Engineered fusion molecules at chelator lipid interfaces imaged by reflection interference contrast microscopy (RICM)

In molecular biology, biotechnology, and protein-engineering, the expression of histidine fusion proteins is a very powerful technique for the identification and one-step purification based on the interaction of the histidine stretch with immobilized metal complexes. By synthesis of a novel class of...

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Bibliographic Details
Published in:Biosensors & bioelectronics 1995, Vol.10 (9-10), p.805-812
Main Authors: Gritsch, Stefan, Neumaier, Klaus, Schmitt, Lutz, Tampé, Robert
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Chelating Agents
Histidine - chemistry
Lipid Bilayers - chemistry
membranes
Microscopy, Interference - methods
molecular recognition
Molecular Sequence Data
pattern formation
protein immobilization
protein-engineering
Recombinant Fusion Proteins - chemistry
self-assembly
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Summary:In molecular biology, biotechnology, and protein-engineering, the expression of histidine fusion proteins is a very powerful technique for the identification and one-step purification based on the interaction of the histidine stretch with immobilized metal complexes. By synthesis of a novel class of chelator lipids, this technique was combined with the concept of self-assembly leading to interfaces for immobilization and orientation of histidine-tagged biomolecules (Schmitt et al., 1994). Here, these chelator lipid layers were transferred onto solid substrate by vesicle fusion and Langmuir-Blodgett-techniques. Specific binding of a peptide containing an oligohistidine sequence to these functionalized interfaces was demonstrated by reflection interference contrast microscopy (RICM). Due to the phase separation behaviour of lipid mixtures, the chelator lipid interface could be further structured in two dimensions. Binding and organization of histidine-tagged molecules at these two-dimensional recognition arrays was imaged by RICM with a layer thickness resolution of 0·2 nm, and 0·5 μm laterally. Specific docking can be triggered by adding nickel ions and disrupted by EDTA. This concept opens up possibilities for reversible immobilization, enrichment and organization of histidine fusion proteins at interfaces and their application in biosensing.
ISSN:0956-5663
1873-4235
DOI:10.1016/0956-5663(95)99219-B