title: Antibody engineering reveals the important role of J segments in the production efficiency of llama single-domain antibodies in Saccharomyces cerevisiae.
abstract: Variable domains of llama heavy-chain antibodies (VHH) are becoming a potent tool for a wide range of biotechnological and medical applications. Because of structural features typical of their single-domain nature, they are relatively easy to produce in lower eukaryotes, but it is not uncommon that some molecules have poor secretion efficiency. We therefore set out to study the production yield of VHH. We computationally identified five key residues that are crucial for folding and secretion, and we validated their importance with systematic site-directed mutations. The observation that all key residues were localised in the V segment, in proximity of the J segment of VHH, led us to study the importance of J segment in secretion efficiency. Intriguingly, we found that the use of specific J segments in VHH could strongly influence the production yield. Sequence analysis and expression experiments strongly suggested that interactions with chaperones, especially with the J segment, are a crucial aspect of the production yield of VHH.
authors.affiliation: Biomolecular Imaging Group, Department of Biology, Faculty of Science,  Utrecht University, Padualaan 8, 3584 CH, Utrecht,  The Netherlands.
authors.forename: A
authors.initials: A
authors.lastname: Gorlani
authors.forename: D Lutje
authors.initials: DL
authors.lastname: Hulsik
authors.forename: H
authors.initials: H
authors.lastname: Adams
authors.forename: G
authors.initials: G
authors.lastname: Vriend
authors.forename: P
authors.initials: P
authors.lastname: Hermans
authors.forename: T
authors.initials: T
authors.lastname: Verrips
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D002161:Camelids, New World; D022801:Complementarity Determining Regions / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D007135:Immunoglobulin Variable Region / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D011487:Protein Conformation; D011489:Protein Denaturation; D015202:Protein Engineering / Q000379:methods*; D058849:Protein Refolding; D017433:Protein Structure, Secondary; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013696:Temperature
chemical_list: D000906:Antibodies; D022801:Complementarity Determining Regions; D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region