Interspecies exchange mutagenesis of the first epidermal growth factor-like domain of human factor VII
| Autor: | Sampath Sridhara, Morris A. Blajchman, Bryan J. Clarke, A. Pyke, Robert F. Kelley, V. Williamson |
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| Rok vydání: | 2005 |
| Předmět: |
Recombinant Fusion Proteins
Protein Array Analysis Biology Protein Engineering Transfection law.invention Cell Line Thromboplastin Tissue factor chemistry.chemical_compound Structure-Activity Relationship Epidermal growth factor Mutant protein law Animals Humans Blood Coagulation chemistry.chemical_classification Factor VII Epidermal Growth Factor Biological activity Hematology Fusion protein Molecular biology Amino acid Protein Structure Tertiary Biochemistry chemistry Amino Acid Substitution Recombinant DNA Rabbits |
| Zdroj: | Journal of thrombosis and haemostasis : JTH. 3(6) |
| ISSN: | 1538-7933 |
| Popis: | Summary. The first epidermal growth factor-like (EGF1) domain of human factor VII (FVII) is essential for binding to tissue factor (TF). We hypothesized that the previously observed increased coagulant activity of rabbit plasma (i.e. FVII) with human TF might be explained by the five non-conserved amino acids in the rabbit vs. the human FVII EGF1 domain. Accordingly, we ‘rabbitized’ the human FVII EGF1 domain either by exchanging the entire EGF1 domain creating human FVII(rabEGF1) or by the single amino acid substitutions S53N, K62E, P74A, A75D and T83K. After transient expression in HEK293 cells, the recombinant FVII (rFVII) mutant proteins were analyzed for biological activity and binding affinity to human TF by competitive enzyme-linked immunosorbent assay (ELISA). Biological activity of the unpurified rFVII mutant proteins was either depressed or statistically unchanged vs. rFVII(WT). However, three of six rFVII mutant proteins had increased affinity for human TF in the rank order rFVII(rabEGF1) (3.3-fold) > rFVII(K62E) (2.9-fold) > rFVII(A75D) (1.7-fold). The mutant protein rFVII(K62E) was then permanently expressed and purified. Fully activated, purified rFVIIa(K62E) had a twofold greater clotting activity and 2.8-fold greater direct FVIIa amidolytic activity when compared with rFVIIa(WT). Quantitation of the affinity of TF binding by surface plasmon resonance indicated that the KD of purified rFVII(K62E) for human soluble TF (sTF) was 1.5 nm compared with 7.5 nm for rFVII(WT), i.e. fivefold greater affinity. We conclude that substitution of selected amino acid residues of the FVII EGF1 domain facilitated the creation of human rFVII chimeric proteins with both enhanced biological activity and increased affinity for TF. |
| Databáze: | OpenAIRE |
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