pharmaceutics Article Enhanced Production of ECM Proteins for Pharmaceutical Applications Using Mammalian Cells and Sodium Heparin Supplementation Javier Garcia-Pardo * , Sergi Montané , Francesc Xavier Avilés , Sebastian Tanco and Julia Lorenzo * Institut de Biotecnologia i Biomedicina and Departament de Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona, 08193 Bellaterra, Barcelona, Spain * Correspondence: javiergarciapardo@msn.com (J.G.-P.); julia.lorenzo@uab.cat (J.L.); Tel.: +34-93-586-8936 (J.G.-P.); +34-93-586-8957 (J.L.) Abstract: The yields of soluble ECM proteins recombinantly produced with mammalian cells can be significantly enhanced by exploiting the stabilizing properties of heparin. Here, we propose a simple and straightforward scalable protocol for the mammalian cell production of ECM proteins with affinity for heparin, using heparin as a supplement. As proof of concept, we have demonstrated the high-level expression of four biomedically relevant human enzymes such as carboxypeptidase Z (CPZ), carboxypeptidase A6 (CPA6), beta-galactoside alpha-2,6-sialyltransferase 2 (ST6GAL1) and thrombin-activable fibrinolysis inhibitor (TAFI). We found a strong linear correlation between the isoelectric point (pI) of a protein and the improvement in protein expression levels upon heparin addition, providing a reference for selecting novel protein targets that would benefit from heparin supplementation. Finally, we demonstrated the compatibility of this approach with a three-step purification strategy that includes an initial heparin affinity purification step. Using CPZ as a Citation: Garcia-Pardo, J.; Montané, representative example, we performed a preparative purification of this enzyme. The purified protein S.; Avilés, F.X.; Tanco, S.; Lorenzo, J. is enzymatically active and can be used for pharmaceutical applications as well as for high-throughput Enhanced Production of ECM functional and structural studies. Proteins for Pharmaceutical Applications Using Mammalian Cells Keywords: ECM proteins; sodium heparin; heparin binding; enhanced protein expression; car- and Sodium Heparin boxypeptidases; mammalian cells; HEK 293F cells; carboxypeptidase A6; carboxypeptidase Z; Supplementation. Pharmaceutics 2022, thrombin-activable fibrinolysis inhibitor 14, 2138. https://doi.org/10.3390/ pharmaceutics14102138 Academic Editor: Dong Hee Na 1. Introduction Received: 31 July 2022 Accepted: 4 October 2022 The rapid progress of the biopharmaceutical industry has created a constant need to Published: 8 October 2022 produce a wide variety of structurally complex recombinant proteins [1–4]. To achieve the production of functional proteins it is often required to incorporate post-translational Publisher’s Note: MDPI stays neutral modifications, molecular chaperones and/or co-factors to support their elaborated folding with regard to jurisdictional claims in and enzymatic activity [1,5]. Thus, the use of mammalian expression platforms has been published maps and institutional affil- increasing because they are able to produce complex eukaryotic proteins that are otherwise iations. problematic to express in other systems [2,3,5]. In particular, the utilization of suspension- grown mammalian host cell lines provides a highly homogeneous and scalable platform to produce pharmaceutical proteins under native-like conditions [3,6]. Examples of re- Copyright: © 2022 by the authors. cently approved pharmaceutical products obtained with this system include monoclonal Licensee MDPI, Basel, Switzerland. antibodies, hormones, clotting factors, and enzymes [1,7]. Moreover, mammalian cells are This article is an open access article of great importance for research purposes, to produce structural complex recombinant distributed under the terms and proteins in structural biology at a laboratory scale [6]. However, despite recent advances in conditions of the Creative Commons the field, this expression system still suffers from significant limitations, especially with Attribution (CC BY) license (https:// “difficult-to-express” proteins [8,9]. This is the case of several secreted proteins that bind to creativecommons.org/licenses/by/ the extracellular matrix (ECM) [10,11]. 4.0/). Pharmaceutics 2022, 14, 2138. https://doi.org/10.3390/pharmaceutics14102138 https://www.mdpi.com/journal/pharmaceutics Pharmaceutics 2022, 14, 2138 2 of 14 The ECM is a multifunctional scaffolding structure that provides structural and bio- chemical support to cells and their secreted protein factors. The ECM is built up of numer- ous macromolecules such as collagens, elastin, microfibrillar proteins, proteoglycans (e.g., hyaluronan) and non-collagenous glycoproteins [11,12]. In addition to these structural components, many secreted molecules can strongly bind to the ECM scaffolding proteins due to their heparin-binding properties [13]. Among ECM-binding proteins, several types of growth factors, proteases and relevant signaling molecules have been described so far [11,14]. The affinity of these proteins to the ECM is often mediated by their specific bind- ing to heparan sulfate proteoglycans and other heparin-like molecules [15,16]. Through this binding, the ECM protects such proteins from proteolytical degradation [17] and can fine-tune their biological responses [18]. Not surprisingly, ECM and ECM-associated factors are involved in human diseases, since they mediate essential biological processes such as cell adhesion, cell communication and differentiation [19,20]. Therefore, recent thera- peutic strategies have focused on utilizing ECM molecules for the development of novel therapeutics [21]. However, the production of ECM-binding proteins as soluble molecules is often still a difficult task due to their inherent capacity for ECM binding and aggregation. Here we describe a simple and highly efficient method for expressing ECM-binding proteins in suspension-grown mammalian cells. This approach combines transient expres- sion in Human Embryonic Kidney 293F (HEK 293F) cells with the addition of sodium heparin two days after transfection to improve the protein stability of ECM-bound pro- teins. To test the robustness of this pipeline, the expression of seven different biomedically relevant secreted enzymes was evaluated. A robust enhanced expression of all ECM- binding proteins was observed, demonstrating that the addition of heparin enables the soluble accumulation, and thus higher production yields, of those ECM/heparin-binding molecules. We also show that this approach allows protein purification of soluble and active enzymes such as carboxypeptidase Z (CPZ), an ECM-bound enzyme involved in C-terminal neuropeptide processing. All in all, we report a cost-effective protein expres- sion system that boosts the recombinant production of ECM-binding proteins suitable for pharmaceutical applications. 2. Materials and Methods 2.1. Plasmids and Cell Culture Plasmids for mammalian expression of carboxypeptidase D (CPD, UniProtKB acces- sion: O75976), carboxypeptidase Z (CPZ, UniProtKB accession: Q66K79), carboxypeptidase O (CPO, UniProtKB accession: Q8IVL8), thrombin-activable fibrinolysis inhibitor (TAFI, UniProtKB accession: Q96IY4) and beta-galactoside alpha-2,6-sialyltransferase 1 (ST6GAL1, UniProtKB accession: P15907) were obtained by subcloning the respective genes into the pTriEx-7 (Merck Millipore, Burlington, MA, USA) expression vector. All these constructs express the protein of interest fused to an N-terminal IgM signal sequence plus an N- terminal Strep-tag®(Trp-Ser-His-Pro-Gln-Phe-Glu-Lys), as previously described [22–24]. The plasmid pcDNA3.1(-)hCPA6-HA-H6 was generously provided by Prof. Peter Lyons (Andrews University, MI, USA) and was constructed by subcloning human pro-CPA6 gene (UniProtKB accession: Q8N4T0) into the EcoRI/BamHI sites of pcDNA3.1(-) [13]. The pOPINF-α-gal expression vector was kindly provided by Dr. J. L. Corchero (Universitat Autònoma de Barcelona, Spain). In this vector, α-gal (UniProtKB accession: P06280) pro- duction is under the control of CMV enhancer and chicken b-actin (CBA) promoter and includes an additional 6xHis tag at the protein C-terminus [25]. FreeStyle™ 293F cells (HEK 293F cells, Thermo Fisher Scientific, Waltham, MA, USA) were grown in FreeStyle 293 expression medium (Thermo-Fischer Scientific) in flasks on a rotary shaker (120 rpm) at 37 ◦ C and in a humidified atmosphere with 8% CO2 . For maintenance, the cell culture was diluted each 48–72 h, to maintain the cells at a density between 0.2 × 106 and 3.0 × 106 cells/mL. Since the FreeStyle 293 expression medium is a ready-to-use medium, no additional supplementation is required. Pharmaceutics 2022, 14, 2138 3 of 14 2.2. Step-by-Step Protein Expression Procedure E.1 Dilute HEK 293F cells at a density of 0.5 × 106 cells/mL into a final volume of 450 mL into a 2-L shaker flask. Note 1: This protocol is optimized for a large-scale expression of heparin-binding proteins but is suitable for any scale of expression with slight modifications. To adapt this protocol, all the volumes and quantities of reagents should be scaled proportionally. Note 2: It is widely accepted that cell culture flasks can accommodate a minimum of 1/10 of their nominal volume to a maximum of 1/4 of their nominal volume of suspension culture. For larger expression volumes, we recommend using multiple culture flasks. E.2. Incubate the cells for 24 h in an orbital shaker incubator at 37 ◦ C, 120 rpm and in a humidified atmosphere with 8% CO2 until cells reach a density of 1.0 × 106 cells/mL. E.3. Pipette a total of 500 µg of DNA into 50 mL of FreeStyle 293 expression medium and vortex the sample for 30 s (1 µg of DNA per mL of final cell culture). Note 3: Here we used three different mammalian expression vectors such as pTriEx-7, pcDNA3.1 or pOPINF. However, any other vector that allows for an efficient mammalian protein expression to the extracellular medium can be used for this protocol. It is ad- visable to use plasmid DNA of the highest quality/purity and suitable for cell culture. Typically, DNA should be sterile and endotoxin-free, as well as free from other typical DNA extraction contaminants. E.4. Add 1.5 mL of a filter-sterilized, 1.0 mg/mL polyethylenimine (PEI linear, 25,000 Da, Polysciences, Warrington, PA, USA) in water solution to the DNA mix and vortex vigorously for 30 s. The final DNA–PEI ratio in the transfection mix is 1:3 (w/w). E.5. Incubate the mixture at room temperature for 15–20 min to enable DNA/PEI complex formation. E.6. Gently, add the DNA/PEI transfection mix to the cells. Note 4: Under optimal conditions, cells are transfected when they reach a density of about 1.0 × 106 cells/mL. E.7. Following transfection, incubate the cells at 37 ◦ C, 120 rpm and in a humidified atmosphere with 8% CO2 in an orbital shaker incubator. E.8. At 48 h post-transfection, add 5 mL of sodium heparin solution (Hospira Prod. Farm. y Hosp., S.L.) to the cells. Note 5: We typically use a sterile sodium heparin solution with 5000 IU/mL (equiv- alent to 50 mg/mL heparin with a molecular weight of 12,000–15,000 Da), derived from porcine intestinal mucosa and suitable for cell culture. E.9. Incubate the cells in an orbital shaker for additional 5–10 days at 37 ◦ C, 120 rpm and in a humidified atmosphere with 8% CO2. Note 6: The optimal expression time to obtain maximum protein yield should be determined experimentally for each protein of interest. E.10. Harvest the cells by centrifugation at 3000× g for 5 min. Use the supernatant immediately for the downstream protein purification or store it at −80 ◦ C until further use. 2.3. Optimized Protein Purification Protocol P.1. Defrost the medium (if necessary) and add a cocktail of EDTA-free protease inhibitors (highly recommendable, depending on the protein). Note 7: The protocol of purification described here was optimized for the purification of CPZ, and, therefore, for other heparin affinity proteins, additional steps can be optimized to assure maximum protein recovery. Note 8: It is advisable to run an SDS-PAGE of the conditioned medium prior to purification to confirm the expression of the target protein. 2.3.1. Purification Step-1 Heparin affinity purification step using a Heparin HyperD® resin (PALL Life Sciences, Port Washington, NY, USA). We recommend following the manufacturer’s protocol with minor modifications as follows. Pharmaceutics 2022, 14, 2138 4 of 14 P.2 Equilibrate 10.0 mL of heparin affinity resin per liter of culture medium using 3 column volumes of resin equilibration buffer (Buffer 1: 100 mM Tris-HCl, 100 mM NaCl, pH 7.4). P.3 Flow the clarified conditioned medium containing the recombinant protein and discard the flow-through. P.4 Wash the resin with 2 column volumes (20 mL) of equilibration Buffer 1. P.5 Elute the recombinant protein by applying an increasing gradient of NaCl up to 1.5 M using the same equilibration buffer. Eluting buffer could be increased stepwise and applied to the column (e.g., 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 and 1.0 M NaCl). Alternatively, a linear NaCl gradient could be applied using an automated FPLC system. Collect a 10 µL sample of each eluate and analyze them by SDS-PAGE. 2.3.2. Purification Step-2 P.6 Equilibrate 5.0 mL of Strep-tag affinity resin (IBA-Lifesciences, Gottingen, Ger- many) per liter of culture medium by washing three times with resin equilibration buffer (Buffer 2: 100 mM Tris-HCl, 150 mM NaCl, pH 8.0). P.7 Load the purest samples from step P.5 into the column and collect 10 µL of each eluted fraction. If small aggregates are present, the protein can be filtered through a 0.22 µm filter. P.8 Wash the resin with 25 mL of equilibration Buffer 2. P.9 Elute the recombinant protein with 12 column volumes of elution buffer (100 mM Tris-HCl, 150 mM NaCl, pH 8.0 and 2.5 mM d-desthiobiotin (IBA-Lifesciences Gottingen, Germany)). Collect a 10 µL sample of each eluate and analyze by SDS-PAGE. 2.3.3. Purification Step-3 P.10 Equilibrate the size exclusion chromatography column with gel filtration buffer (25 mM Tris-HCl, 150 mM NaCl, pH 8.0). P.11 Load the purest samples from step P.9 into the column and collect 10 µL of each eluted fraction. If small aggregates are present, the protein can be filtered through a 0.22 µm filter. P.12 Run samples from this step on an SDS-PAGE and Coomassie stain for analysis. In this step, all the purest fractions can be pooled and stored at −80 ◦ C. Note 9: This purification step is useful to perform a desalting and buffer exchange of our sample. It is also useful to remove possible macromolecules and small contaminants present in the sample after the second purification step (e.g., d-desthiobiotin). Note 10. The quality of our protein can also be tested by assessing its biological activity. It is also highly advisable to monitor the progress of the purification by measuring the activity (e.g., enzymatic activity) of our sample in the expression medium and after each purification step. 2.4. Cytotoxicity Experiments For the cytotoxicity experiments, HEK 293F cells were seeded into 96-well plates using per well 3.0 × 103 cells prepared in 200 µL of FreeStyle 293 expression medium. Three wells were filled only with 200 µL of FreeStyle 293 expression medium and were used as plate blank (see below). The 96-well plates were incubated for 24 h before sodium heparin (Hospira Prod. Farm. y Hosp, S.L.) was added at the indicated concentration (ranging from 0 to 2000 IU/mL); each condition was tested in triplicate. The growth inhibitory effect was measured after 24 and 72 h of treatment by the XTT assay [26,27]. The XTT assay is based on the ability of metabolically active cells (i.e., viable cells) to cleave the tetrazolium salt XTT into the orange formazan dye. The amount of formazan formed can be evaluated by reading the absorbance at a wavelength between 450 and 500 nm. Briefly, aliquots of 20 µL of XTT solution (2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H- tetrazolium-5-carboxanilide) were added to each well. After 4 h, the color formed was quantified by a spectrophotometric plate reader (Perkin Elmer Victor3 V, Waltham, MA, Pharmaceutics 2022, 14, 2138 5 of 14 USA) at 490 nm. Cell cytotoxicity was evaluated in terms of cell growth inhibition in treated cultures and expressed as % of the control condition (i.e., 0 IU/mL sodium heparin). For this calculation, the absorbance of wells containing only the XTT reagent and FreeStyle 293 expression medium (the plate blank) was subtracted from all wells. The results were expressed as the percentage of cell viability relative to control cells (which were considered 100%) and used to plot dose–response curves. 2.5. Enzyme Activity Assays The carboxypeptidase activity of CPZ was assayed with the fluorescent substrate dansyl-Phe-Ala-Arg as previously described [23]. In brief, a 100 µL reaction mix, containing 0.2 mM of dansyl-Phe-Ala-Arg in 100 mM Tris-acetate, pH 7.5, 100 mM NaCl was incubated with a final concentration of 100 nM of CPZ for 60 min at 37 ◦ C. After incubation, reactions were stopped by adding 50 µL of 0.5 M HCl. Then, 1 mL of chloroform was added to each reaction and the tubes were gently mixed and centrifuged for 2 min at 300× g. After centrifugation, 0.5 mL of the chloroform phase was transferred to new tubes and completely dried overnight at 25 ◦ C. Finally, dried samples containing mainly the product generated in the enzymatic reaction were resuspended with 200 µL of PBS containing 0.1% of Triton X-100. The amount of product generated was determined by fluorescence at 395 nm upon excitation at 350 nm, using a 96-well plate spectrofluorometer. 2.6. Statistical Analyses To identify changes in protein expression levels between heparin-supplemented and control cultures as a function of time, the data (expressed as mean ± SEM) was analyzed using a two-way analysis of variance (ANOVA), followed by Sidak’s multiple comparison tests. These multiple comparisons were limited to comparing the mean protein levels in the presence and absence of heparin for the different time points, as this is the main interest of our study. To evaluate the effect of heparin on cell viability, a one-way ANOVA followed by Sidak’s multiple comparisons test was performed. In all cases, an α level (level of significance) of 5% was used. All statistical analyses were performed using GraphPad Prism version 6.01 [28]. 3. Results 3.1. Heparin as an Additive to Enhance Recombinant Protein Expression The recombinant production of ECM-bound molecules can present challenges, partic- ularly in the medium-to-large scale necessary for biopharmaceutical applications. ECM binding is typically mediated by their interactions with heparan sulfate proteoglycans (HSP), which are highly sulfated polysaccharides with structural similitude to the anti- coagulant molecule heparin [15]. Thus, it is not surprising that many of the ECM-bound proteins are traditionally classified as heparin-binding proteins. Upon binding to hep- arin, these secreted molecules are protected from aggregation and proteolytic degradation, which often leads to increased yields of the full-active protein in the extracellular culture medium [13,29]. This approach outlines a detailed strategy for the transient expression of ECM-binding proteins with affinity for heparin in HEK 293F cells (Figure 1A and Materials and Methods section). In our experience, enhanced amounts of recombinant protein expression were typically obtained after supplementation with 50 IU/mL of sodium heparin two days after transfection. At this concentration, no toxic effects of heparin on HEK 293F cells were observed up to 72 h (Figure 1B–D). 3.2. Enhanced Expression of Biomedically Relevant ECM/Heparin-Binding Proteins by Heparin Supplementation Using this expression pipeline, we successfully expressed the human carboxypepti- dase Z (CPZ), which is a human ECM-bound metalloenzyme involved in neuropeptide processing. This protein is extremely difficult to express in the absence of heparin sup- Pharmaceutics 2022, 14, 2138 6 of 14 plementation [23]. In the absence of heparin, most of the produced CPZ in adherent cell Pharmaceutics 2022, 14, 6 of 14 cultures is bound to the ECM due to its heparin-binding properties (Figure 2A) [23,29]. As shown in Figure 2B, heparin addition after two days post-transfection resulted in a notable accumulation of the enzyme in the extracellular medium that was up to five-fold higher compared to the unsupplemented expression. Figure 1. Schematic representation of the optimized pipeline for the production of ECM proteins with Figure affinity1.by Schematic heparin. (A) representation HEK 293F cellsof were the optimized seeded at apipeline forofthe cell density 0.5production of ECM × 106 cells/mL. Afterproteins 24 h, with affinity by heparin. (A) HEK 293F cells were seeded at a cell density of 0.5 cells were transfected with the DNA/PEI mix, and after 48 h, the cell culture was supplemented with× 10 6 cells/mL. After 24 h, cells were transfected with the DNA/PEI mix, and after 48 h, the cell culture was supplemented 50 IU/mL of sodium heparin to enhance protein production. After the appropriate incubation time with 50 IU/mL of sodium heparin to enhance protein production. After the appropriate incubation (typically between 7 and 10 days), the cell culture can be recovered, centrifuged and the conditioned time (typically between 7 and 10 days), the cell culture can be recovered, centrifuged and the con- medium (CM) used for protein purification. (B,C) The cytotoxicity of sodium heparin against HEK ditioned medium (CM) used for protein purification. (B,C) The cytotoxicity of sodium heparin 293F cells was evaluated after (B) 24 and (C) 72 h of treatment with heparin at a concentration up against HEK 293F cells was evaluated after (B) 24 and (C) 72 h of treatment with heparin at a con- to 2000 IU/mL. centration FiftyIU/mL. up to 2000 IU/mLFifty was IU/mL the highest was concentration of sodium heparin the highest concentration that heparin of sodium did not cause that did notable cytotoxicity against HEK 293F cells. (D) Representative images not cause notable cytotoxicity against HEK 293F cells. (D) Representative images of HEK of HEK 293F cells 293Fafter cells 72 h incubation, with or without 50 IU/mL of sodium heparin. In (B) and after 72 h incubation, with or without 50 IU/mL of sodium heparin. In (B) and (C), data are shown(C), data are shown mean±±SD asasmean SDofofthree threeindependent independent experiments. experiments. One-way One-wayANOVAANOVAfollowedfollowed bybySidak’s Sidak’s multiple multiple comparisonstest comparisons test to to evaluate evaluate the theeffect effectofofheparin heparin onon cellcell viability. TheThe viability. asterisk represents asterisk a significant represents a signifi- difference cant in means difference in meansbetween heparin between treatment heparin and control treatment cells when and control cellsanwhen α of 0.05 an αisof considered. 0.05 is consid- ered. 3.2. Enhanced Expression of Biomedically Relevant ECM/Heparin-Binding Proteins by Heparin Supplementation Using this expression pipeline, we successfully expressed the human carboxypepti- As shown in Figure 2B, heparin addition after two days post-transfection resulted in a notable accumulation of the enzyme in the extracellular medium that was up to five-fold higher compared to the unsupplemented expression. Pharmaceutics 2022, 14, 2138 7 of 14 Figure 2. Enhanced expression of human CPZ in HEK 293F cells after heparin supplementation. (A) Electrostatic surface potential distribution of the catalytic domain of human CPZ (AlphaFold Figure 2. Enhancedmodelexpression of human AF-Q66K79-F1). Blue andCPZred in HEKpositive indicate 293F cells after heparin and negative supplementation. charge potential, respectively. (A) Electrostatic surfaceThepotential distribution isoelectric point of the catalytic (pI) of the full-length domain CPZ is indicated. (B) of human CPZ Representative (AlphaFold immunoblots of the model AF-Q66K79-F1). Blue and red indicate positive and negative charge potential, respectively. human CPZ expression over time in HEK 293F cells in the absence (black bars) and presence (greyThe iso- bars) electric point (pI) of theoffull-length heparin as additive CPZ(50isIU/mL). CPZ levels indicated. (B) were determined using Representative an anti-Strep-tag antibody. immunoblots of the human (C) Representative relative expression levels of human CPZ over time in HEK 293F cells. Values were CPZ expression over time in HEK 293F cells in the absence (black bars) and presence (grey bars) of normalized to the maximum CPZ signal detected in the conditioned medium in absence of heparin. heparin as additive (50 are Values IU/mL). mean ± CPZSEM oflevels were experiments. 3 independent determined usingANOVA Two-way an anti-Strep-tag antibody. (C) was performed, followed Representative relative by Sidak’s multiple comparison test, which was limited to comparing the mean protein levelsValues expression levels of human CPZ over time in HEK 293F cells. in the were normalized to the maximum CPZ signal presence and absence of heparindetected in thetime for the different conditioned medium points. The two-way ANOVAin absence suggestedof heparin. that CPZ expression levels depended both on the presence/absence of heparin and the days of expression. Values are mean ± SEM of 3 independent experiments. Two-way ANOVA was performed, followed The asterisk represents a significant difference in means between absence and presence of heparin by Sidak’s multiplewhencomparison test, which was limited to comparing the mean protein levels in the an α of 0.05 is considered. presence and absence of heparin for the different time points. The two-way ANOVA suggested that CPZ expression levels To demonstrate depended the on both robustness of our approach onof the presence/absence different heparin ECM-binding proteins, and the days of expres- we further tested the expression of six additional biomedically relevant enzymes (Figure 3). sion. The asterisk represents a significant difference in means between absence and presence of hep- Three of such proteins (TAFI, CPA6 and ST6GAL1) were expected to have heparin/ECM- arin when an α of 0.05 is considered. binding properties based on previous findings (Figure 3A–C) [30–35]. After heparin sup- plementation, CPA6 protein levels in the conditioned medium were increased about 55-fold after 5 days post-transfection. Similarly, in the case of TAFI, the amount of secreted protein To demonstrate the robustness of our approach on different ECM-binding proteins, raised to 6-fold after 5 days post-transfection as a result of heparin addition. The smallest we further testedimprovement the expression of sixforadditional was observed ST6GAL1, forbiomedically which only a 2-foldrelevant enhancementenzymes of protein(Figure 3). Three of such proteins levels (TAFI, was achieved. CPA6 and Interestingly, ST6GAL1ST6GAL1) were displays a very lowexpected to have pI in comparison to the hepa- other four heparin-binding proteins tested. As expected, our approach did not increase the rin/ECM-bindingextracellular properties based on previous findings (Figure 3A–C) [30–35]. After hep- levels of other proteins without heparin affinity properties, such as human arin supplementation, CPD [22],CPA6 protein Olevels carboxypeptidase in theα-galactosidase [24] or human conditioned medium (α-gal) were [25] (Figure increased 3E,F). about 55-fold after 5 days post-transfection. Similarly, in the case of TAFI, the amount of secreted protein raised to 6-fold after 5 days post-transfection as a result of heparin addi- tion. The smallest improvement was observed for ST6GAL1, for which only a 2-fold en- hancement of protein levels was achieved. Interestingly, ST6GAL1 displays a very low pI in comparison to the other four heparin-binding proteins tested. As expected, our ap- proach did not increase the extracellular levels of other proteins without heparin affinity Pharmaceutics 2022, 14, 8 of 14 Pharmaceutics 2022, 14, 2138 8 of 14 Figure 3. Comparative expression profiles of a set of biomedically relevant enzymes upon heparin Figure 3. Comparative expression profiles of a set of biomedically relevant enzymes upon heparin supplementation. Immunoblots supplementation. and relative Immunoblots andexpression levels were relative expression determined levels for threefor were determined different three different heparin-binding proteins: (A) heparin-binding CPA6,(A) proteins: (B)CPA6, TAFI (B) andTAFI (C) ST6GAL1. The expression and (C) ST6GAL1. was evaluated The expression was evaluated in in the absencethe (black absencebars) (black or bars) in theorpresence in the presence (grey of (grey bars) bars) of 50 IU/mL 50 IU/mL of sodium of sodium heparin heparin added 72 h added after transfection, 72 h after transfection, as described as described in the in the experimental experimental section. section. The expression The expression profiles profiles of (D) of (D) CPD, CPD, (E) CPO and (F) α-galactosidase (α-gal) were analyzed as control proteins. In all cases, the values were (E) CPO and (F) α-galactosidase (α-gal) were analyzed as control proteins. In all cases, the values normalized to the maximum protein signal detected in the conditioned medium in absence of hep- were normalized to the maximum protein signal detected in the conditioned medium in absence arin. Values are mean ± SEM of 3 independent experiments. In all cases, structural models showing of heparin. Values the electrostatic ± SEMpotential are meansurface of 3 independent distributionexperiments. are presented. InThe all cases, structural coordinates used models are: CPA6 (Al- showing the phaFold electrostatic surface model potential distribution AF-Q8N4T0-F1), TAFI (PDBare presented. code The coordinates 3LMS), ST6GAL1 used4JS1), (PDB code are: CPA6 CPD domain II (AlphaFold (AlphaFold model model AF-O75976-F1), AF-Q8N4T0-F1), TAFI (PDB code CPO (PDB ST6GAL1 3LMS), code 5MRV) andcode (PDB α-gal (6IBR). 4JS1), CPD Alldomain the structural II (AlphaFold representations were generated model AF-O75976-F1), CPOwith(PDBPymol code [36]. 5MRV) Two-way ANOVA and α-gal was (6IBR). performed, All followed by the structural Sidak’s multiple comparison test, which was limited to comparing the mean protein levels in the representations were generated with Pymol [36]. Two-way ANOVA was performed, followed by presence and absence of heparin for the different time points. The two-way ANOVA suggested that Sidak’s multiple comparison test, which was limited to comparing the mean protein levels in the CPA6, TAFI and ST6GAL1 expression levels depended both on the presence/absence of heparin and presence andthe absence days ofofexpression. heparin forFor theCPD different time points. and CPO, The two-way the two-way ANOVAANOVA indicatessuggested that only that the time of CPA6, TAFIexpression and ST6GAL1 expression significantly levels the influences depended levels ofboth on theof expression presence/absence of heparin these proteins. Meanwhile, α-gal ex- and the dayspression levels are of expression. Forindependent CPD and CPO, of thethe presence/absence two-way ANOVA of heparin andthat indicates the only expression the timetime. The of expression asterisk represents significantly a significant influences difference the levels in means between of expression of theseabsence andMeanwhile, proteins. presence of heparin α-gal when an α of 0.05 is considered. expression levels are independent of the presence/absence of heparin and the expression time. The asterisk represents a significant difference in means between absence and presence of heparin when an α of 0.05 is considered. Pharmaceutics 2022, 14, 9 of 14 3.3. Our Approach Is Compatible with a Straightforward Purification of ECM/Heparin-Binding Pharmaceutics 2022, 14, 2138 9 of 14 Proteins As described above, a typical procedure for the expression of CPZ was applied. After optimizing the expression 3.3. Our Approach Is Compatibleconditions, we produced with a Straightforward large amounts Purification of (2 L) of conditioned medium to test whether ECM/Heparin-Binding the expressed protein is of high quality and suitable for protein Proteins purification. After 8above, As described days apost-transfection, typical procedure for thethe conditioned expression medium of CPZ was from the culture applied. After was recovered optimizing andtheCPZ purified expression throughwe conditions, anproduced optimized three-step large amountspurification protocol, in- (2 L) of conditioned volving medium twoto affinity and the test whether oneexpressed size exclusion protein chromatographies is of high quality and (Figure 4A). suitable forFigure protein4B dis- purification. plays an SDS-PAGEAfter 8 showing days post-transfection, the presence the conditioned of CPZ medium frommedium in the conditioned the culture and the was recovered and CPZ purified through an optimized three-step purification purity of CPZ after each purification step. Heparin has high negative charge [37], which protocol, involving two affinity and one size exclusion chromatographies (Figure 4A). Figure 4B can affect the electrostatic binding of proteins to ionic exchangers during their purifica- displays an SDS-PAGE showing the presence of CPZ in the conditioned medium and the tion. In order to determine the effect of this additive on protein purification, we performed purity of CPZ after each purification step. Heparin has high negative charge [37], which a first affinity can affect purificationbinding the electrostatic of CPZ of using proteinsheparin to ionicas a ligand.during exchangers Interestingly, even though their purification. sodium In order heparin was added to determine during the effect protein of this additiveexpression on protein to purification, solubilize the wesecreted performed protein, this a first affinity purification of CPZ using heparin as a ligand. Interestingly, even though first additive did not interfere with a typical heparin affinity purification. After the step, eluted sodium fractions heparin containing was added duringCPZ protein protein were to expression pooled andthe solubilize loaded intoprotein, secreted a Strep-tag this additive affinity did not resin and interfere further with ausing purified typicalsize heparin affinitychromatography exclusion purification. After(SEC) the first step, 4B). (Figure eluted fractions containing CPZ protein were pooled and loaded into The final purified protein showed a high purity (Figure 4B). Approximately, a yield of 2– a Strep-tag affinity resinofand 3 mg further active purified protein perusing liter size exclusion of cell culture chromatography was obtained(SEC) (Figure(Figure 4C).4B). Theabsence In the final of purified protein showed a high purity (Figure 4B). Approximately, a yield of 2–3 mg of heparin, such challenges can result in low yields of protein production or even in the ab- active protein per liter of cell culture was obtained (Figure 4C). In the absence of heparin, sence suchofchallenges detectable canlevels resultofinprotein secretion low yields [23,38]. of protein production or even in the absence of detectable levels of protein secretion [23,38]. Figure 4. Purification of human CPZ. (A) Scheme showing the protocol followed for the purification of CPZ. Figure For protein purification, 4. Purification of human the extracellular CPZ. (A) Scheme medium was the showing collected afterfollowed protocol 8 days post-transfection for the purification of CPZ. For protein purification, the extracellular medium was collected heparin and the recombinant protein was purified in three purification steps: (I) affinity after 8 days chro- post-transfec- matography, tion (II) affinity chromatography and the recombinant using Strep-tag protein was purified in three purification purificationresin and(I) steps: (III) size exclusion heparin affinity chro- chromatography matography, (SEC), achromatography (II) affinity protocol optimized previously using by our Strep-tag group [23]. purification (B) and resin An aliquot of exclusion (III) size the initial conditioned chromatography medium, (SEC), as well optimized a protocol as from the previously elution of each by purification our group [23].step, (B) wereAn visualized aliquot of the on SDS-PAGE initial conditioned by Coomassie medium, as staining. well as(C) Thethe from purified protein elution shows of each the carboxypeptidase purification activity step, were visualized on against dansyl-Phe-Ala-Arg, a fluorescent CPB-like peptide substrate SDS-PAGE by Coomassie staining. (C) The purified protein shows the carboxypeptidase [23,39]. The activity valuesactivity were normalized against to the maximum dansyl-Phe-Ala-Arg, activity detected a fluorescent CPB-likein the presence peptide of the enzyme. substrate [23,39]. Values The are mean values activity ± SEM were of 3 independent normalized reactions. activity detected in the presence of the enzyme. Values are mean to the maximum ± SEM of 3 independent reactions. Pharmaceutics 2022, 14, 2138 10 of 14 Pharmaceutics 2022, 14, 10 of 14 3.4. Considerations 3.4. Considerationsforfor Selecting Selecting ECM/Heparin-Binding ECM/Heparin-Binding Proteins Proteins Surfaceelectrostatics Surface electrostaticsclearly clearlyplays playsaamajor majorrole roleininheparin/ECM–protein heparin/ECM–protein interactions [40]. interactions Typically, [40]. thesethese Typically, proteins are enriched proteins in basic are enriched amino in basic amino acids acidssuch such asasarginine arginineand andlysine. ly- As shown sine. Asin Figure shown in 2A, the2A, Figure modeled structure the modeled of the of structure catalytic domain the catalytic of human domain CPZ CPZ of human serves as a prototypical serves example ofexample as a prototypical the characteristic electrostatic of the characteristic surface potential electrostatic distribution surface potential found in distri- heparin-binding bution proteins. This model found in heparin-binding clearly proteins. displays This model a large clearly number displays of basic a large number residues of (i.e., Arg and basic Lys) clustered residues (i.e., Arg at a highly and polarizedatprotein Lys) clustered a highly surface, which polarized mediate protein ECMwhich surface, binding [29]. mediate Next, we ECM binding [29]. performed Next, weanalysis an accurate performed of an allaccurate members analysis of theofM14 all members family of of proteases. the M14 family of proteases. Similar electrostatic surface potential distribution Similar electrostatic surface potential distribution patterns were observed for other enzymes patterns were observed from thisfor otherfamily protein enzymes from such as this protein family such carboxypeptidase as carboxypeptidase A3 (CPA3), CPA6 or TAFI, A3all(CPA3), of them with CPA6 or TAFI, all of them with proven ECM/heparin-binding proven ECM/heparin-binding properties. Overall, these proteins have a marked bias properties. Overall, these towards proteins a basic amino acid content and thus display the most basic isoelectric points (pIs)most have a marked bias towards a basic amino acid content and thus display the among all basic membersisoelectric of thepoints (pIs) among M14 family all members of proteases (Table ofS1)the M14 [41]. family It is of proteases also common (Table in these S1) proteins for [41]. It is also common in these proteins for the local arrangement at the the local arrangement at the surface of basic amino acids as heparin-binding sites and a clear surface of basic amino acids as heparin-binding sites and a clear polarization of basic residues at the pro- polarization of basic residues at the protein surface (Figure 5A) [30,41,42]. Such clustering tein surface (Figure 5A) [30,41,42]. Such clustering of basic amino acids rarely occurs in of basic amino acids rarely occurs in other extracellular MCPs without heparin-binding other extracellular MCPs without heparin-binding properties (Figure 5B). properties (Figure 5B). Figure 5. Correlation between protein pI and the heparin-driven enhancement of protein expres- Figure 5. Correlation between protein pI and the heparin-driven enhancement of protein expres- sion.(A,B) sion. (A,B)Comparative Comparative electrostatic electrostatic surface surface potential potential distribution distribution betweenbetween the catalytic the catalytic domain ofdomain of (A) (A) TAFI TAFI andand (B) (B) CPO, CPO, as representative as representative examples examples of heparin-binding of heparin-binding and non-heparin-binding and non-heparin-binding proteins. proteins.TheTheputative putativeheparin-binding heparin-binding region of TAFI region is indicated of TAFI as “heparin-binding is indicated site” [30]. as “heparin-binding site” [30]. By contrast, CPO lacks the typical surface clustering of basic residues necessary for heparin binding. By contrast, CPO lacks the typical surface clustering of basic residues necessary for heparin binding. (C) Correlation plot showing the expression fold change upon heparin supplementation and the (C) Correlation plot showing the expression fold change upon heparin supplementation and the predicted protein pI for all the expression constructs evaluated in this report. Note that higher pIs predicted are protein associated withpIan forincreased all the expression constructs enhancement evaluated in protein in thisbyreport. production Note the cells thatheparin upon higher pIs are associated with an increased enhancement in protein production by the cells upon heparin addition. The upper right quadrant (highlighted in green) encircles the ECM/heparin-binding proteins that were produced at levels ≥ 1.5-fold, all of them with a pI higher than 6.3. Pharmaceutics 2022, 14, 2138 11 of 14 In an effort to identify common physicochemical features that affect protein expression, we analyzed the relationship between the enhancement in protein expression observed in Figure 3 and the pI of these proteins. When plotting the expression level fold change upon heparin supplementation versus protein pI, a clear correlation between these two variables is observed (Figure 5C). This finding suggests that the overall pI of a protein is one of the key parameters that can be used to predict the particular response of a protein to heparin supplementation. More interestingly, this correlation also provides a reference for selecting novel protein targets that would benefit from the present protein expression approach. 4. Discussion The initial and often most used and challenging step necessary for the bioproduction of pharmaceutically relevant proteins is their recombinant protein expression. Here, we described a straightforward and efficient protein expression pipeline that was able to provide a robust enhancement of the recombinant expression levels for proteins with proven ECM/heparin-binding properties. The proof of concept experiments on CPZ, CPA6, TAFI and ST6GAL1 show the advantages of heparin supplementation, which further demonstrates the robustness and suitability of this approach for the production of novel pharmaceutical protein targets. These selected proteins are novel enzymes with relevant physiological functions. For instance, CPZ is a secreted zinc-containing exopeptidase that functions in the extracellular processing of neuropeptides and growth factors [23]. CPA6 is also an ECM-bound enzyme with carboxypeptidase activity. Deletion of part of the CPA6 gene has been associated with the development of the Duane syndrome, which suggests that this enzyme plays a pivotal role in the migration and axonal guidance during embryonic development [13]. TAFI is an important metalloenzyme involved in coagulation regulation and forms a molecular link between coagulation and fibrinolysis [42]. ST6GAL1 is a key player in cancer development since it catalyzes the addition of α2,6-linked sialic acids to terminal N-glycans [43]. ST6GAL1 upregulation has been associated with numerous types of cancer including pancreatic, prostate, breast and ovarian tumors [43]. Notably, heparin supplementation led to an increase in the extracellular protein levels between 2- and 55-fold when compared to the expression in the absence of heparin (see Figures 2 and 3). It has been shown that the formation of heparin-bound protein complexes strongly correlates with protein stabilization and solubilization [15]. This might explain the observed increase in the recombinant protein yields during expression. Furthermore, we showed that the improvement in protein yield is particularly good for proteins with basic pIs, observing a strong correlation between pI and the fold-change improvement in expression levels induced by heparin (Figure 5C). We evaluated our approach using suspension HEK 293F cells, one of the most utilized expression hosts for recombinant protein expression. However, in addition to HEK 293-derived cell lines, our approach can be applied to other common mammalian protein expression systems, such as CHO cells [1]. In addition, this approach could be extended to other common expression systems that are able to express recombinant proteins in the extracellular media. The latter might include insect cell expression host systems (e.g., Sf9 cells, Sf21, Tn-368 and High-Five™) or yeast expression systems (e.g., Saccharomyces cerevisiae or PichiaPink) [1]. In summary, it is possible that our heparin supplementation approach and the findings presented here can be extended to multiple expression platforms with similar results. Previous studies have demonstrated that heparin strongly binds to a wide range of pharmaceutically relevant proteins [15,42,44]. Thus, it is not surprising that several ECM-bound proteins can be released from the matrix to the culture medium upon heparin binding [13,23,29]. Moreover, heparin and heparin derivatives are commonly used in heparin affinity chromatography as a very effective and simple method to purify a wide range of proteins [45,46]. We have also demonstrated that heparin supplementation is compatible with a purification workflow that includes an affinity purification step that uses heparin as a ligand. Using CPZ, a proven case of a difficult-to-produce protein, we demonstrated that this workflow is able to purify proteins with a high degree of purity and Pharmaceutics 2022, 14, 2138 12 of 14 in the active form (Figure 4B), obtaining the highest protein yield reached for CPZ reported so far (about 2–3 mg/L of cell culture). The presence of heparin in the cell culture media does not interfere with the binding of proteins to a heparin affinity resin. Furthermore, such a purification step is a good tool to remove the additive from the recombinant protein solution. Previous attempts to express CPZ using either insect cells or mammalian cells resulted in extremely low levels of CPZ expression in the cell culture media and, as a result, failed protein purification attempts [23,38,47]. Similarly, the expression levels of several phar- maceutically relevant proteins are still too low or extremely difficult to express. Thus, our approach can be very useful to produce ECM proteins with affinity by heparin as well as other secreted basic proteins for which their expression remained elusive. To our knowledge, this is the first comprehensive study of the use of heparin supplementation as an enhancer of the protein expression of ECM/heparin-binding proteins. Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/pharmaceutics14102138/s1, Table S1: ECM/heparin-binding properties of human metallocarboxypeptidases from the M14 family of proteases. Author Contributions: J.G.-P., F.X.A., J.L. and S.T. designed the conceptual framework of the study and experiments. J.G.-P. and S.M. performed the experiments and contributed to data acquisition, method development, data analysis and preparation of the manuscript figures. F.X.A. contributed to the revision of the manuscript, general scientific background and attainment of financing and equipment. J.G.-P. wrote the manuscript with the contributions and comments from all authors. All authors have read and agreed to the published version of the manuscript. Funding: This work was funded by the Ministerio de Ciencia, Innovación y Universidades, Spain, grant RTI2018-098027-B-C22 and PID2021-127983OB-C22. J.G.-P. is funded by the Spanish Ministry of Science and Innovation with a postdoctoral grant Juan de la Cierva Incorporacion (IJC2019-041039-I). S.T. is funded by a Maria Zambrano fellowship from the Ministerio de Universidades, Spain. Data Availability Statement: The authors declare that all the data presented in this study are available in the paper or in the supplementary information. Additional raw data supporting the findings of this study are available from the corresponding author upon reasonable request. Conflicts of Interest: The authors declare no conflict of interest. References 1. Tripathi, N.K.; Shrivastava, A. 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