Chemistry: analytical and immunological testing – Nuclear magnetic resonance – electron spin resonance or other...
Reexamination Certificate
2001-04-30
2004-05-11
Le, Long V. (Department: 1641)
Chemistry: analytical and immunological testing
Nuclear magnetic resonance, electron spin resonance or other...
C436S086000, C436S089000, C436S501000, C436S518000, C436S811000, C435S007100
Reexamination Certificate
active
06734023
ABSTRACT:
TECHNICAL FIELD
The present invention relates generally to a method of screening for protein stability. More particularly, the present invention relates to a quantitative method of screening for protein stability that can be employed in a high throughput setting using relatively small amounts of pure and impure protein samples.
Table of Abbreviations
BSA
bovine serum albumin
CD
circular dichroism
DSC
differential scanning calorimetry
ESI
electrospray ionization
FE
fraction exchanged
FIV
feline immunodeficiency virus
GdmCl
guanidinium monochloride
IPTG
isopropylthio-&bgr;-D-galactosidase
H/D
hydrogen/deuterium
HX
hydrogen exchange
&lgr;
6-85
monomeric &lgr; repressor
MALD
matrix assisted laser desorption/ionization
MBP
maltose binding protein
MS
mass spectrometry
MS-HX
mass spectrometry-hydrogen exchange
m/z
mass-to-charge ratio
NMR
nuclear magnetic resonance
SIV
simian immunodeficiency virus
SUPREX
stability of unpurified proteins from rates of
H/D exchange
Amino Acid Abbreviations
Single-Letter Code
Three-Letter Code
Name
A
Ala
Alanine
V
Val
Valine
L
Leu
Leucine
I
Ile
Isoleucine
P
Pro
Proline
F
Phe
Phenylalanine
W
Trp
Tryptophan
M
Met
Methionine
G
Gly
Glycine
S
Ser
Serine
T
Thr
Threonine
C
Cys
Cysteine
Y
Tyr
Tyrosine
N
Asn
Asparagine
Q
Gln
Glutamine
D
Asp
Aspartic Acid
E
Glu
Glutamic Acid
K
Lys
Lysine
R
Arg
Arginine
H
His
Histidine
Functionally Equivalent Codons
Amino Acid
Codons
Alanine
Ala
A
GCA GCC GCG GCU
Cysteine
Cys
C
UGC UGU
Aspartic Acid
Asp
D
GAC GAU
Glumatic acid
Glu
E
GAA GAG
Phenylalanine
Phe
F
UUC UUU
Glycine
Gly
G
GGA GGC GGG GGU
Histidine
His
H
CAC CAU
Isoleucine
Ile
I
AUA AUC AUU
Lysine
Lys
K
AAA AAG
Methionine
Met
M
AUG
Asparagine
Asn
N
AAC AAU
Proline
Pro
P
CCA CCC CCG CCU
Glutamine
Gln
Q
CAA CAG
Threonine
Thr
T
ACA ACC ACG ACU
Valine
Val
V
GUA GUC GUG GUU
Tryptophan
Trp
W
UGG
Tyrosine
Tyr
Y
UAC UAU
Leucine
Leu
L
UUA UUG CUA CUC
CUG CUU
Arginine
Arg
R
AGA AGG CGA CGC
CGG CGU
Serine
Ser
S
ACG AGU UCA UCC
UCG UCU
BACKGROUND ART
Natural proteins differ from most polymers in that they predominantly populate a single, ordered three-dimensional structure in solution. It has long been recognized that this ordered structure can be transformed to an approximate random chain by changes in temperature, pressure or solvent conditions (Neurath et al., (1944)
Chem. Rev
. 34: 157-265). The ability to induce protein unfolding, and subsequent refolding, has allowed scientists to analyze the physical chemistry of the folding reaction in vitro (Schellman, (1987)
Annu. Rev. Biophys. Bio
. 16: 115-37). These investigations have shed light on the kinetics and thermodynamics of conformational changes in proteins and are of biological interest for two important reasons. First, they aid in the understanding of how proteins, which start their existence in the cell in a disordered state, manage to rapidly transform into a single, folded, functional conformation. Second, they elucidate the nature of functionally significant structural fluctuations present in proteins once folding equilibrium is reached.
The function of a protein is contingent on the stability of its native conformation. Consequently, in the field of protein biochemistry, stability measurements are frequently performed to establish a polypeptide as a stably folded protein and to study the physical forces that lead to its folding (Schellman, (1987)
Annu. Rev. Biophys. Bio
. 16: 115-37). Stability measurements also provide important biological information; a decrease in stability can be a sign of misfolding, which in some proteins leads to disease (Dobson, (1999)
Trends Biochem. Sci
. 24: 329-32) while an increase in stability can be indicative of ligand binding (Schellman, (1975)
Biopolymers
14: 999-1018). Despite their utility, stability measurements currently necessitate time-consuming experiments with pure protein samples. In proteomic experiments (Blackstock & Weir, (1999)
Trends Biotechnol
. 17: 121-27), where a large number of polypeptides often need to be analyzed, stability measurements are not practical.
Recent studies have demonstrated that hydrogen exchange coupled with electrospray ionization (ESI) mass spectrometry can qualitatively distinguish native-like proteins from unfolded polypeptides in partially purified samples (Rosenbaum et al., (1999)
J. Am. Chem. Soc
. 121: 9509-13) and can be used to study the kinetics and thermodynamics of folding (Miranker et al., (1996)
FASEB J
. 10: 93-101; Denq & Smith, (1999)
Anal. Biochem
. 276: 150-60). However, these studies did not disclose the quantitative analysis of native-like proteins.
Stability measurements are frequently performed to establish a polypeptide as a stably folded protein and to study the forces that lead to its folding. Conventional denaturation methods used for the analysis of protein stability have at least the following identified experimental limitations: 1) stability can only be measured under conditions where the protein is partially unfolded; 2) the energetics of localized fluctuations can not be easily measured; 3) many proteins cannot be analyzed because they aggregate during the course of denaturation; 4) stability measurements cannot be obtained in complex mixtures, extracts or living cells; 5) relatively large amounts of protein are required for analysis; and 6) denaturation experiments are time-consuming and not amenable to high-throughput analysis. These constraints drastically limit the number of biological problems that can be addressed through stability measurements.
Thermodynamic stability is an important biological property that has evolved to an optimal level to fit the functional needs of proteins. Therefore, investigating the stability of proteins is important not only because it affords information about the physical chemistry of folding, but also because it can provide important biological insights. A proper understanding of protein stability is also useful for technological purposes. The ability to rationally make proteins of high stability, low aggregation or low degradation rates will be valuable for a number of applications. For example, proteins that can resist unfolding can be used in industrial processes that require enzyme catalysis at high temperatures (Van den Burg et al., (1998)
Proc. Natl. Acad. Sci. U.S.A
. 95(5): 2056-60); and the ability to produce proteins with low degradation rates within the cell can help to maximize production of recombinant proteins (Kwon et al., (1996)
Protein Eng
. 9(12): 1197-202).
Stability measurements can also be used as probes of other biolological phenomena. The most basic of these phenomena is biological activity. The ability of proteins to populate their native states is a universal requirement for function. Therefore, stability can be used as a convenient, first level assay for function. For example, libraries of polypeptide sequences can be tested for stability in order to select for sequences that fold into stable conformations and might potentially be active (Sandberg et al., (1995)
Biochem
. 34: 11970-78).
Changes in stability can also be used to detect binding. When a ligand binds to the native conformation of a protein, the global stability of a protein is increased (Schellman, (1975)
Biopolymers
14: 999-1018; Pace & McGrath, (1980)
J. Biol. Chem
. 255: 3862-65; Pace & Grimsley, (1988)
Biochem
. 27: 3242-46). The binding constant can be measured by analyzing the extent of the stability increase. This strategy has been used to analyze the binding of ions and small molecules to a number of proteins (Pace & McGrath, (1980)
J. Biol. Chem
. 255: 3862-65; Pace & Grimsley, (1988)
Biochem
. 27: 324246; Schwartz, (1988)
Biochem
. 27: 8429-36; Brandts & Lin, (1990)
Biochem
. 29: 6927-40; Straume & Freire, (1992) Anal. Biochem. 203: 259-68; Graziano et al., (1996)
Biochem
. 35: 13386-92; Kanaya et al., (1996)
J. Biol. Chem
. 271: 32729-36).
The linkage between stability and binding has recently been implemented as a method to detect ligand binding (U.S. Pat. No. 5,679,582 to Bowie & Pakula). This method, however, does not take advantage of the high sensitivit
Fitzgerald Michael C.
Ghaemmaghami Sina
Oas Terence G.
Powell Kendall D.
Cheu Changhwa J
Duke University
Jenkins & Wilson & Taylor, P.A.
Le Long V.
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