Engineering Fluorogen Activating Proteins into Self-Assembling Materials
| Autor: | Zachary Drennen, Wilson S. Meng, Yi Wen, Christopher Szent-Gyorgyi, Wen Liu, Alan S. Waggoner, Matthew J. Saunders |
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| Rok vydání: | 2013 |
| Předmět: |
Recombinant Fusion Proteins
Molecular Sequence Data Biomedical Engineering Pharmaceutical Science Bioengineering Peptide Sequence (biology) Protein Engineering Fluorescence Article Amphiphile Rosaniline Dyes Amino Acid Sequence Peptide sequence Fluorescent Dyes Pharmacology chemistry.chemical_classification Organic Chemistry Protein engineering Spectrometry Fluorescence chemistry Biochemistry FOS: Biological sciences Biophysics Immunoglobulin Light Chains Oligopeptides 69999 Biological Sciences not elsewhere classified Single-Chain Antibodies Biotechnology Conjugate |
| Zdroj: | Bioconjugate Chemistry. 24:803-810 |
| ISSN: | 1520-4812 1043-1802 |
| Popis: | We present herein characteristics of a conjugate in which dL5, a fluorogen-activating protein (FAP) and AEAEAKAK, an amphiphilic peptide are combined to form a solid-phase fluorescence-detection platform. The FAP dL5 is a covalently linked dimer of two identical light chain variable fragments which activates the fluorescence of the fluorogen malachite green (MG). The amphiphilic peptide of sequence AEAEAKAK is a building block of stimuli-responsive materials that undergoes sol-gel phase transition at high ionic strengths. We hypothesize that the novel bi-functional protein containing both the FAP and the amphiphile, termed dL5_EAK, co-assembles with the self-assembling peptide [AEAEAKAK]2 (EAK16-II) to form an insoluble membrane composite whereby the fluorescence enhancement function of the FAP domain remains intact. Denaturing polyacrylamide electrophoresis indicated that greater than 78% of dL5_EAK incorporates into the EAK16-II membrane. Conversely, less than 32% of dL5 without the EAK sequence associates with the insoluble fraction of EAK16-II in buffers. Membranes containing dL5_EAK and EAK16-II exhibited at least 4-fold higher fluorescence intensity compared to mixtures containing dL5 and EAK16-II. Scanning electron microscopy revealed the presence of particulates, presumably FAPs, scattering on the membrane fibrils. The evidence suggests a system of materials that can be developed into in situ-forming local sensors by immobilizing dL5 into coacervate, on which MG can be detected. It is envisioned that dL5 membranes can be established in diseased locales to monitor infiltration and migration of inflammatory cells marked with antibodies conjugated to MG. |
| Databáze: | OpenAIRE |
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