Native, engineered and de novo designed ligands targeting the SARS-CoV-2 spike protein.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the deadly coronavirus disease 2019 (Covid-19) and is a concerning hazard to public health. This virus infects cells by establishing a contact between its spike protein (S-protein) and host human angiotensin-converting enzyme 2 (hACE2) receptor, subsequently initiating viral fusion. The inhibition of the interaction between the S-protein and hACE2 has immediately drawn attention amongst the scientific community, and the S-protein was considered the prime target to design vaccines and to develop affinity ligands for diagnostics and therapy. Several S-protein binders have been reported at a fast pace, ranging from antibodies isolated from immunised patients to de novo designed ligands, with some binders already yielding promising in vivo results in protecting against SARS-CoV-2. Natural, engineered and designed affinity ligands targeting the S-protein are herein summarised, focusing on molecular recognition aspects, whilst identifying preferred hot spots for ligand binding. This review serves as inspiration for the improvement of already existing ligands or for the design of new affinity ligands towards SARS-CoV-2 proteins. Lessons learnt from the Covid-19 pandemic are also important to consolidate tools and processes in protein engineering to enable the fast discovery, production and delivery of diagnostic, prophylactic, and therapeutic solutions in future pandemics.

Tryptophan tags and de novo designed complementary affinity ligands for the expression and purification of recombinant proteins.

A common strategy for the production and purification of recombinant proteins is to fuse a tag to the protein terminal residues and employ a "tag-specific" ligand for fusion protein capture and purification. In this work, we explored the effect of two tryptophan-based tags, NWNWNW and WFWFWF, on the expression and purification of Green Fluorescence Protein (GFP) used as a model fusion protein. The titers obtained with the expression of these fusion proteins in soluble form were 0.11mgml-1 and 0.48mgml-1 for WFWFWF and NWNWNW, respectively. A combinatorial library comprising 64 ligands based on the Ugi reaction was prepared and screened for binding GFP-tagged and non-tagged proteins. Complementary ligands A2C2 and A3C1 were selected for the effective capture of NWNWNW and WFWFWF tagged proteins, respectively, in soluble forms. These affinity pairs displayed 106M-1 affinity constants and Qmax values of 19.11±2.60ugg-1 and 79.39ugg-1 for the systems WFWFWF AND NWNWNW, respectively. GFP fused to the WFWFWF affinity tag was also produced as inclusion bodies, and a refolding-on column strategy was explored using the ligand A4C8, selected from the combinatorial library of ligands but in presence of denaturant agents.

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.