Tag-specific Affinity Purification of Recombinant Proteins by us-ing

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Tag-specific Affinity Purification of Recombinant Proteins by us-ing Molecularly Imprinted Polymers Lidia N Gómez-Arribas, Javier Lucas Urraca, Elena Benito-Peña, and Maria C. Moreno-Bondi Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b05731 • Publication Date (Web): 21 Feb 2019 Downloaded from http://pubs.acs.org on February 21, 2019

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Analytical Chemistry

Tag-specific Affinity Purification of Recombinant Proteins by using Molecularly Imprinted Polymers Lidia N. Gómez-Arribas, Dr. Javier L. Urraca*, Dr. Elena Benito-Peña*, Prof. María C. MorenoBondi* Chemical Optosensors and Applied Photochemistry Group (GSOLFA), Department of Analytical Chemistry, Faculty of Chemistry, Complutense University of Madrid, 28040 Madrid, Spain ABSTRACT: Epitope tagging is widely used to fuse a known epitope to proteins for which no affinity receptor is available by using recombinant DNA technology. One example is FLAG epitope (DYKDDDDK), which provides better purity and recoveries than the favourite poly-histidine tag. However, purification requires using anti-FLAG antibody resins, the high cost and non-reusability of which restrict widespread use. One cost-effective solution is provided by the use of bioinspired anti-FLAG molecularly imprinted polymers (MIPs). This work describes the development of MIPs, based on the epitope approach, synthesized from the tetrapeptide DYKD as template that affords purification of FLAG-derived recombinant proteins. Polymer was optimized by using a combinatorial approach to select the functional monomer(s) and cross-linker(s) resulting in the best specific affinity towards FLAG and the peptide DYKD. The imprinted resin obtained was used to purify mCherry proteins tagged with either FLAG or DYKD epitopes from crude cell lysates. Both mCherry variants were highly efficiently purified (R ≥ 95%, RSD ≤ 15%, n = 3) and impurities were removed. Unlike existing antibody-based resins, the proposed tag-imprinting strategy provides a general method for meeting the growing demand for efficient, inexpensive, versatile materials for tagged proteins purification.

Recombinant DNA technology is one of the most powerful techniques among those which have supported salient advances in life science and industrial research.1 Thus, the production of therapeutic protein-based vaccines, industrial enzymes and new biomaterials has benefited from advances in the expression of recombinant proteins in various systems including bacteria, yeasts and mammalian cells. However, this technique is rather expensive to implement owing to the high cost of downstream processing, which includes isolation and purification of proteins. One effective alternative is provided by genetic coding of proteins with universal epitope tags, which enables selective extraction by use of tag-specific affinity resins, tag-dependent aggregation or precipitation reactions.2 In fact, epitope tags are also useful tools for structural and functional proteomics initiatives.2 No universal tag for the expression of proteins in any host has so far been identified; however, one of the most versatile and cost-effective protein purification methods uses poly-histidine (His-) tags.3 The capture step relies on the affinity of four or more histidine residues for immobilized nickel ion via coordinated bonds. This approach, however, is subject to a number of limitations, such as co-purification of proteins with external HIS residues, metal leaching from the sorbent or interference with proper target protein folding and activity,3,4 that can be partially circumvented by replacing the His-tag with a smaller epitope tag such as FLAG, c-Myc or hemagglutinin (HA). These alternative tags typically consist of 8–12 amino acids and allow the simple, expeditious purification of recombinant proteins by using immunoaffinity resins, all with a high yield and purity. 3,5

FLAG-tag, which is a DYKDDDDK peptide, provides superior purity and recoveries of fused proteins by virtue of its being more hydrophilic than other common epitope tags and thus less likely to denature or inactivate the proteins to which it is fused.3 FLAG-tag can be easily purified by using immunoaffinity resins functionalized with anti-FLAG tag M1 or M2 monoclonal antibodies.6 However, the high cost of these sorbents, and their limited reusability, have so far hindered widespread use. One alternative strategy is provided by Molecularly Imprinted Polymers (MIPs). These synthetic materials have emerged as effective substitutes for biological recognition elements such as antibodies and enzymes. MIPs contain well-defined molecularly engineered receptor sites that enable the selective binding of target molecules even in complex mixtures.7,8 These polymers are intrinsically more stable and robust than its natural counterparts and, in principle, can be reused several times without altering its selective recognition properties —which results in decreased costs.9 By analogy with the recognition of antigens by the immune system, where a small part of the entire molecule may by itself be responsible for the whole interaction, epitope imprinting10-20 is the most viable alternative to well-established antibody-based resins in terms of affinity and capacity. In the proposed approach, a small peptide fragment (the epitope) rather than the whole protein is templated and subsequently added to a protein by using recombinant DNA technology. To date, the His-tag label has only been used for protein purification with MIPs21 (see Section S1, Table S1, Supporting Information).

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Monomers

Page 2 of 8

Cross-linkers

HEMA

1-ALPP

TFMAA

2-DAEM

Epitope templates

MBA

TRIM

PEGDMA

D

Y

K D

D

D

D

K

DYKD tag EAMA

MAA

DVB

Combinatorial MIP library

EDMA

FLAG® tag

EDMA

Crude cell lysis extract

× 40

EAMA

D

Y

(2)

K D

(1) Polymerization

Removal of silica beads

(4)

(3) Removal of template ABDV

Prepolymerization mixture

Selective binding of tagged proteins Silica beads

Figure 1. Workflow of the production of DYKD and FLAG tag-imprinted resins, and purification of tagged mCherryFLAG by MISPE. Herein we report the first molecularly imprinted polymer based on the epitope approach and synthesized from the tetrapeptide DYKD as template that affords purification of FLAG-derived recombinant proteins. Using a combinatorial screening design for polymer synthesis allowed the best template, functional monomer(s) and cross-linker(s) for the formulation to be identified. The optimum composition was used to prepare microparticles from porous silica beads acting as sacrificial scaffolds in the polymerization process.22 For the proof of concept study, two different N-terminus tagged fluorescence proteins (mCherry-DYKD and mCherry-FLAG) were designed and expressed in E. coli cells in order to assess the ability of the resulting MIP as an immune-like affinity resin for purifying crude cell extracts of both mCherry variants by using molecularly imprinted solid-phase extraction (MISPE).

EXPERIMENTAL SECTION Combinatorial MIP library. Silica microspheres (SiliaSphere™ PC, 40–75 µm diameter, 500 Å pore size, SiliCycle, www.silicycle.com) were used as sacrificial scaffolds for polymerization. Methacrylic acid (MAA, Sigma-Aldrich), 2(trifluoromethyl)acrylic acid (TFMAA, Sigma-Aldrich), 1-allyl piperazine (1-ALLP, Sigma-Aldrich), N,N-diethylaminoethylmethacrylate (2-DAEM, Sigma-Aldrich), N-(2-aminoethyl)methacrylamide hydrochloride (EAMA·HCl, Polysciences) 2-hydroxyethylmethacrylate (HEMA, Acros) were used as functional monomers. Ethyleneglycol dimethacrylate (EDMA), trimethylolpropane dimethacrylate (TRIM), divinylbenzene (DVB), N,N-methylenebisacrylamide (MBA) and poly(ethyleneglycol) dimethacrylate (PEGDMA) were purchased from Sigma-Aldrich and used as cross-linkers. Dimethylformamide (DMF, GPR Rectapur) was used as porogen. The initiator, 2-2′-azo-bis-(2,4-dimethylvaleronitrile) (ABDV), was obtained from Wako Pure Chemicals, Ltd. The

structures of the monomers and cross-linkers are shown in figure S1. The MIP/NIP libraries were developed by using the compositions shown in Tables S2 and S3 in the Supporting Information. The peptide DYKD (Aspartic acid-Tyrosine-LysineAspartic acid from Peptide Sciences) was used as template. The volume of porogen (DMF) used in each polymer synthesis was that need for a VDMF/(VDMF + Vtotal monomers) ratio of ca. 0.57. The pre-polymerization solutions were transferred to 27 mL borosilicate glass vials containing silica microspheres that were used as sacrificial scaffolds to synthesize the polymers. The mixture was mixed under stirring until the silica beads were freely flowing and then purged with argon for 10 min. Copolymerization was carried out at 70 0C for 24 h. The silica was dissolved by adding 3 x 150 mL of a 3 M aqueous solution of ammonium hydrogen difluoride (NH4HF2, Alfa Aesar) and shaking the mixture for 24 h after each addition . Non-imprinted polymers (NIPs) were prepared under identical conditions but using no template. The polymers were thoroughly washed to pH ~ 7 with water on a shaking table, and then with 1 L of a methanol (MeOH, Fisher Scientific)/trifluoroacetic acid (TFA, HPLC grade Alfa Aesar) (1%, v/v) mixture and 0.5 L of methanol. The resulting polymer beads were dried in a vacuum oven at 50 0C for 24 h prior characterization and use. Then, the powders were allowed to settle in methanol/water (80:20, v/v) to remove fine particles. The peptide DYKDDDDK (FLAG, Peptide Sciences) and aspartic acid (D, Acros Organic) were used as alternative templates for MIP synthesis (Table S3, polymers MSP9 and MSP10, respectively), using EAMA as functional monomer and EDMA as cross-linker.

Molecularly imprinted solid-phase extraction (MISPE) for peptide analysis. Solid-phase extraction cartridges (1 mL, Varian, Palo Alto, CA, USA) were packed with 25 mg of


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Analytical Chemistry MIP or the corresponding NIP. The cartridges were equilibrated with 10 mL of MeOH and 10 mL of HEPES buffer (100 mM, pH 7.5). The peptide solution, containing 1 mL of DYKD and 5 mg L–1 FLAG, was percolated at a constant flow rate of 0.4 mL min–1 with the aid of an 8-channel Ismatec ISM936D peristaltic pump (Oak Harbor, WA, US). Then, the cartridges were rinsed with 3 mL of water to remove non-specifically retained compounds and the peptides were eluted with 1 mL H2O/tetrabutylammonium sulphate (TBA, Sigma-Aldrich) 1% (w/v). The eluates from the MISPE column were injected into the RRLC system for analysis. All analyses were done in triplicate. The imprinting factor (IF) for each peptide was calculated as the ratio of its recovery from the MIP (MSP8) to that from the NIP (NSP8) as described in S8 of the Supplemental Information.

Construction and expression of mCherry Fusion Proteins. For the expression of mCherry-tagged DYKD and mCherry-tagged FLAG, plasmids pQE-T7-2-(mCherry-DYKD) and pQE-T7-2-(mCherry-FLAG) were constructed. The mCherry gene was polymerase chain reaction (PCR)-amplified from vector pmCherry by KOD Xtreme Hot Start DNA Polymerase (Merck Millipore) with oligonucleotides depicted in Table S5. The sense primers FP-mCherry -SacI-M-DYKD (5´GCG AGCTCATGG ATT ACA AGG ATG TGA GCA AGG GCG AGG AGG AT-3´) and FP-mCherry -SacI-M- FLAG (5´- GCG AGC TCA TGG ATT ACA AGG ATG ACG ACG ATA AGG TGA GCA AGG GCG AGG AGG AT -3´) contained a 5´-overhang composed of the DNA sequence encoding for either DYKD or FLAG tags (underlined) to generate the translational fusions DYKD-mCherry and FLAG-mCherry. The PCR products were subcloned at the SacI and KpnI sites of the pQE-T7-2 to generate pQE-T7-2-(mCherry-DYKD) and pQE-T7-2-(mCherryFLAG). Successful cloning was confirmed by DNA sequence analysis. For the overexpression of the mCherry tagged proteins, E. coli One Shot BL21 Star (DE3) cells were transformed with the corresponding plasmids pQE-T7-2-(mCherry-DYKD) and pQET7-2-(mCherry-FLAG) as described elsewhere.23 The mixtures were plated on LB agar plates, supplemented with a 50 mg L–1 kanamycin and incubated at 37 °C for 20 h. Then, manually screened single colonies were grown in LB agar plates containing 15 µL of spread isopropyl β-D-1-thiogalactopyranoside (IPTG) at 37 °C for 20 h to induce protein expression. Selected single colonies were inoculated in TB supplemented with 50 mg L–1 kanamycin at 37 °C under stirring at 230 rpm for 16 h to an optical density at 600 nm (OD600) ranging 0.6–1. Then, IPTG was added to the culture to a final concentration of 1 mM in order to induce protein expression, and cell growth was continued for 4 h. Finally, transformed cells were harvested by centrifugation at 5000 rpm at 4 °C for 10 min, resuspended in lysis buffer (5 mL g–1 cell pellet) and incubated on a rotating mixer at room temperature for 1 h. Insoluble cell debris was removed by centrifugation at 11 000 rpm at 4 °C for 15 min. Lysis extracts were filtered through SPE frits (20 μm pore size, Agilent) and stored at –80 °C until use.

(NSP8) and equilibrated with 10 mL of Tris buffer (20 mM, pH 7.5) (TRIZMA hydrochloride, Sigma Aldrich). Then, a volume of 500 μL of sample (50 μL of cell lysate diluted to 500 μL with Tris buffer, 20 mM, pH 7.5) was percolated through each cartridge at a flow rate of 0.1 mL min–1 with the aid of a 8-channel Ismatec ISM936D peristaltic pump (Oak Harbor, WA, US). The cartridges were rinsed with 3 mL of water to remove nonspecifically retained proteins. Finally, bound proteins were eluted with 3 × 0.5 mL of glycine buffer (0.1 M, pH 3) (Scharlau) and immediately neutralized with 20 μL of Tris buffer (1 M, pH 9) (BioRad). The cartridges were then thoroughly washed with glycine–HCl buffer (0.1 M, pH 3) and water before a new analysis. Eluate samples from the MISPE column were transferred to the wells of 96-microtiter plates (Greiner) and directly measured with a CLARIOstar microplate reader from BMG LabTech (Ortenberg, Germany). Fluorescence signals were recorded at the typical wavelengths for mCherry (exc = 587 nm, em = 610 nm). For quantification in terms of recovery (%), the emission intensity of eluted sample was compared with the signal for the mixture of 50 μL of cell lysate diluted with 450 μL of glycine buffer (0.1 M, pH 3) and neutralized with 20 μL Tris buffer (1 M, pH 9).

RESULTS AND DISCUSSION Preparation of a combinatorial MIP library. In order to ensure a high epitope affinity in the resulting MIPs for FLAG and DYKD tag rebinding, we considered mimicking the pocket of the anti-FLAG M2 monoclonal antibody, which has proved specific for the first four amino acids in the FLAG epitope (i.e., DYKD).24–26 From the molecular imprinting perspective, the motifs of both FLAG and the DYKD (Scheme 1) meet the essential requirements for an ideal template, namely: stability, compatibility with radical polymerization27 and presence of charged basic and acidic groups capable of engaging in multiple ionic interactions with a broad palette of functional monomers.28 A combinatorial library was designed by screening functional monomers of different polarity in order to ensure the best possible MIP composition. The candidates included the acidic monomers methacrylic acid (MAA), trifluormethacrylic acid (TFMAA), N-(2-aminoethyl)methacrylamide hydrochloride (EAMA), the neutral monomer 2-hydroxyethylmethacrylate (HEMA) and the basic monomers 1-allylpiperazine (1-ALLP) and 2-diethylaminoethylmethacrylate (2-DAEM) in combination with the cross-linkers trimethylolpropane dimethacrylate (TRIM), ethyleneglycol dimethacrylate (EDMA), divinylbenzene (DVB), N,N-methylenebisacrylamide (MBA) and poly(ethyleneglycol) dimethacrylate (PEGDMA). Polymerization was accomplished by using ABDV as initiator and dimethylformamide (DMF) as porogen. Non-imprinted polymers (NIPs) were also prepared under identical conditions except that no template was used. Figure 1 shows the ingredients of the synthetic process and Table 1 the composition of the combinatorial library (for details, see Section 2, Figure S1, and Tables S2 and S3, Supporting Information).

More details on the construction and expression of mCherry fusion proteins are provided in the Supporting Information.

Molecularly imprinted solid-phase extraction (MISPE) for mCherry Fusion Protein purification. Solid-phase ex-

Table 1. Composition of the combinatorial MIP library.

traction cartridges (1 mL, Varian, Palo Alto, CA, USA) were packed with 25 mg of MIP (MSP8) or the corresponding NIP


Analytical Chemistry Functional monomers (FM)

Crosslinker (CL)

T:FM:CL

MSP1

DYKD

MAA/ TFMAA

TRIM

0.5:2:2:30

MSP2

DYKD

MAA/ TFMAA

DVB

0.5:2:2:30

MSP3

DYKD

MAA/ TFMAA

PEGDMA

0.5:2:2:30

MSP4

DYKD

MAA/ TFMAA

MBA

0.5:2:2:30

MSP5

DYKD

MAA/ TFMAA/ HEMA

EDMA

0.5:2:2:4:30

MSP6

DYKD

1-ALLP

EDMA

0.5:4:20

MSP7

DYKD

2-DAEM

EDMA

0.5:4:20

MSP8

DYKD

EAMA

EDMA

0.5:3:20

MSP9

FLAG

EAMA

EDMA

0.5:3:20

MSP10

D

EAMA

EDMA

0.5:3:20

Using an appropriate template molecule in MIP syntheses is crucial. This led us to examine the influence of the template used in the imprinting reaction on peptide capture in MIPs prepared with the templates DYKD (MSP8), FLAG (MSP9) and aspartic acid (MSP10). Extractions were performed as described above and washing with water used before elution to minimize non-specific interactions between the peptides and imprinted materials. As can be seen from Figures 3c and 3d, recovery of the target epitopes from the polymers based on aspartic acid (MSP10) were negligible (RMSP10-DYKD = 3%, RSD  2 %) or very low (RMSP10-FLAG = 30%, RSD 1%), and so was that from the NIP (RNSP8-DYKD = 5%, RSD  1%). Interestingly, the polymers obtained with FLAG as template (MSP9) exhibited lower affinity for DYKD (RMSP9-DYKD < 25%, RSD  2%) than those synthesized with the shorter peptide (RMSP8-DYKD = 64%, RSD  7%). This result can be ascribed to DYKD not fitting accurately in the larger binding sites produced by the FLAG motif.30 In contrast, the polymers prepared with DYKD (MSP8) or FLAG (MSP9) as template exhibited extraction recoveries exceeding 89% for the FLAG tag (RMSP8-FLAG= 89%, RSD  6%; RMSP9-FLAG= 94%, RSD  6%) and minimal adsorption in the NIP (RNSP8-FLAG= 27%, RSD  4%). a) 100

b) 100

80

80

Recovery / %

Silica microbeads 40–75 m in size were used as sacrificial scaffolds for the polymerization reaction in order to improve mass transfer of the recombinant proteins to the MIP recognition sites.29 Organic polymer microspheres were only obtained for the MIPs prepared from MAA+TFMAA as functional monomers and TRIM (MSP1) or PEGDMA (MSP3) as cross-linkers —or EAMA as functional monomer and EDMA as cross-linker (MSP8). The scanning electron micrographs of MSP8 microparticles in Figure 2 confirm the porosity and structural fidelity of the resulting materials.

 2%; RMSP8-FLAG = 92%, RSD  2%). However, differences between the MIP and NIP were only significant with the DYKD peptide (RNSP8-DYKD = 16.5%, RSD  5%; RNSP8-FLAG = 87.5%, RSD  2%), possibly because of the hydrophobicity of the FLAG tag (log PFLAG = 6.47) relative to the DYKD peptide (log PDYKD = – 2.22) favouring non-specific interactions with the polymer matrix.

Figure 2. Scanning electron micrographs of MIPs obtained as spherical polymers after silica etching. The micrographs correspond to MSP8, which was obtained by using DYKD, EAMA and EDMA in a 0.5:3:20 mole proportion.

Evaluation of the synthetized MIP/NIP polymers by MISPE. The retention performance of DYKD and FLAG peptides in imprinted microspheres was assessed by packing an amount of 25 mg of MIP or NIP in a 1 mL gravity flow cartridge. All cartridges were conditioned with HEPES buffer (100 mM, pH 7.5) and then loaded with 1 mL of an aqueous solution containing 5 mg L–1 DYKD and FLAG in the buffer. Peptides were eluted with 1 mL of 1% (w/v) TBA to disrupt electrostatic interactions between the polymer and peptides by ion-pairing. Eluted fractions were analysed by HPLC-DAD at abs = 220 nm (Section 3 and 7, Supporting Information). Figure 3a shows the recoveries of the two peptides. As can be seen, MSP1 and MSP3 exhibited weak affinity towards the peptides, with retention < 20% (RSD  5%). MSP8 microparticles (Figure 3b) exhibited effective re-binding of both peptides (RMSP8-DYKD = 65.5%, RSD

60 40

20 0

FLAG DYKD

Recovery / %

Template (T)

60 40 20

d) 100

80

80

60 40 20 0

Wash 0 mL Wash 1 mL Wash 3 mL

FLAG DYKD

0

c) 100

Recovery / %

Polymer

Recovery / %

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 8

60 40 20 0

Wash 0 mL Wash 1 mL Wash 3 mL

Figure 3. Extraction recoveries (%) from MIPs (MSP1, MSP3, MSP8) and NIPs (NSP1, NSP3, NSP8) of variable composition for DYKD and FLAG (a,b) and from MIPs (MSP8,9,10) and the corresponding NIP (NSP8), after percolation of 1 mL of 5 mg L–1 DYKD (c) or FLAG(d). These results are consistent with those reported by Minoura31 and Sellergren32 according to which, polymers imprinted with a short peptide efficiently recognize both the template itself and a larger peptide. Uptake and selectivity were greatest with MSP8-imprinted polymer microspheres for both tags; thus, the imprinting factor (IF) was 12.8 for DYKD and 3.3 for FLAG (see Section 8, Supporting Information). The promising results obtained encouraged us to check whether MSP8 was in


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fact a suitable resin for improved downstream purification of FLAG and DYKD tagged proteins captured from E.coli cell lysis extracts.

Construction of the mCherry-tagged proteins. We used the synthesis of a genetically modified fluorescent protein labelled with either FLAG or DYKD tag as a benchmark method. For this purpose, we selected mCherry, a monomeric far-red fluorescent protein that expresses itself efficiently in E. coli; also, it is non-toxic to the bacterial system and possesses a high photostability.33 Both epitopes were fused to the N-terminus instead of a C-terminal in order to ensure efficient translation initiation and facilitate tag removal. 34 Two different vectors were constructed by using plasmid pmCherry as DNA template and plasmid pQE-T7-2 as host for the final plasmid constructions [pQE-T7-2-(mCherry-DYKD) and pQE-T7-2-(mCherry-FLAG), respectively]. The bacterial strains and constructed primers are shown in Tables S4 and S5 (see Sections S4 and S5, Supporting Information). The recombinant process workflow is illustrated in Scheme 1.

Scheme 1. Simplified scheme of recombinant plasmid pQE-T7-2-mCherry-tag construction (tag = DYKD or FLAG, respectively).

Kan

mCherry

pQE-T7-2

Digestion: KpnI SacI NotI

Kan

pmCherry

Amp

a) PCR: FP-mCherry-SacI-M-tag RP-mCherry-KpnI-ST b) Digestion: KpnI SacI

pQE-T7-2

mCherry

mCherry-FLAG and 27.2 kDa for mCherry-DYKD proteins, Table S7) and the experimental SDS-PAGE values was observed. However, this is a frequent occurrence and has been ascribed to aberrant migration of some proteins during the electrophoretic process.37 Fluorescent mCherry proteins were easily detected by measuring fluorescence emission under ambient conditions. Figures S3b and S3c (Section S9, Supporting Information) show an extract of the lysis of each protein and the corresponding emission spectrum. As can be seen, no difference in fluorescence profile between the proteins was observed irrespective of the particular N-terminus tag.38 The amount of mCherry-tagged protein present in each lysis extract was quantified by native PAGE electrophoresis under the conditions described in Section S9 (Supporting Information). Fluorescence bands were directly viewed (Figure S4) and manually excised to determine the protein content with the bicinchoninic acid assay (BCA). The concentrations of mCherryDYKD and mCherry-FLAG in the corresponding cell lysis extracts were 0.94 and 0.50 mg mL–1, respectively.

Purification of the mCherry-tagged proteins by MISPE. The crude cell lysis extracts (1/10, v/v) of BL21 E. coli cells transformed with either mCherry-DYKD or mCherryFLAG were processed in MSP8 and NSP8 cartridges previously equilibrated with binding buffer. After washing with 3 mL of water, the mCherry variants were eluted with 3 × 0.5 mL of eluent buffer (EB) (viz., 0.1 M glycine–HCl buffer, pH 3), which is commonly used to purify FLAG-tagged proteins.39 The cartridges were thoroughly washed with EB and water for further reuse. Eluates were immediately neutralized with 20 μL of Tris buffer (1 M, pH 9), transferred to 96-well plates and measured with a microplate reader (exc = 587 nm; em = 610 nm). Robust recoveries (%) were obtained (> 4 cycles) before harsher cleaning was required.32 For quantification in terms of recovery (%), the emission intensity of each eluate was compared with that of an extract of the corresponding cell lysate (50 μL) that was diluted with 450 μL of EB and neutralized with 20 μL of Tris buffer (1 M, pH 9).

a)

pQE-T7-2mCherrytag

E. coli extract

Lysis of E.Coli for the purification of mCherry-FLAG and mCherry-DYKD

For overexpression of the mCherry variants, the plasmids pQE-T7-2-(mCherry-DYKD) and pQE-T7-2-(mCherry-FLAG) were transformed into E. coli One Shot cells.35,36 Experimental details on the molecular cloning and protein expression are described in Sections 5 and 6 of the Supporting Information. The target proteins were identified by using SDS-PAGE electrophoresis to compare induced and non-induced cell cultures (Figure S3a, Section S9, Supporting Information). A mismatch between the theoretical molecular weight values (27.7 kDa for

Recovery / %

Transfection of E. coli BL21 with pQE-T7-2-mCherry-FLAG pQE-T7-2-mCherry-DYKD

b) 100

DNA ligase T4

Recovery / %

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry

80 60

40 20 0

1st elution 2nd elution 3rd elution

100

80 60 40

20 0

1st elution 2nd elution 3rd elution

Figure 4. Recoveries (%) obtained after each elution by percolating 500 μL of diluted lysis extract of mCherryDYKD (a) or mCherry-FLAG (b) (1:10, v/v) and following the whole MISPE process. Figures 4a and 4b show the extraction recoveries obtained for the mCherry-DYKD and mCherry-FLAG lysate extracts, respectively. Total recoveries of up to 95% (RSD < 15%) were obtained for mCherry-DYKD in MSP8 (n = 4, 25 mg MIP); by contrast, recoveries from NSP8 were negligible (R = 1.5%, RSD < 13%). The results were similar for mCherry-FLAG (n = 3, 25 mg MIP), with total recoveries of 103% (RSD < 9%) in MSP8 and 1.5% (RSD < 8%) in NSP8. Whereas most mCherry-DYKD was recovered in the first elution with 0.5 mL of EB, extracting mCherry-FLAG required using larger volumes (2 × 0.5 mL) of



disrupting solvent. As also previously found by Schwark et al.,32 MISPE purification not only afforded high, specific capture, but also facilitated gentle, quantitative release of the protein in its functional form. a) Marker (kDa)

b)

1

2

3

4

5

Marker (kDa) 96

96

1

2 3

4 5

66

66 48 44

48 44

32

32 26

26

*

*

18.5

18.5

c)

1 2 3

4 5

Figure 5. SDS-PAGE electrophoresis gel of a diluted cell lysis extract from mCherry-DYKD (a), mCherry-FLAG (b) and Western blot analysis of mCherry-FLAG (c): before MISPE (lane 1), washing solution (MISPE lane 2, NISPE lane 4) and elution solution (MISPE lane 3, NISPE lane 5). Purified mCherry-tagged proteins are denoted by an arrowhead (►) and coeluted proteins (superoxide dismutase [Fe]) with an asterisk. SDS-PAGE confirmed that MISPE enabled the selective extraction of the tagged proteins from cell lysis extracts. Figures 5a and 5b (lanes 1) show the profile of the diluted lysate. MISPE provided substantial sample clean-up. Analysis of the elution solutions confirmed the presence of either mCherryDYKD (Figure 5a, lane 3) or mCherry-FLAG (Figure 5b, lane 3) depending on the particular lysate. No signals for the eluates from NSP8 (Figure 5a and 5b, lanes 5) were observed, however. These results confirm that the MIP allowed the interfering matrix components to be removed during the loading and washing steps without compromising retention of the target protein. Also, both proteins were actively retained by the MIP but not by the NIP, which testifies to the usefulness of the epitope imprinting approach for the selective extraction of proteins labelled with DYKD or FLAG tags. The mCherryFLAG cell lysates were also purified using ANTI-FLAG M2 affinity gel (Section 10, Supporting Information), which provided much lower recoveries (R = 58%, RSD < 12%) than with the MIP column. The binding capacity (Q, mg protein g–1 polymer) of the imprinted polymers for both mCherry-tagged proteins was estimated with the BCA assay (Section 10, Supporting Information) and found to be 1.78 mg g–1 for mCherryDYKD and 1.03 mg g–1 for mCherry-FLAG. A Western blot test (see Figure 5c) was carried out with anti-FLAG M2 monoclonal antibodies to confirm the identity of mCherry-FLAG in the different solutions. The analysis of the lysate (lane 1), and of the washing and eluted fractions from the MIP (lanes 2 and 3) and NIP cartridges (lanes 4 and 5), confirmed the presence of mCherry-FLAG in the cell lysis extract and MIP eluate only. The fractions eluted from the MIP column assessed with SDSPAGE gels (Figure 5a, Lane 3 and Figure 5b, Lane 3) were analysed by MALDI-TOF/TOF mass spectrometry following “in-

gel” trypsin digestion. The MASCOT search details are described in Section S10 of the Supporting Information (Figures S6 and S7). Peptide Mass Fingerprint (PMF) analysis of the mCherry-tagged protein bands revealed a total of 25 peptides matched for mCherry-DYKD protein (MASCOT score 203; sequence coverage 85%). Peptide LSFPEGFKWER was confirmed with high confidence (p < 0.05; ion score = 76) by MS/MS analysis. A total of 28 peptides were matched in mCherry-FLAG protein (MASCOT score = 302; sequence coverage = 73%). Peptides LSFPEGFKWER (ion score = 62) and GEEDNMAIIKEFMR (methionine oxidation, ion score = 80) were confirmed with a high confidence by MS/MS analysis. These analyses unraveled the identity of the lower bands coeluting with the mCherry-tagged proteins (denoted by asterisks in Figures 5a and 5b, Lanes 3). An E. coli protein known as superoxide dismutase [Fe] (SODF_ECO57, UniProt accession number P0AGD5, 21.3 kDa) was obtained in both cases and potential protein degradation discarded. Also, PMF analysis allowed a sequence coverage of 94% to be calculated. Selected peptides were subjected to MS/MS analysis and confirmed as described in Section 10, Figures S8 and S9 (Supporting Information). The presence of this protein was also observed with the ANTI-FLAG M2 affinity gel (Section 10, Supporting Information). In fact, it was described previously by Britton et al.,40 who found co-elution of an E.coli protein together with mCherry but did not attempt identification.

CONCLUSIONS In conclusion, a novel MIP for the highly selective purification of FLAG or DYKD tagged recombinant proteins based on the epitope imprinting approach was developed. Using MIPs instead of immunoaffinity columns3 considerably reduces assay costs while increasing the recovery yields and purity. Also, our results show that the joint use of MIPs and a new fusion tag (DYKD) provides a cost-effective protein purification method requiring no large fusion tags. This antibody-like material fills the existing gap between polymers and antibodies.

ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge on the ACS Publications website at DOI:. Experimental section includes: tables for combinatorial MIP library synthesis, chromatographic conditions for peptide analysis and procedures of the molecular cloning and expression of mCherry-tagged proteins. Results section includes purification, quantification and identification of mCherry variants by gel electrophoresis (native and SDS-PAGE), Western Blot (ECL-WB) and MALDI-TOF mass spectrometry.

AUTHOR INFORMATION Corresponding Author * [email protected] * [email protected] * [email protected]

Author Contributions The manuscript was written through contributions of all authors. / All authors have given approval to the final version of the manuscript.

Funding Sources


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Analytical Chemistry The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was funded by the Spanish Ministry of Economy and Competitiveness (Grant CTQ2015-69278-C2-1-R/AIE). The authors are grateful to the Proteomics Unit of the Complutense University of Madrid, which is a member of ProteoRed, PRB3-ISCIII.

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Crude cell lysis extract EDMA

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(40) Britton, J.; Dyer, R. P.; Majumdar, S.; Raston, C. L.; Weiss, G. A. Ten-Minute Protein Purification and Surface Tethering for Continuous-Flow Biocatalysis. Angew. Chem. Int. Ed. 2017, 56, 1–6.

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