Cláudia S.M. Fernandes

A Tailor-Made “Tag–Receptor” Affinity Pair for the Purification of Fusion Proteins

A novel affinity “tag–receptor” pair was developed as a generic platform for the purification of fusion proteins. The hexapeptide RKRKRK was selected as the affinity tag and fused to green fluorescent protein (GFP). The DNA fragments were designed, cloned in Pet‐21c expression vector and expressed in E. coli host as soluble protein. A solid‐phase combinatorial library based on the Ugi reaction was synthesized: 64 affinity ligands displaying complementary functionalities towards the designed tag. The library was screened by affinity chromatography in a 96‐well format for binding to the RKRKRK‐tagged GFP protein. Lead ligand A7C1 was selected for the purification of RKRKRK fusion proteins. The affinity pair RKRKRK‐tagged GFP with A7C1 emerged as a promising solution (Ka of 2.45×105 M−1). The specificity of the ligand towards the tag was observed experimentally and theoretically through automated docking and molecular dynamics simulations.

Mild and cost-effective green fluorescent protein purification employing small synthetic ligands.

The green fluorescent protein (GFP) is a useful indicator in a broad range of applications including cell biology, gene expression and biosensing. However, its full potential is hampered by the lack of a selective, mild and low-cost purification scheme. In order to address this demand, a novel adsorbent was developed as a generic platform for the purification of GFP or GFP fusion proteins, giving GFP a dual function as reporter and purification tag. After screening a solid-phase combinatorial library of small synthetic ligands based on the Ugi-reaction, the lead ligand (A4C7) selectively recovered GFP with 94% yield and 94% purity under mild conditions and directly from Escherichia coli extracts. Adsorbents containing the ligand A4C7 maintained the selectivity to recover other proteins fused to GFP. The performance of A4C7 adsorbents was compared with two commercially available methods (immunoprecipitation and hydrophobic interaction chromatography), confirming the new adsorbent as a low-cost viable alternative for GFP purification.

Retroviral particles are effectively purified on an affinity matrix containing peptides selected by phage-display

Retroviral particles are expensive to manufacture, mostly due to the downstream processing steps which result in low recoveries (≈30%) and concentration factors. In this work, a dodecapeptide phage‐display library was panned against retrovirus like particles expressing the envelope protein Ampho4070A (VLPs‐AMPHO) and VLPs without the target protein, used as a negative control (VLPs). A depletion/selection panning protocol was successfully used to deal with the structural complexity of the target, and a total of three distinct peptide sequences displaying preferential binding towards VLPs‐AMPHO were found. Peptide 3 (CAAALAKPHTENHLLT), which appeared as one lead candidate, was synthesized and immobilized onto two purification matrices, cross‐linked agarose and magnetic particles. The matrices selectively bound VLPs‐AMPHO and in both cases recovery yields higher than 90% were obtained when employing mild elution conditions, while maintaining viral particle morphology and size.

A theoretical and experimental approach toward the development of affinity adsorbents for GFP and GFP-fusion proteins purification.

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.

Biobased Monoliths for Adenovirus Purification

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.

Engineered Protein Variants for Bioconjugation

Abstract The possibility to covalently modify biological entities, namely proteins, nucleic acids, sugars, is possible through bioconjugation, creating a boundary between biology and chemistry. The field of protein bioconjugation, in particular, has been extremely active and rendered several methods to attain protein conjugates with drugs, enzymes, inorganic or organic materials, dyes and fluorescent reporter molecules, and polymers. The initial bioconjugation methods took advantage of the reactive side chains of naturally occurring amino acids and lately evolved toward the introduction of reactive amino acids at precise locations through DNA recombinant technologies. In addition, unnatural amino acids also provide a source of site-specific and selective bioconjugation tools. Bioconjugation derives from a profound cross talk between disciplines because modern organic chemistry has progressed enormously toward chemoselective reactions performed under physiological conditions.

Magnetic fishing of recombinant green fluorescent proteins and tagged proteins with designed synthetic ligands

ABSTRACT 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.