Fast conversion of scFv to Fab antibodies using type IIs restriction enzymes.
Single chain variable fragment (scFv) antibody libraries are widely used for developing novel bioaffinity reagents, although Fab or IgG molecules are the preferred antibody formats in many final applications. Therefore, rapid conversion methods for combining multiple DNA fragments are needed to attach constant domains to the scFv derived variable domains. In this study we describe a fast and easy cloning method for the conversion of single framework scFv fragments to Fab fragments using type IIS restriction enzymes. All cloning steps excluding plating of the Fab transformants can be done in 96 well plates and the procedure can be completed in one working day. The concept was tested by converting 69 scFv clones into Fab format on 96 well plates, which resulted in 93% success rate. The method is particularly useful as a high-throughput tool for the conversion of the chosen scFv clones into Fab molecules in order to analyze them as early as possible, as the conversion can significantly affect the binding properties of the chosen clones.
Department of Biochemistry/Biotechnology, University of Turku, Turku, Finland. Electronic address: hansan@utu.fi.
Hanna
H
Sanmark
Department of Biochemistry/Biotechnology, University of Turku, Turku, Finland.
Tuomas
T
Huovinen
Department of Biochemistry/Biotechnology, University of Turku, Turku, Finland.
Tero
T
Matikka
Department of Biochemistry/Biotechnology, University of Turku, Turku, Finland.
Tiina
T
Pettersson
Department of Biochemistry/Biotechnology, University of Turku, Turku, Finland.
Maria
M
Lahti
Department of Biochemistry/Biotechnology, University of Turku, Turku, Finland.
Urpo
U
Lamminmäki
D000915:Antibody Affinity / Q000276:immunology; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular / Q000379:methods*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry*; D051100:Glial Cell Line-Derived Neurotrophic Factor / Q000276:immunology*; D005982:Glutathione Transferase / Q000276:immunology*; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000235:genetics / Q000276:immunology*; D008969:Molecular Sequence Data; D019151:Peptide Library; D017422:Sequence Analysis, DNA; D057127:Single-Chain Antibodies / Q000235:genetics / Q000276:immunology*
D051100:Glial Cell Line-Derived Neurotrophic Factor; D007140:Immunoglobulin Fab Fragments; D019151:Peptide Library; D057127:Single-Chain Antibodies; D005982:Glutathione Transferase; D015252:Deoxyribonucleases, Type II Site-Specific
Antibody; Fab; High-throughput cloning; IIs restriction enzyme; scFv