture, where the pellet was washed 5 times. It was noted that after the third round of washing the purity of the protein didn't change, indicating that 3 wash cycles are suitable for protein purification (Figure 2A ). This procedure was repeated for Trx-RTX-GFP ( Figure 2B ) and SpyT002-MBP-RTX-GFP ( Figure 2C) , where 3.3 mg and 1.5 mg of purified protein was achieved respectively. For SpyT002-MBP-RTX-GFP, after every round of washing the pellet size decreased significantly, and for this reason the yield of this protein is lower than the others.

Spy peptide-protein interaction depends on two factors; SpyT:SpyC ratio and their concentrations in the reaction. In general, a 1.5 to 3-fold excess ratio is recommended for one partner, with a 2h reaction time and 10 µM concentration of the limiting protein at room temperature. This time can be increased to 16h for crowded surfaces [13] . To achieve the maximum reaction in short time, the ratio and temperature were increased to a 3.5-fold molar excess and 30 °C respectively. Although higher temperature such as 37 °C accelerates the reaction, it may not suitable for some proteins due to stability concerns. The remaining factor was binding partner concentration. The Coated SpyC proteins were divided into two groups. In one group the concentration of the SpyC was constant at 100 µg/ml (1.5µM). In another group, The SpyC was ½ serially diluted from 100 µg/ml (1.5µM) to 0.78 µg/ml (~10 nM) and then coated onto ELISA wells to determine threshold of reaction at low concentration. [14] . For coating, PBS buffer was used with simple dilution, without need of protein dialysis. Due to the sensitivity of the RTX to calcium, skimmed milk was not used as blocking agent.

As the Spy peptide-protein interaction occurs in a pH range from 4-8 [13] , SpyT fusion proteins were diluted in PBS-T buffer and ½ serially diluted from 500 to 3.9 µg/ml.

To assess the maximum possible signal at different concentrations of reacted SpyT002-GFP fusion protein to the coated SpyC, the purified Trx-RTX-GFP test protein was ½ serially diluted from 100 to 0.78 µg/ml (from A-H) and was used as positive control. The signal of the positive control was approximately 3 (OD 450nm) in all concentrations. At a constant concentration of SpyC (100 µg/ml), the signal of SpyT at concentrations ranging from 500 to 15µg/ml was similar to the positive control. The signal decreased significantly at 7.8 µg/ml (86 nM) and 3.9 µg/ml (43nM) of SpyT concentration. The SpyT lysate signal was somewhat lower due to impurity, but followed pure protein pattern ( Figure 4A ). In serially diluted SpyC, the signal was maximum at 200 nM concentration of SpyC ( Figure 4B ). At lower concentrations the signal decreased to about 40% of positive control at 12nM. According to the result of the SpyT purified and lysate samples, for 100 µg/ml concentration of SpyC, the minimum concentration of SpyT that is required for a high reaction rate in 2h at 30 °C is ~173 nM of SpyT protein (in this work ~15 µg/ml) ( Figure 4C ). However, it is possible to use lower concentrations, but with a slower reaction rate. Serial dilution of SpyC results showed that the minimum concentration of SpyC required for a high reaction rate is between 100 and 200 nM (~6.25-12.5µg/ml) and in this case at least ~350 to 700nM of SpyT (in this work ~31-62.5µg/ml) is required ( Figure 4D ). These results match the original Spy002 article, which reports that the reaction rate in 100 nm SpyC is ~70% [9] .

The Spy tag system has been successfully used for basic research and applied science, such as cell inner/outer localization [15] , enzyme thermal stabilization [16] , enzyme immobilization [17] , vaccine development [18, 19] and protein detection [20] and purification [21] . Since the development of this method in 2012, its applications are expanding along with its potential [22] . In this work we have combined a non-chromatographic method with the spy catcher002 protein to generate a potentially cost-effective universal capture method for immobilization of SpyT-fusion recombinant proteins from unpurified feeds. The RTX-mediated protein purification takes 1 to 2 hours, without any specific equipment. As mentioned above, we produced ~6 mg of purified fusion protein in one example, which is theoretically suitable for the generation of hundreds of ELISA wells. Conversely, the sample size is also small, which subsequently decreases the required culture volume. In addition, this system is sensitive to small amounts of recombinant protein, which is ideal for low-expressing recombinant proteins or in mammalian transient expression systems. However, low concentrations of recombinant protein (100nM for both partners) may show different results due to reaction rate dependence of Spy variant 002 on protein concentration. Recently, variant 003 of this method has been developed [23] . Increased sensitivity of this method to even sub-nanomolar concentrations may be possible by applying Spy003. As both ELISA and Spy tag-protein are compatible with urea, it is possible to use this method with urea to dissolve inclusion bodies and continue process steps with immobilized protein. This method is also helpful for the study of SpyT fusion vaccines and for studies of immunization or in labon chip methods. 

Phage Display in the Quest for New Selective Recognition Elements for Biosensors

Phage display screening of therapeutic peptide for cancer targeting and therapy

Protein engineering approaches for antibody fragments: directed evolution and rational design approaches

Advance in phage display technology for bioanalysis

Methods for Selecting Phage Display Antibody Libraries

Development of a Phage Display Panning Strategy Utilizing Crude Antigens: Isolation of MERS-CoV Nucleoprotein human antibodies

Integrating SpyCatcher/SpyTag covalent fusion technology into phage display workflows for rapid antibody discovery

Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin

Evolving Accelerated Amidation by SpyTag/SpyCatcher to Analyze Membrane Dynamics

A designed, phase changing RTX-based peptide for efficient bioseparations

Column-Free Purification Methods for Recombinant Proteins Using Self-Cleaving Aggregating Tags

Quantitation of Nucleic Acids and Proteins

Insider information on successful covalent protein coupling with help from SpyBank

Catching a SPY: Using the SpyCatcher-SpyTag and Related Systems for Labeling and Localizing Bacterial Proteins

SpyTag/SpyCatcher Cyclization Enhances the Thermostability of Firefly Luciferase

Target-Specific Covalent Immobilization of Enzymes from Cell Lysate on SiO2 Nanoparticles for Biomass Saccharification

A Universal Plug-and-Display Vaccine Carrier Based on HBsAg VLP to Maximize Effective Antibody Response

Surface display of classical swine fever virus E2 glycoprotein on gram-positive enhancer matrix (GEM) particles via the SpyTag/SpyCatcher system

Rapid analysis of protein expression and solubility with the SpyTag-SpyCatcher system

Spy&Go purification of SpyTag-proteins using pseudo-SpyCatcher to access an oligomerization toolbox

Secrets of a covalent interaction for biomaterials and biotechnology: SpyTag and SpyCatcher

Approaching infinite affinity through engineering of peptide-protein interaction

The authors declare no conflicts of interest.

1SpyC002-F-EcoRI

• Spy Tag-Protein covalent interaction is rapid and specific method for protein immobilization. • By utilization of Non-Chromatographic method and SpyCatcher protein it is possible rapidly develop a universal solid support for SpyTag protein purification.• This method is highly simple and applicable to other proteins.