Journal of Molecular Biology
Regular ArticleThe de Novo Design of an Antibody Combining Site: Crystallographic Analysis of the VL Domain Confirms the Structural Model
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Improvement of antibody affinity by introduction of basic amino acid residues into the framework region
2018, Biochemistry and Biophysics ReportsCitation Excerpt :Thus, the approach of this study could be applied to a variety of antibodies. With conventional affinity maturation strategies, such as directed evolution, which often attempt affinity enhancement by improving the shape complementarities of the paratopes and epitopes [17,18], it is essential to identify structural data and / or hot-spots. In contrast, because our strategy does not involve shape complementarities, precise characterization of the Fab and its antigen is not required.
Malachite green mediates homodimerization of antibody V<inf>L</inf> domains to form a fluorescent ternary complex with singular symmetric interfaces
2013, Journal of Molecular BiologyCitation Excerpt :Amino acid contact areas above a cutoff of 2 Å2 were subsequently mapped to CDRs that had been identified on the SACS server [74]. Variable domain complexes in Fig. 3 and Fig. S3, identified by their crystal structure PDB ID, are as follows: 2C1P, anti-finrozole (drug candidate) [75]; 3CFB, anti-trans-stilbene (blue fluorescent complex) [76]; 1FLR, anti-fluorescein (quenched fluorescent dye) [77]; 1LVE, LEN myeloma VL dimer [78]; 1QAC, LEN Q89L mutant VL dimer [24]; 2Q1E, amyloidogenic mutant VL dimer [79]; 2RHE, Bence-Jones VL dimer [80]; and 1IVL, synthetic VL dimer [81]. Equilibrium titration assays employed 150 pM dL5* dimer or 300 nM L5* monomer (150 nM dimer equivalent).
Combinatorial design of an anticalin directed against the extra-domain b for the specific targeting of oncofetal fibronectin
2013, Journal of Molecular BiologyCitation Excerpt :Moreover, the simple and robust biomolecular architecture of Anticalins provides a particular advantage for the construction of functionalized proteins that enable innovative therapeutic strategies. The structural gene for ED-B was prepared by in-house gene synthesis following a previously described procedure.89 Therefore, a 273-bp nucleotide sequence was back-translated from the amino acid sequence of ED-B18 with preferred E. coli codon usage and further optimized by minimizing mRNA secondary structures.
Extra-domain B in oncofetal fibronectin structurally promotes fibrillar head-to-tail dimerization of extracellular matrix protein
2012, Journal of Biological ChemistryCitation Excerpt :Gene fragments for Fn domains 7, 8, 9, and 10 were amplified from the plasmid pET11b-FN7–10 (34) using Pfu Ultra II Fusion HS DNA Polymerase (Agilent, Santa Clara, CA) and the following primer pairs: 5′-AGT GTA TTC CAT ATG CCA TTG TCT CCA CC-3′ and 5′-CGA CTG TCT AGA CAG TCG ACG CCT TGA AGA CCC TAC TAC CAT AGT CTC-3′ for domain 7; 5′-CAG CAG ACC GCG GTT CCT CCT CCC AC-3′ and 5′-CGA TTG ACA ACT AAC AAC CGG TTA G-3′ for the domain tandem 8–9 (restriction sites for NdeI, BglII, and SacII are underlined; the last primer generated a blunt end). The 273-bp fragment encoding ED-B (17) was obtained by gene synthesis via PCR assembly from 81-bp-long oligodeoxynucleotides according to a published procedure (35). Cloning of the ED-B gene was achieved via BglII and SacII restriction sites, which were appended by PCR amplification using primers 5′-GCA GCT GAC AGA TCT GTC-3′ and 5′-GCT TAC CCC GCG GTC TGC TGG G-3′.
A disulfide-free single-domain V<inf>L</inf> intrabody with blocking activity towards huntingtin reveals a novel mode of epitope recognition
2011, Journal of Molecular BiologyCitation Excerpt :Also, contrasting with the conventional hydrophobic interface between the paired variable domains of an antibody, this αHtt-VL/αHtt-VL association involves the predominantly hydrophilic surface on the opposite side of each Ig domain. Notably, crystal structures of other engineered VL domains,38 including the Q89L variant of the myeloma protein LEN,39 revealed a hydrophobic mode of Ig domain pairing involving essentially the same interface as for RHE but with an upside down mutual orientation, which is clearly different (cf. Fig. 7). Antibody fragments expressible in the cellular cytoplasm, so-called intrabodies, have been developed against a host of medically relevant intracellular targets.