Ana Sofia Pina

Studies on the molecular recognition between bioactive peptides and angiotensin-converting enzyme.

High blood pressure or hypertension is a condition affecting many individuals and represents a controllable risk factor for cardiovascular diseases such as coronary heart disease and stroke. A non‐pharmacological approach to manage these includes the application of food components with antihypertensive activity. Milk protein‐derived peptides have been exploited as natural hypotensive agents, namely the peptides Val‐Pro‐Pro (VPP) and Ile‐Pro‐Pro (IPP), already commercialized in functional foods as a potential alternative to synthetic drugs. These bioactive peptides inhibit in vitro and in vivo the Angiotensin I‐converting enzyme (ACE), a protein with an important role in blood pressure regulation. In this work, we attempted to elucidate the possible mode of interaction between the peptides and ACE, including mechanisms of binding to the cofactor Zn2+, and further contrast this with the known mode of inhibition exerted by synthetic drugs (Captopril, Enalaprilat and Lisinopril). The bioactive peptide Ala‐Leu‐Pro‐Met‐His‐Ile‐Arg (ALPMHIR), also known to inhibit the enzyme ACE but with a lower efficiency than VPP and IPP, was utilized in the docking studies for comparison. It was observed that the best docking poses obtained for VPP and IPP were located at the ACE catalytic site with very high resemblance to the drugs mode of interaction, including the coordination with Zn2+. As for ALPMHIR, the best docking poses were located in the narrow ACE channel outside the catalytic site, representing higher affinity energies and fewer resemblances with the interaction established by drugs. Copyright

Bio-recognition and detection using liquid crystals.

Liquid crystals (LCs) are used extensively by the electronics industry as display devices. Advances in the understanding of the liquid crystalline phase and the chemistry therein lead to the development of LC exhibiting faster switching speed with greater twist angle. This in turn lead to the emergence of liquid crystal displays, rendering dial-and-needle based displays (such as those used in various meters) and cathode ray tubes obsolete. In this article, we review the history of LC and their emergence as an invaluable material for display devices and the more recent discovery of their use as sensing elements in biosensors. This new application of LC as tools in the development of fast and simple biosensors is envisaged to gain more importance in the foreseeable future.

Challenges and opportunities in the purification of recombinant tagged proteins

Abstract The purification of recombinant proteins by affinity chromatography is one of the most efficient strategies due to the high recovery yields and purity achieved. However, this is dependent on the availability of specific affinity adsorbents for each particular target protein. The diversity of proteins to be purified augments the complexity and number of specific affinity adsorbents needed, and therefore generic platforms for the purification of recombinant proteins are appealing strategies. This justifies why genetically encoded affinity tags became so popular for recombinant protein purification, as these systems only require specific ligands for the capture of the fusion protein through a pre-defined affinity tag tail. There is a wide range of available affinity pairs “tag-ligand” combining biological or structural affinity ligands with the respective binding tags. This review gives a general overview of the well-established “tag-ligand” systems available for fusion protein purification and also explores current unconventional strategies under development.

Immobilization of enterokinase on magnetic supports for the cleavage of fusion proteins

Magnetic nanobiocatalysts for tag cleavage on fusion proteins have been prepared by immobilizing enterokinase (EK) onto iron oxide magnetic nanoparticles coated with biopolymers. Two different chemistries have been explored for the covalent coupling of EK, namely carbodiimide (EDC coupling) and maleimide activation (Sulfo coupling). Upon immobilization, EK initial activity lowered but EDC coupling lead to higher activity retention. Regarding the stability of the nanobiocatalysts, these were recycled up to ten times with the greater activity losses observed in the first two cycles. The immobilized EK also proved to cleave a control fusion protein and to greatly simplify the separation of the enzyme from the reaction mixture.

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.

Comparison of Fluorescence Labelling Techniques for the Selection of Affinity Ligands from Solid-Phase Combinatorial Libraries

This study reports the comparison of fluorimetric techniques (fluorescence microscopy and spectrofluorimetry on a 96-well format) for the on-bead screening of combinatorial libraries of affinity ligands for chromatographic separations. Two solid-phase libraries of synthetic ligands based on distinct scaffolds were synthesized by combinatorial chemistry. The libraries comprising ligands representing different hydrophobic/hydrophilic properties and sizes were tested for binding to randomly selected biomolecules (labelled with a fluorophore). Fluorescence microscopy was revealed to be a reliable and reproducible technique for the detection of lead ligands which strongly bound the target biomolecule. Results obtained by fluorescence intensity measurements in a 96-well format were less consistent, mainly due to challenges related with the accurate dispensing of the solid support.

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.

Affinity tags in protein purification and peptide enrichment: an overview.

The reversible interaction between an affinity ligand and a complementary receptor has been widely explored in purification systems for several biomolecules. The development of tailored affinity ligands highly specific towards particular target biomolecules is one of the options in affinity purification systems. However, both genetic and chemical modifications on proteins and peptides widen the application of affinity ligand-tag receptor pairs towards universal capture and purification strategies. In particular, this chapter will focus on two case studies highly relevant for biotechnology and biomedical areas, namely, the affinity tags and receptors employed on the production of recombinant fusion proteins and the chemical modification of phosphate groups on proteins and peptides and the subsequent specific capture and enrichment, a mandatory step before further proteomic analysis.

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.