Designed protein pores as components for biosensors

Details Designed protein pores as components for biosensors Designed protein pores as components for biosensors

2001-02-15

2004-11-30

Noguerola, Alex (Department: 1753)

Chemistry: electrical and wave energy

Apparatus

Electrolytic

C204S403080, C930S010000

Reexamination Certificate

active

06824659

ABSTRACT:

BACKGROUND OF THE INVENTION
The field of the invention is metal detection.
Biosensors are analytical devices that convert the concentration of an analyte into a detectable signal by means of a biologically-derived sensing element. Well-known biosensors include commercial devices for sensing glucose. In addition, true biosensors, biomimetric devices, and devices that use living cells have recently been developed. For example, to detect divalent metal cations, true biosensors have been made using the enzyme carsonic anhydrase (Thompson et al., 1993, Anal. Chem. 65: 730-734), the metal binding site of which has been altered (Ippolito et al., 1995, Proc. Natl. Acad. Sci. USA. 92:5017-5020). To monitor HIV antibody levels, the enzyme alkaline phosphatase into which an HIV epitope has been inserted has been utilized (Brennan et al., 1995, Proc. Natl. Acad. Sci. USA 92:5783-5787).
SUMMARY OF THE INVENTION
The invention features a mutant staphylcoccal alpha hemolysin (&agr;HL) polypeptide containing heterologous metal-binding amino acid. The polypeptide assembles into a heteroheptameric pore assembly in the presence of a wild type (WT) &agr;HL polypeptide. Preferably, the metal-binding amino acid occupies a position in a transmembrane channel of the heteroheptameric pore assembly, e.g., an amino acid in the stem domain of WT &agr;HL is substituted with a heterologous metal-binding amino acid. More preferably, metal-binding amino acid projects into the lumen of the transmembrane channel.
By the term “heterologous amino acid” is meant an amino that differs from the amino acid at the corresponding site in the amino acid sequence of WT &agr;HL. By “analyte-binding amino acid” is meant any amino acid having a functional group which covalently or non-covalently binds to an analyte. By “transmembrane channel” is meant the portion or an &agr;HL polypeptide that creates a lumen through a lipid bilayer. The transmembrane channel of an &agr;HL pore assembly is composed of 14 anti-parallel &bgr; strands (the “&bgr; barrel”), two of which are contributed by the stem domain of each &agr;HL polypeptide of the pore. By “stem domain” is meant the portion of an &agr;HL polypeptide which scans approximately amino acids 110 to 150 of SEQ ID NO:1 (see, e.g.,
FIG. 1F
)
An &agr;HL polypeptide containing at least two non-consecutive heterologous metal-binding amino acids in a stem domain of &agr;HL is also within the invention. By “metal-binding amino acid” is meant any amino acid which covalently or noncovalently binds to a metal ion, e.g., Ser, Thr, Met, Tyr, Glu, Asp, Cys, or His. Unnatural amino acids, such as 1,2,3 triazole-3-alanine and 2-methyl histidine, which have altered pK
a
values, sceric properties, and arrangement of N atoms resulting in different abilities to bind metal ions, can also be introduced to confer metal-responsiveness. Preferably, the heterologous amino acids project into the lumen of the transmembrane channel, i.e., the amino acids occupy two or more of the following positions of SEQ ID NO:1: 11, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147 or 149. Alternatively, the heterologous amino acids are located on the outside of the transmembrane channel, i.e., the amino acids occupy two or more of the following positions of SEQ ID NO:1: 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148. The polypeptide may contain at least three non-consecutive heterologous metal-binding amino acids in the stem domain. Preferably, the polypeptide contains at least 4 non-consecutive heterologous metal-binding amino acids in the stem domain; more preferably, the amino acids occupy positions 123, 125, 133, and 135 of SEQ ID NO:1; more preferably, each these positions are occupied by the heterologous metal-binding amino acid His; and most preferably, the polypeptide is the &agr;HL mutant 4H, as described below.
To facilitate separation and purification of mutant analyte-responsive &agr;HL polypeptides, the polypeptide may also contain a heterologous amino acid, e.g., a Cys residue, at a site distant from the stem domain, e.g., at position 292 of SEQ ID NO:1.
The invention also features a heteromeric pore assembly containing a metal-responsive (M) &agr;HL polypeptide, e.g., a pore assembly which contains a wild type (WT) staphylococcal &agr;HL polypeptide and a metal-responsive &agr;HL polypeptide in which a heterologous metal-binding amino acid of the metal-responsive &agr;HL polypeptide occupies a position in a transmembrane channel of the pore structure. For example, the ratio of WT and M &agr;HL polypeptides is expressed by the formula WT
7−n
M
n
, where n is 1, 2, 3, 4, 5, 6, or 7; preferably the ratio of &agr;HL polypeptides in the heteroheptamer is WT
7−n
4H
n
; most preferably, the ratio is WT
6
4H
1
. Homomeric pores in which each subunit of the heptomer is a mutated &agr;HL polypeptide (i.e., where n=7) are also encompassed by the invention.
Also within the invention is a digital biosensor device comprising a heteromeric &agr;HL pore assembly. The device detects binding of a metal ion to a heterologous amino acid through a single channel (single current) or through two or more channels (macroscopic current). Rather than containing a heterologous amino acid substitution, the metal-responsive &agr;HL polypeptide in the device may contain a chelating molecule associated with an amino acid in the stem domain.
The analyte-responsive &agr;HL polypeptides (and pore assemblies containing such polypeptides) can be used in a method of detecting the presence of an analyte, e.g., a metal such as a divalent Group IIB and transition metal. Zn(II), Co(II), Cu(II), Ni(II), or Cd(II) can be detected using the methods described herein. For example, a detection method may include the steps of (a) contacting the sample to be analyzed with an analyte-responsive &agr;HL pore assembly, and (b) detecting an electrical current in a digital mode through a single channel (single current) or two or more channels (macroscopic current). A modulation or perturbation in the current detected compared to a control current measurement, i.e., a current detected in the absence of the analyte indicates the presence (and concentration) of the analyte.
The invention also includes a method of identifying an unknown analyte in a mixture of analytes which includes the following steps: (a) contacting the mixture with an analyte-responsive &agr;HL pore assembly; (b) detecting an electrical current in a digital mode through a single channel (or through two or more channels) to determine a mixture current signature; and (c) comparing the mixture current signature to a standard current signature of a known analyte. A concurrence of the mixture current signature with the standard current signature indicates the identity of the unknown analyte in the mixture.
Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims. All references cited herein are incorporated by reference in their entirety.


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