Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Reexamination Certificate
2000-09-25
2003-05-27
Achutamurthy, Ponnathapu (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
C435S069100, C435S222000, C435S252300, C435S320100, C435S471000, C510S350000, C524S267000, C536S063000
Reexamination Certificate
active
06569663
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to serine protease variants and compositions comprising the variants which have decreased immunogenicity relative to their corresponding wild-type serine proteases.
BACKGROUND OF THE INVENTION
Enzymes make up the largest class of naturally occurring proteins. One class of enzyme includes proteases which catalyze the hydrolysis of other proteins. This ability to hydrolyze proteins has been exploited by incorporating naturally occurring and protein engineered proteases into cleaning compositions, particularly those relevant to laundry applications.
In the cleaning arts, the mostly widely utilized of these proteases are the serine proteases. Most of these serine proteases are produced by bacterial organisms while some are produced by other organisms, such as fungi. See Siezen, Roland J. et al., “Homology Modelling and Protein Engineering Strategy of Subtilases, the Family of Subtilisin-Like Serine Proteases”,
Protein Engineering
, Vol. 4, No. 7, pp. 719-737 (1991). Unfortunately, the efficacy of the wild-type proteases in their natural environment frequently does not translate into the unnatural cleaning composition environment. Specifically, protease characteristics such as, for example, thermal stability, pH stability, oxidative stability and substrate specificity are not necessarily optimized for utilization outside the natural environment of the enzyme.
Several approaches have been employed to alter the wild-type amino acid sequence of serine proteases with the goal of increasing the efficacy of the protease in the unnatural wash environment. These approaches include the genetic redesign of proteases to enhance thermal stability and to improve oxidation stability under quite diverse conditions.
However, because such genetically engineered proteases are foreign to mammals, they are potential antigens. As antigens, these proteases cause immunological and allergic responses (herein collectively described as immunological responses) in mammals. In fact, sensitization to serine proteases has been observed in environments wherein humans are regularly exposed to the proteases. Such environments include manufacturing facilities, where employees are exposed to the proteases through such vehicles as uncontrolled dust or aerosolization. Aerosolization can result by the introduction of the protease into the lung, which is the route of protease exposure which causes the most dangerous response. Protease sensitization can also occur in the marketplace, where consumers′ repeated use of products containing proteases may cause an allergic reaction.
Furthermore, while genetic engineering has been prominent in the continuing search for more highly effective proteases for use in laundry applications, genetically engineered proteases have been minimally utilized in personal care compositions and light duty detergents. A primary reason for the absence of engineered proteases in products such as, for example, soaps, gels, body washes, and shampoos, is due to the aforementioned problem of human sensitization leading to undesirable immunological responses. It would therefore be highly advantageous to provide a personal care composition which provides the cleansing properties of engineered proteases with minimized provocation of immunological responses.
One approach toward alleviating the immunological activity of a protease is through the redesign of one or more epitopes of the protease. Epitopes are those amino acid regions of an antigen which evoke an immunological response through the binding of antibodies or the presentation of processed antigens to T cells via a major histocompatibility complex protein (MHC). Changes in the epitopes can affect their efficiency as an antigen. See Walsh, B. J. and M. E. H. Howden, “A Method for the Detection of IgE Binding Sequences of Allergens Based on a Modification of Epitope Mapping”,
Journal of Immunological Methods
, Vol. 121, pp. 275-280 (1989).
The present inventors have discovered that those serine proteases commonly known as subtilisins, including subtilisin BPN′, have a prominent epitope region at amino acid positions 70-84 corresponding to BPN′. The present inventors have herein genetically redesigned such subtilisins to alleviate the immunogenic properties attributed to this epitope region. In so doing, the present inventors have discovered subtilisins which evoke a decreased immunological response yet maintain their activity as an efficient cleansing protease. Accordingly, the present proteases are suitable for use in several types of compositions including, but not limited to, laundry, dish, hard surface, skin care, hair care, beauty care, oral, and contact lens compositions.
SUMMARY OF THE INVENTION
The present invention relates to variants of serine proteases having decreased immunogenicity relative to their corresponding wild-type proteases. More particularly, the present invention relates to variants having a modified amino acid sequence of a wild-type amino acid sequence, wherein the modified amino acid sequence comprises a substitution of one or more of positions 70-84 corresponding to subtilisin BPN′. The invention further relates to mutant genes encoding such variants and cleaning and personal care compositions comprising such variants.
DETAILED DESCRIPTION OF THE INVENTION
The essential components of the present invention are herein described below. Also included are non-limiting descriptions of various optional and preferred components useful in embodiments of the present invention.
The present invention can comprise, consist of, or consist essentially of any of the required or optional components and/or limitations described herein.
All percentages and ratios are calculated by weight unless otherwise indicated.
All percentages are calculated based on the total composition unless otherwise indicated.
All component or composition levels are in reference to the active level of that component or composition, and are exclusive of impurities, for example, residual solvents or by-products, that may be present in commercially available sources.
All documents referred to herein, including all patents, patent applications, and printed publications, are hereby incorporated by reference in their entirety.
As used herein, abbreviations will be used to describe amino acids. Table I provides a list of abbreviations used herein:
TABLE I
Amino Acid
Three-letter Abbreviation
One-letter Abbreviation
Alanine
Ala
A
Arginine
Arg
R
Asparagine
Asn
N
Aspartic Acid
Asp
D
Cysteine
Cys
C
Glutamine
Gln
Q
Glutamic Acid
Glu
E
Glycine
Gly
G
Histidine
His
H
Isoleucine
Ile
I
Leucine
Leu
L
Lysine
Lys
K
Methionine
Met
M
Phenylalanine
Phe
F
Proline
Pro
P
Serine
Ser
S
Threonine
Thr
T
Tryptophan
Trp
W
Tyrosine
Tyr
Y
Valine
Val
V
Definitions
As used herein, the term “mutation” refers to alterations in gene sequences and amino acid sequences produced by those gene sequences. Mutations may be deletions, substitutions, or additions of amino acid residues to the wild-type protein sequence.
As used herein, the term “wild-type” refers to a protein, herein specifically a protease, produced by unmutated organisms.
As used herein, the term “variant” means a protein, herein specifically a protease, having an amino acid sequence which differs from that of the wild-type protease.
As referred to herein, while the variants of the present invention are not limited to those of subtilisin BPN′, all amino acid numbering is with reference to the amino acid sequence for subtilisin BPN′ E. J. which is represented by SEQ ID NO:1. The amino acid sequence for subtilisin BPN′ is further described by Wells, J. A., E. Ferrari, D. J. Henner, D. A. Estell, and E. J. Chen,
Nucleic Acids Research
, Vol. II, 7911-7925 (1983), incorporated herein by reference.
Variants of the Present Invention
The present inventors have discovered an epitope region in serine proteases which corresponds to positions 70-84 of subtilisin BPN′. The present inventors have further discovered that one or more amino acid substitut
Correa Paul Elliott
Rubingh Donn Nelton
Sikorski Elizabeth Ellen
Achutamurthy Ponnathapu
Kendall Dara M.
Moore William W
Peebles Brent M.
The Procter & Gamble & Company
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