Purification, sequence and crystallization of an anti-tissue factor Fab and its use for the crystallization of tissue factor

• Published in: Journal of crystal growth Vol. 122; no. 1; pp. 253 - 264
• Main Authors: Ruf, Wolfram, Stura, Enrico A., LaPolla, Robert J., Syed, Rashid, Edgington, Thomas S., Wilson, Ian A.
• Format: Journal Article Conference Proceeding
• Language: English
• Published: Amsterdam Elsevier B.V 01.01.1992; Elsevier
• Subjects: Antigen-antibody reactions, antigen-antibody complexes, antibody-complement and others. Study of affinity. Antigen presentation; Biological and medical sciences; Fundamental and applied biological sciences. Psychology; Fundamental immunology; Molecular immunology; Crystal growth; Purification; Nucleotide sequence; Peptide fragment; Tissue factor; Monoclonal antibody; Aminoacid sequence; Immune complex
• ISSN: 0022-0248; 1873-5002
• DOI: 10.1016/0022-0248(92)90254-G

The crystallization of proteins as Fab-antigen complexes may be advantageous in the crystallization of biologically important proteins, since many different monoclonal antibodies are often available against a given protein. From each of these, Fab fragments can be generated providing new opportunities for crystallization of the protein as a complex. Here we report the screening and identification of a suitable Fab for the crystallization of the soluble extracellular domain of tissue factor, the receptor responsible for the cellular initiation of the coagulation protease cascade. From six specific Fabs, three have been identified that crystallize readily as free Fabs. The refinement of the purification procedure for one of these Fabs (TF8-5G9) was required to provide material which reliably formed complex crystals with the tissue factor extracellular domain. A further improvement in the quality of the complex crystals was achieved by enzymatic removal of sialic acid from tissue factor resulting in a significant reduction of its charge heterogeneity.