title: Construction of a Semisynthetic Human VH Single-Domain Antibody Library and Selection of Domain Antibodies against α-Crystalline of Mycobacterium tuberculosis.
abstract: The use of human variable heavy (VH) domain antibodies has been on the rise due to their small scaffold size and simple folding mechanism. A highly diverse library is largely dependent on the diversity introduced within the complementarity-determining region (CDR) cassettes. Here we introduced diversity with the use of a single framework diversifying all three CDRs using tailored codons consisting of degenerate trinucleotides (NNK). The length of the degeneracy in the CDRs was also taken into consideration based on the most frequently occurring length of CDRs and the canonical confirmation for each antibody subfamily. The semisynthetic human VH domain genes were assembled in a single pot using a temperature cascading process. The affinity selection process with Mycobacterium tuberculosis (MTb) α-crystalline was done using a semiautomated process. Enrichment of target-specific clones was observed with successful identification of monoclonal VH domain antibodies for MTb α-crystalline. In short, the semisynthetic library generated was able to select monoclonal VH domain antibodies against full MTb α-crystalline protein with complete semisynthetic CDRs displayed on a single scaffold. The library has the potential to be applied for the isolation of antibodies against other pathogenic proteins.
authors.affiliation: Institute for Research in Molecular Medicine, Universiti Sains Malaysia, Minden, Penang, Malaysia.
authors.forename: Nur Hidayah
authors.initials: NH
authors.lastname: Hairul Bahara
authors.affiliation: Institute for Research in Molecular Medicine, Universiti Sains Malaysia, Minden, Penang, Malaysia.
authors.forename: Siang Tean
authors.initials: ST
authors.lastname: Chin
authors.affiliation: Institute for Research in Molecular Medicine, Universiti Sains Malaysia, Minden, Penang, Malaysia ADAPT Research Cluster, Centre for Research Initiatives-Clinical & Health Sciences, Universiti Sains Malaysia, Kubang Kerian, Kelantan, Malaysia.
authors.forename: Yee Siew
authors.initials: YS
authors.lastname: Choong
authors.affiliation: Institute for Research in Molecular Medicine, Universiti Sains Malaysia, Minden, Penang, Malaysia ADAPT Research Cluster, Centre for Research Initiatives-Clinical & Health Sciences, Universiti Sains Malaysia, Kubang Kerian, Kelantan, Malaysia theamsoon@usm.my.
authors.forename: Theam Soon
authors.initials: TS
authors.lastname: Lim
mesh_terms: D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology*; D001426:Bacterial Proteins / Q000276:immunology*; D045744:Cell Line, Tumor; D022801:Complementarity Determining Regions / Q000276:immunology; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000276:immunology*; D008969:Molecular Sequence Data; D009169:Mycobacterium tuberculosis / Q000276:immunology*; D019151:Peptide Library
chemical_list: D000911:Antibodies, Monoclonal; D001426:Bacterial Proteins; D022801:Complementarity Determining Regions; D007135:Immunoglobulin Variable Region; D019151:Peptide Library
keywords: antigen screening; domain antibody; phage display