Generation and Use of Antibody Fragments for Structural Studies of Proteins Refractory to Crystallization

Stahl, Stephen J.; Watts, Norman R.; Wingfield, Paul T.

doi:10.1007/978-1-62703-992-5_35

Cited by 2 publications

(3 citation statements)

References 18 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The methods used were as described previously (23,57). In brief, rabbits homozygous for immunoglobulin allotypes V H al and C K b9 were immunized with purified rHBeAg, and spleen and bone marrow were collected and processed for total RNA preparation.…”

Section: Selection Of Anti-rhbeag Chimeric Rabbit/human Fab By Phage mentioning

confidence: 99%

See 1 more Smart Citation

Chimeric rabbit/human Fab antibodies against the hepatitis Be-antigen and their potential applications in assays, characterization, and therapy

Zhuang

Watts

Palmer

et al. 2017

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

Hepatitis B virus (HBV) infection afflicts millions worldwide, causing cirrhosis and liver cancer. HBV e-antigen (HBeAg), a clinical marker for disease severity, is a soluble variant of the viral capsid protein. HBeAg is not required for viral replication but is implicated in establishing immune tolerance and chronic infection. The structure of recombinant e-antigen (rHBeAg) was recently determined, yet to date, the exact nature and quantitation of HBeAg still remain uncertain. Here, to further characterize HBeAg, we used phage display to produce a panel of chimeric rabbit/human monoclonal antibody fragments (both Fab and scFv) against rHBeAg. Several of the Fab/scFv, expressed in , had unprecedentedly high binding affinities ( ∼10 m) and high specificity. We used Fab/scFv in the context of an enzyme-linked immunosorbent assay (ELISA) for HBeAg quantification, which we compared with commercially available kits and verified with seroconversion panels, the WHO HBeAg standard, rHBeAg, and patient plasma samples. We found that the specificity and sensitivity are superior to those of existing commercial assays. To identify potential fine differences between rHBeAg and HBeAg, we used these Fabs in microscale immunoaffinity chromatography to purify HBeAg from individual patient plasmas. Western blotting and MS results indicated that rHBeAg and HBeAg are essentially structurally identical, although HBeAg from different patients exhibits minor carboxyl-terminal heterogeneity. We discuss several potential applications for the humanized Fab/scFv.

show abstract

Section: Selection Of Anti-rhbeag Chimeric Rabbit/human Fab By Phage mentioning

confidence: 99%

“…This was used for characterization and for crystallization screening. Alternatively, immune complexes were applied to a nickel-Sepharose 6 Fast Flow column and processed as described previously (57).…”

Section: Preparation Of Immune Complexes For Structural Studiesmentioning

confidence: 99%

Chimeric rabbit/human Fab antibodies against the hepatitis Be-antigen and their potential applications in assays, characterization, and therapy

Zhuang

Watts

Palmer

et al. 2017

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

show abstract

“…An interesting approach to tackle difficult targets is the use of crystallization chaperones: proteins that increase the crystallization probability of the target by binding to it with high affinity, thereby stabilizing its structure and/or conformation and hence providing a different surface that may be involved in crystal lattice interactions (Koide, 2009). Originally, antibody fragments such as Fab or Fv were used to facilitate the crystallization of membrane proteins (Hunte & Michel, 2002;Uysal et al, 2009;Stahl et al, 2014). Because of the inherent difficulties with the production of Fab and Fv fragments, several alternatives have been explored.…”

Section: Introductionmentioning

confidence: 99%

Nanobody-aided crystallization of the transcription regulator PaaR2 from Escherichia coli O157:H7

et al. 2021

View full text Add to dashboard Cite

paaR2–paaA2–parE2 is a three-component toxin–antitoxin module found in prophage CP-993P of Escherichia coli O157:H7. Transcription regulation of this module occurs via the 123-amino-acid regulator PaaR2, which forms a large oligomeric structure. Despite appearing to be well folded, PaaR2 withstands crystallization, as does its N-terminal DNA-binding domain. Native mass spectrometry was used to screen for nanobodies that form a unique complex and stabilize the octameric structure of PaaR2. One such nanobody, Nb33, allowed crystallization of the protein. The resulting crystals belong to space group F432, with unit-cell parameter a = 317 Å, diffract to 4.0 Å resolution and are likely to contain four PaaR2 monomers and four nanobody monomers in the asymmetric unit. Crystals of two truncates containing the N-terminal helix–turn–helix domain also interact with Nb33, and the corresponding co-crystals diffracted to 1.6 and 1.75 Å resolution.

show abstract

Generation and Use of Antibody Fragments for Structural Studies of Proteins Refractory to Crystallization

Cited by 2 publications

References 18 publications

Chimeric rabbit/human Fab antibodies against the hepatitis Be-antigen and their potential applications in assays, characterization, and therapy

Chimeric rabbit/human Fab antibodies against the hepatitis Be-antigen and their potential applications in assays, characterization, and therapy

Nanobody-aided crystallization of the transcription regulator PaaR2 from Escherichia coli O157:H7

Contact Info

Product

Resources

About