Biomimetic ligands have emerged to overcome disadvantages inherent in biological ligands. In particular, the Ugi reaction can generate scaffolds where molecular diversity can be introduced, allowing the synthesis and screening of ligand libraries in a high-throughput manner against a variety of biological targets. Two adsorbents bearing Ugi-based synthetic ligands, coined A4C7 and A7C1, were previously developed for the selective recovery of green fluorescent protein (GFP) and RKRKRK-tagged GFP directly from Escherichia coli crude extracts. This work describes, for the first time, the in situ synthesis of Ugi-based ligands on magnetic beads and their application in the magnetic recovery of cognate proteins.

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The green fluorescent protein (GFP) is widely employed to report on a variety of molecular phenomena, but its selective recovery is hampered by the lack of a low-cost and robust purification alternative. This work reports an integrated approach combining rational design and experimental validation toward the optimization of a small fully-synthetic ligand for GFP purification. A total of 56 affinity ligands based on a first-generation lead structure were rationally designed through molecular modeling protocols. The library of ligands was further synthesized by solid-phase combinatorial methods based on the Ugi reaction and screened against Escherichia coli extracts containing GFP. Ligands A4C2, A5C5 and A5C6 emerged as the new lead structures based on the high estimated theoretical affinity constants and the high GFP binding percentages and enrichment factors. The elution of GFP from these adsorbents was further characterized, where the best compromise between mild elution conditions, yield and purity was found for ligands A5C5 and A5C6. These were tested for purifying a model GFP-fusion protein, where ligand A5C5 yielded higher protein recovery and purity. The molecular interactions between the lead ligands and GFP were further assessed by molecular dynamics simulations, showing a wide range of potential hydrophobic and hydrogen-bond interactions.

Retroviral vectors gained popularity toward other viral vectors as they integrate their genome into hosts' genome, a characteristic required for the modification of stem cells. However, the production of viable particles for gene therapy is hampered by the low ratio of infectious to non-infectious viral particles after purification, low titers and limited number of competent viral receptors. We have developed de novo two fully synthetic triazine-based ligands that can selectively bind retroviral particles pseudotyped with amphotropic murine leukemia virus envelope (AMPHO4070A). A 78-membered library of triazine-based ligands was designed in silico and was virtually screened against the modeled structure of the AMPHO4070A protein. Ligands displaying the highest energy of binding were synthesized on cross-linked agarose and experimentally tested. Adsorbents containing ligands A5A10 and A10A11 showed selectivity toward viral particles containing the target protein (VLP-AMPHO), binding 19 ± 5 $μ$g/g support and 47 ± 13 $μ$g/g support, respectively. The elution conditions for both ligands were mild and with high recovery yields (80-100{%}), in comparison with common purification practices. These results were based on a lab-scale experimental setting with VLP integrity being confirmed through TEM. In particular, the elution buffer containing 12 mM imidazole allowed the recovery of intact amphotropic viral particles.

Adenoviruses are important platforms for vaccine development and vectors for gene therapy, increasing the demand for high titers of purified viral preparations. Monoliths are macroporous supports regarded as ideal for the purification of macromolecular complexes, including viral particles. Although common monoliths are based on synthetic polymers as methacrylates, we explored the potential of biopolymers processed by clean technologies to produce monoliths for adenovirus purification. Such an approach enables the development of disposable and biodegradable matrices for bioprocessing. A total of 20 monoliths were produced from different biopolymers (chitosan, agarose, and dextran), employing two distinct temperatures during the freezing process (−20 °C and −80 °C). The morphological and physical properties of the structures were thoroughly characterized. The monoliths presenting higher robustness and permeability rates were further analyzed for the nonspecific binding of Adenovirus serotype 5 (Ad5) preparations. The matrices presenting lower nonspecific Ad5 binding were further functionalized with quaternary amine anion-exchange ligand glycidyltrimethylammonium chloride hydrochloride by two distinct methods, and their performance toward Ad5 purification was assessed. The monolith composed of chitosan and poly(vinyl) alcohol (50:50) prepared at −80 °C allowed 100% recovery of Ad5 particles bound to the support. This is the first report of the successful purification of adenovirus using monoliths obtained from biopolymers processed by clean technologies.

Affinity‐triggered assemblies rely on affinity interactions as the driving force to assemble physically‐crosslinked networks. WW domains are small hydrophobic proteins binding to proline‐rich peptides that are typically produced in the insoluble form. Previous works attempted the biological production of the full WW domain in tandem to generate multivalent components for affinity‐triggered hydrogels. In this work, an alternative approach was followed by engineering a 13‐mer minimal version of the WW domain that retains the ability to bind to target proline‐rich peptides. Both ligand and target peptides were produced chemically and conjugated to multivalent polyethylene glycol, yielding two components. Upon mixing, they together form soft biocompatible affinity‐triggered assemblies, stable in stem cell culture media, and displaying mechanical properties in the same order of magnitude as for those hydrogels formed with the full WW protein in tandem.

Currently most economical and technological bottlenecks in protein production are placed in the down-stream processes. With the aim of increasing the efficiency and reducing the associated costs, variousaffinity ligands have been developed. Affitins are small, yet robust and easy to produce, proteins derivedfrom the archaeal extremophilic “7 kDa DNA-binding” protein family. By means of combinatorial pro-tein engineering and ribosome display selection techniques, Affitins have shown to bind a diversity oftargets. In this work, two previously developed Affitins (anti-lysozyme and anti-IgG) were immobilizedonto magnetic particles to assess their potential for protein purification by magnetic fishing. The opti-mal lysozyme and human IgG binding conditions yielded 58 mg lysozyme/g support and 165 mg IgG/gsupport, respectively. The recovery of proteins was possible in high yield (≥95{%}) and with high purity,namely ≥95{%} and 81{%}, when recovering lysozyme from Escherichia coli supernatant and IgG from humanplasma, respectively. Static binding studies indicated affinity constants of 5.0 × 104M−1and 9.3 × 105M−1for the anti-lysozyme and anti-IgG magnetic supports. This work demonstrated that Affitins, which canbe virtually evolved for any protein of interest, can be coupled onto magnetic particles creating novelaffinity adsorbents for purification by magnetic fishing.

The effect of kefir grains on the proteolysis of major milk proteins in milk kefir and in a culture of kefir grains in pasteurized cheese whey was followed by reverse {phase-HPLC} analysis. The reduction of kappa-, alpha-, and beta-caseins {(CN)}, alpha-lactalbumin {(alpha-LA)}, and beta-lactoglobulin {(beta-LG)} contents during 48 and 90 h of incubation of pasteurized milk {(100mL)} and respective cheese whey with kefir grains (6 and 12 g) at 20 degrees C was monitored. Significant proteolysis of {alpha-LA} and kappa-, alpha-, and beta-caseins was observed. The effect of kefir amount (6 and 12 {g/100mL)} was significant for {alpha-LA} and alpha- and {beta-CN.} {alpha-Lactalbumin} and {beta-CN} were more easily hydrolyzed than {alpha-CN.} No significant reduction was observed with respect to {beta-LG} concentration for 6 and 12 g of kefir in {100mL} of milk over 48 h, indicating that no significant proteolysis was carried out. Similar results were observed when the experiment was conducted over 90 h. Regarding the cheese whey kefir samples, similar behavior was observed for the proteolysis of {alpha-LA} and {beta-LG:} {alpha-LA} was hydrolyzed between 60 and 90% after 12h (for 6 and 12 g of kefir) and no significant {beta-LG} proteolysis occurred. The proteolytic activity of lactic acid bacteria and yeasts in kefir community was evaluated. Kefir milk prepared under normal conditions contained peptides from proteolysis of {alpha-LA} and kappa-, alpha-, and beta-caseins. Hydrolysis is dependent on the kefir:milk ratio and incubation time. {beta-Lactoglobulin} is not hydrolyzed even when higher hydrolysis time is used. Kefir grains are not appropriate as adjunct cultures to increase {beta-LG} digestibility in whey-based or whey-containing foods.

The simultaneous separation and quantification of two casein peptides {(IPP}, {VPP)} presenting potent inhibitory activity of angiotensin-converting-enzyme {(ACE)} and casein in fermented milks was developed. Gradient elution was carried out at a flow-rate of 1 {mL/min}, using a mixture of two solvents. Solvent A was 0.1% {TFA} in water and solvent B was acetonitrile-water-trifluoracetic acid 95:5:0.1. The effluent was monitored by {UV} detector at 214 nm. Calibration curves were constructed in the interval of 0.01-1.0 {mg/mL} for {VPP}, 0.005-1.0 {mg/mL} for {IPP}, and 0.05-3.0 {mg/mL} for casein. R2 invariably exceeded 0.999. The detection limits were 0.004 for {VPP}, 0.002 {mg/mL} for {IPP}, and 0.02 {mg/mL} for casein. Repeatability of the method was evaluated by six consecutive injections of two standard solutions containing {VPP}, {IPP}, and casein. The {RSD} values for concentration were all below 5.08%. Recovery studies were carried out to determine the accuracy of the method. Recoveries ranged between 88 and 98.2%. The methodology was applied, not only, for the monitorization of {VPP}, {IPP}, and casein in commercial fermented milks labeled as presenting antihypertensive properties, but also, in milk with different degrees of fermentation by L. Helveticus, and in other commercial functional fermented milks, such as, those presenting cholesterol lowering properties.