An improved fluorescent tag and its nanobodies for membrane protein expression, stability assay, and purification

Cai, Hongmin; Yao, Hailin; Li, Tingting; Hutter, Cedric A. J.; Li, Yanfang; Tang, Yanhong; Seeger, Markus A.; Li, Dianfan

doi:10.1038/s42003-020-01478-z

Cited by 26 publications

(34 citation statements)

References 95 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Bio-layer interferometry analysis ( Fig 1B ) with RBD immobilized and SR31 as the analyte showed a K D of 5.6 nM and an off-rate of 1 × 10 −3 s -1 . SR31, but not an irrelevant sybody (Sb66) that targets a green fluorescent protein [ 41 ], could bind to S expressed on the surface of HEK293T cells based on a cell surface staining assay measured by flow cytometry ( Fig 1C ). In addition, SR31, but not Sb66, could pull-down S protein expressed on the surface of SARS-CoV-2 pseudoviruses ( Fig 1D ).…”

Section: Resultsmentioning

confidence: 99%

A high-affinity RBD-targeting nanobody improves fusion partner’s potency against SARS-CoV-2

Yao

Cai

et al. 2021

PLoS Pathog

Self Cite

View full text Add to dashboard Cite

A key step to the SARS-CoV-2 infection is the attachment of its Spike receptor-binding domain (S RBD) to the host receptor ACE2. Considerable research has been devoted to the development of neutralizing antibodies, including llama-derived single-chain nanobodies, to target the receptor-binding motif (RBM) and to block ACE2-RBD binding. Simple and effective strategies to increase potency are desirable for such studies when antibodies are only modestly effective. Here, we identify and characterize a high-affinity synthetic nanobody (sybody, SR31) as a fusion partner to improve the potency of RBM-antibodies. Crystallographic studies reveal that SR31 binds to RBD at a conserved and ‘greasy’ site distal to RBM. Although SR31 distorts RBD at the interface, it does not perturb the RBM conformation, hence displaying no neutralizing activities itself. However, fusing SR31 to two modestly neutralizing sybodies dramatically increases their affinity for RBD and neutralization activity against SARS-CoV-2 pseudovirus. Our work presents a tool protein and an efficient strategy to improve nanobody potency.

show abstract

Section: Resultsmentioning

confidence: 99%

A high-affinity RBD-targeting nanobody improves fusion partner’s potency against SARS-CoV-2

Yao

Cai

et al. 2021

PLoS Pathog

Self Cite

View full text Add to dashboard Cite

show abstract

“…Thus, the feasibility of these approaches relies on high-throughput methods to assess the functionality, subcellular localization, and stability of the mutants [21,22,32,55]. In this regard, combining mutagenesis with the use of a fluorescent protein (FP) as a fusion tag has contributed to speeding up this task [22,24]. However, FP tagging can interfere with crucial protein parameters, such as protein activity, complex formation, and subcellular localization, among others [56][57][58].…”

Section: Systematic Vs Random Mutagenesis Approachmentioning

confidence: 99%

“…Given that the atomic resolution of membrane proteins requires extraction of the target protein from its native environment, the use of particular detergents and/or lipid combinations is highly relevant for the purification of fully functional membrane proteins [19,20]. However, identifying optimal detergent(s) and buffer conditions for protein stability is often difficult and timeconsuming [21,22], although the use of protein-Green Fluorescent Protein (GFP)-fusion constructs facilitates this task [23,24].…”

Section: Introductionmentioning

confidence: 99%

“…Protein instability can arise from protein amino acid composition or the presence of multiple conformational or oligomeric states [25]. To overcome stability issues, constructs of target protein orthologues or engineered sequences, including fusion constructs, deletions, and/or single point mutations can be screened [24,26,27]. Nevertheless, selecting the most suitable constructs can be time-consuming.…”

Section: Introductionmentioning

confidence: 99%

“…Alternatively, high-throughput screens suitable for the identification of stabilizing molecules such as detergents, lipids, antibodies, and ligands (substrates, inhibitors, agonists, etc.) can facilitate the identification of critical additives for membrane protein stability [21,22,24,28].…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Membrane Protein Stabilization Strategies for Structural and Functional Studies

2021

View full text Add to dashboard Cite

Accounting for nearly two-thirds of known druggable targets, membrane proteins are highly relevant for cell physiology and pharmacology. In this regard, the structural determination of pharmacologically relevant targets would facilitate the intelligent design of new drugs. The structural biology of membrane proteins is a field experiencing significant growth as a result of the development of new strategies for structure determination. However, membrane protein preparation for structural studies continues to be a limiting step in many cases due to the inherent instability of these molecules in non-native membrane environments. This review describes the approaches that have been developed to improve membrane protein stability. Membrane protein mutagenesis, detergent selection, lipid membrane mimics, antibodies, and ligands are described in this review as approaches to facilitate the production of purified and stable membrane proteins of interest for structural and functional studies.

show abstract

Structural insights into the binding of nanobodies LaM2 and LaM4 to the red fluorescent protein mCherry

Wang

et al. 2021

Protein Science

View full text Add to dashboard Cite

Red fluorescent proteins (RFPs) are powerful tools used in molecular biology research. Although RFP can be easily monitored in vivo, manipulation of RFP by suitable nanobodies binding to different epitopes of RFP is still desired. Thus, it is crucial to obtain structural information on how the different nanobodies interact with RFP. Here, we determined the crystal structures of the LaM2‐mCherry and LaM4‐mCherry complexes at 1.4 and 1.9 Å resolution. Our results showed that LaM2 binds to the side of the mCherry β‐barrel, while LaM4 binds to the bottom of the β‐barrel. The distinct binding sites of LaM2 and LaM4 were further verified by isothermal titration calorimetry, fluorescence‐based size exclusion chromatography, and dynamic light scattering assays. Mutation of the residues at the LaM2 or LaM4 binding interface to mCherry significantly decreased the binding affinity of the nanobody to mCherry. Our results also showed that LaM2 and LaM4 can bind to mCherry simultaneously, which is crucial for recruiting multiple operation elements to the RFP. The binding of LaM2 or LaM4 did not significantly change the chromophore environment of mCherry, which is important for fluorescence quantification assays, while several GFP nanobodies significantly altered the fluorescence. Our results provide atomic resolution interaction information on the binding of nanobodies LaM2 and LaM4 with mCherry, which is important for developing detection and manipulation methods for RFP‐based biotechnology.

show abstract

An improved fluorescent tag and its nanobodies for membrane protein expression, stability assay, and purification

Cited by 26 publications

References 95 publications

A high-affinity RBD-targeting nanobody improves fusion partner’s potency against SARS-CoV-2

A high-affinity RBD-targeting nanobody improves fusion partner’s potency against SARS-CoV-2

Membrane Protein Stabilization Strategies for Structural and Functional Studies

Structural insights into the binding of nanobodies LaM2 and LaM4 to the red fluorescent protein mCherry

Contact Info

Product

Resources

About