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Binding proteins as biosensors

a technology of binding proteins and biosensors, applied in the field of biotechnology, can solve problems such as the inability the inability of protein and the associated reporter group to bind diagnostically important sugars, and the difficulty of patient compliance,

Inactive Publication Date: 2007-12-06
HSIEH HELEN VIVIAN +5
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Currently, most diabetics use the “finger stick” method to monitor their blood glucose levels and patient compliance is problematic due to pain caused by frequent (several times per day) sticks.
This method is associated with difficulties including the influence of oxygen levels, inhibitors in the blood and problems with electrodes.
In addition, detection results in consumption of the analyte that can cause difficulties when measuring low glucose concentrations.
However, this mutant protein and the associated reporter group do not bind diagnostically important sugars in mammalian bodily fluids.
Specific mutations of sites and / or attachment of certain reporter groups may act to modify a binding constant in an unpredictable way.
Additionally, a biosensor containing reporter groups may have a desirable binding constant, but not result in an easily detectable signal change upon analyte binding.
It is not currently possible to predict these interactions within proteins using existing computational methods, nor is it possible to employ rational design methodology to optimize the choice of reporter probes.
It is currently not possible to predict the effect on either the binding constant or the selectivity based on the position of any reporter group, or amino acid substitution in the protein (or vice-versa).
The prior art details numerous problems associated with the immobilization of biological molecules.
Therefore, problems result including an inability to maintain calibration of the sensing device and signal drift.
In general, binding proteins require orientational control to enable their use, thus physical absorption and random or bulk covalent surface attachment or immobilization strategies as taught in the literature generally are not successful.

Method used

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example 1

Method for the Expression and Purification of Mutant Proteins without Histidine Tags

[0050] GGBP is coded by the Mg1B-1 gene in E. coli. This protein was altered by introducing the amino acid cysteine at various positions through site-directed mutagenesis of the Mg1B-1 gene. These proteins were then expressed in E. coli and purified.

[0051] Cassette mutagenesis of Mg1B-1 was accomplished as follows. The wild-type Mg1B-1 gene was cloned into a pTZ18R vector (Dr. Anthony Cass, Imperial College, London, England). Mutant plasmids were generated from this parent plasmid using cassette mutagenesis producing randomized amino acid sequences, essentially as described by Kunkel (1991) and cloned in E. coli JM109 (Promega Life Science, Madison, Wis.). Mutant plasmids were identified by sequencing. The mutant protein was induced in JM109 and purified as described below. An E. coli JM109 colony containing the mutant plasmid was grown overnight at 37° C. with shaking (220 rpm) in LB broth contain...

example 2

Expression and Purification of Mutant GGBPs Containing Histidine Tags

[0053] GGBP mutants were engineered by either site-directed mutagenesis or cassette mutagenesis. Site-directed mutagenesis (QuikChange, Stratagene, La Jolla, Calif.) was performed to alter individual amino acids in the pQE70 vector by replacing one amino acid with another, specifically chosen amino acid. The cassette mutagenesis method (Kunkel 1991) was performed to randomize amino acids in a specified region of the GGBP gene. The mutated cassettes were then subcloned into the pQE70 expression vector.

[0054] The pGGBP-His plasmid contained the GGBP gene cloned into the pQE70 expression vector (Qiagen, Valencia, Calif.). This construct places six histidine residues on the C-terminus of the GGBP gene. E. coli strain SG13009 was used to overexpress mutant GGBP-His following standard procedures (Qiagen). After overexpression of a 250 mL culture, the cells were collected by centrifugation (6000 rpm) and resuspended in ...

example 3

Labeling of Mutant GGBPs

[0055] An aliquot of mutant GGBP containing cysteine (4.0 nmol) in PBS was treated with 2 mM dithiothreitol (5 μL, 10 nmol) for 30 min. A stock solution of N,N′-dimethyl-N-(iodoacetyl)-N′-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)ethylenediamine (IANBD amide, 0.5 mg) was prepared in DMSO (100 μL, 11.9 mM) and 3.36 μL (40 nmol) was added to the protein. The reaction proceeded at room temperature for 4 h on a Dynal rotamix in the dark. The labeled protein was purified by gel filtration on a NAP-5 column (Amersham Pharmacia). The labeling ratios were determined using an estimated extinction coefficient (50 mM−1 cm−1) for GGBP that was calculated in GeneWorks 2.45 (IntelliGenetics), ε478 (IANBD amide)=25 mM−1 cm−1), and a measurement of O.D. for a standard solution of IANBD amide at 280 nm and 478 nm. The dye concentration in the protein was calculated as Cdye=A478 / ε478. The absorbance of protein at 280 nm was calculated as Aprot(280)=Atotal(280)−Adye(280), where Adye(...

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Abstract

The invention is directed to compositions of mutated binding proteins containing reporter groups, analyte biosensor devices derived therefrom, and their use as analyte biosensors both in vitro and in vivo.

Description

[0001] This application is a continuation-in-part of U.S. patent application Ser. No. 10 / 040,077 filed Jan. 4, 2002, the entire contents of which are incorporated by reference herein.BACKGROUND OF THE INVENTION [0002] 1. Field of the Invention [0003] The invention is in the field of biotechnology. Specifically, the invention is directed to compositions of mutated binding proteins containing reporter groups, analyte biosensor devices derived therefrom, and their use as analyte biosensors both in vitro and in vivo. [0004] 2. Description of Relevant Art [0005] Monitoring glucose concentrations to facilitate adequate metabolic control in diabetics is a desirable goal and would enhance the lives of many individuals. Currently, most diabetics use the “finger stick” method to monitor their blood glucose levels and patient compliance is problematic due to pain caused by frequent (several times per day) sticks. As a consequence, there have been efforts to develop non-invasive or minimally in...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): G01N33/566G01N33/53C07K14/245G01N21/78G01N33/543G01N33/58G01N33/66
CPCC07K14/245G01N33/543G01N33/66G01N33/5438G01N33/54373
Inventor HSIEH, HELEN VIVIANPITNER, J. BRUCEAMISS, TERRY J.NYEZ, COLLEEN M.SHERMAN, DOUGLAS B.WRIGHT, DAVID J.
Owner HSIEH HELEN VIVIAN
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