High-affinity target binding engineered via fusion of a single-domain antibody fragment with a ligand-tailored SH3 domain.
Monoclonal and recombinant antibodies are ubiquitous tools in diagnostics, therapeutics, and biotechnology. However, their biochemical properties lack optimal robustness, their bacterial production is not easy, and possibilities to create multifunctional fusion proteins based on them are limited. Moreover, the binding affinities of antibodies towards their antigens are suboptimal for many applications where they are commonly used. To address these issues we have made use of the concept of creating high binding affinity based on multivalent target recognition via exploiting some of the best features of immunoglobulins (Ig) and non-Ig-derived ligand-binding domains. We have constructed a small protein, named Neffin, comprised of a 118 aa llama Ig heavy chain variable domain fragment (VHH) fused to a ligand-tailored 57 aa SH3 domain. Neffin could be readily produced in large amounts (>18 mg/L) in the cytoplasm of E. coli, and bound with a subpicomolar affinity (K(d) 0.54 pM) to its target, the HIV-1 Nef protein. When expressed in human cells Neffin could potently inhibit Nef function. Similar VHH-SH3 fusion proteins could be targeted against many other proteins of interest and could have widespread use in diverse medical and biotechnology applications where biochemical robustness and strong binding affinity are required.
Department of Virology, Haartman Institute, University of Helsinki, Helsinki, Finland.
Annika
A
Järviluoma
Tomas
T
Strandin
Sebastian
S
Lülf
Jérôme
J
Bouchet
Anna R
AR
Mäkelä
Matthias
M
Geyer
Serge
S
Benichou
Kalle
K
Saksela
D019380:Anti-HIV Agents / Q000737:chemistry / Q000494:pharmacology*; D004926:Escherichia coli; D057809:HEK293 Cells; D006801:Humans; D007700:Kinetics; D011485:Protein Binding; D051572:Proto-Oncogene Proteins c-hck / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000494:pharmacology*; D061905:Single-Domain Antibodies / Q000096:biosynthesis / Q000737:chemistry / Q000494:pharmacology*; D054311:nef Gene Products, Human Immunodeficiency Virus / Q000037:antagonists & inhibitors* / Q000096:biosynthesis; D018909:src Homology Domains
D019380:Anti-HIV Agents; D011993:Recombinant Fusion Proteins; D061905:Single-Domain Antibodies; D054311:nef Gene Products, Human Immunodeficiency Virus; C514837:nef protein, Human immunodeficiency virus 1; C496631:HCK protein, human; D051572:Proto-Oncogene Proteins c-hck