Enzyme-mediated site-specific antibody-protein modification using a ZZ domain as a linker.
A ZZ domain (ZZ) and alkaline phosphatase (AP), luciferase (Luc), or glucose oxidase (GOD) were conjugated using Sortase A (SrtA) from Staphylococcus aureus. The specific peptidyl linker for SrtA was genetically fused to the C-terminus of ZZ, and the other linker was fused to the N-terminus of AP, Luc, or GOD, respectively. The resultant proteins were obtained separately by bacterial expression. The recombinant peptide-tagged ZZ and AP, Luc, or GOD were site-specifically conjugated by SrtA through the extra peptidyl linkers in vitro. The SrtA reaction had little influence on either the antibody-binding activity of the ZZ moiety or the enzymatic activity of AP, Luc, or GOD moieties of the conjugates. Additionally, antibody-ZZ-proteins were yielded easily by mixing antibody with ZZ-AP, ZZ-Luc, or ZZ-GOD, allowing their use in an enzyme-linked immunosorbent assay. These results suggest that the enzymatic approach with SrtA facilitates the construction of ZZ-proteins. Furthermore, mixing antibody and ZZ-proteins produces a wide variety of antibody-ZZ-proteins.
Department of Chemical Science and Engineering, Graduate School of Engineering, and Organization of Advanced Science and Technology, Kobe University, Nada, Kobe 657-8501, Japan.
Takayuki
T
Sakamoto
Shiori
S
Sawamoto
Tsutomu
T
Tanaka
Hideki
H
Fukuda
Akihiko
A
Kondo
D000469:Alkaline Phosphatase / Q000032:analysis / Q000378:metabolism; D019881:Aminoacyltransferases / Q000235:genetics / Q000378:metabolism; D000906:Antibodies / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D000941:Antigens / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D001236:Aspergillus oryzae; D001426:Bacterial Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D003546:Cysteine Endopeptidases / Q000235:genetics / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D004926:Escherichia coli; D005656:Fungal Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D015870:Gene Expression; D005949:Glucose Oxidase / Q000032:analysis / Q000378:metabolism; D018796:Immunoconjugates / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D008156:Luciferases / Q000032:analysis / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics / Q000276:immunology; D015202:Protein Engineering / Q000379:methods*; D017434:Protein Structure, Tertiary / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D013211:Staphylococcus aureus
D000906:Antibodies; D000941:Antigens; D001426:Bacterial Proteins; D005656:Fungal Proteins; D018796:Immunoconjugates; D011993:Recombinant Fusion Proteins; D005949:Glucose Oxidase; D008156:Luciferases; D019881:Aminoacyltransferases; C120542:sortase A; D000469:Alkaline Phosphatase; D003546:Cysteine Endopeptidases