Affibody-beta-galactosidase immunoconjugates produced as soluble fusion proteins in the Escherichia coli cytosol.
Recombinant immunoconjugates constitute a novel class of immunoassay reagents produced by genetic fusion between an antigen recognizing moiety and a reporter enzyme or fluorescent protein, obviating the need for chemical coupling. In this work, we describe the construction, Escherichia coli production and characterization of recombinant beta-galactosidase (beta-gal)-based immunoconjugates directed to human immunoglobulin A (IgA). As the antigen recognizing moieties, either monovalent or dimeric (head-to-tail) versions of an IgA-specific affibody (Z(IgA1)) were used, previously selected in vitro from a protein library based on combinatorial engineering of a single staphylococcal protein A domain. To increase the likelihood of proper presentation on the assembled homotetrameric enzyme surface, the affibody moieties were linked to the N-terminus of the enzyme subunits via a heptapeptide linker sequence. The two resulting immunoconjugates Z(IgA1)-beta-gal and (Z(IgA1))(2)-beta-gal, containing four and eight affibody moieties per enzyme, respectively, could be expressed as soluble and proteolytically stable proteins intracellularly in E. coli from where they were purified to high purity by a single anion exchange chromatography step. The yields of immunoconjugates were in the range 200-400 mg/l culture. Biosensor-binding studies showed that both the Z(IgA1)-beta-gal and (Z(IgA1))(2)-beta-gal immunoconjugates were capable of selective IgA-recognition, but with an apparent higher binding affinity for the variant containing divalent affibody moieties, presumably due to avidity effects. The applicability of this class of recombinant immunoconjugates was demonstrated by IgA detection in enzyme-linked immunosorbent assay (ELISA) and dot-blot analyses. In addition, using human kidney biopsy samples from a nephropathy patient, IgA depositions in glomeruli could be detected by immunohistochemistry with low background staining of tissue.
Department of Biotechnology, AlbaNova University Center, SE-106 91 Stockholm, Sweden.
Jenny
J
Rönnmark
Caroline
C
Kampf
Anna
A
Asplund
Ingmarie
I
Höidén-Guthenberg
Kenneth
K
Wester
Fredrik
F
Pontén
Mathias
M
Uhlén
Per-Ake
PA
Nygren
D015374:Biosensing Techniques; D003600:Cytosol / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D018796:Immunoconjugates / Q000276:immunology* / Q000302:isolation & purification; D007070:Immunoglobulin A / Q000032:analysis* / Q000276:immunology; D007150:Immunohistochemistry; D011993:Recombinant Fusion Proteins / Q000276:immunology*; D001616:beta-Galactosidase / Q000276:immunology*
D018796:Immunoconjugates; D007070:Immunoglobulin A; D011993:Recombinant Fusion Proteins; D001616:beta-Galactosidase