Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues
Reexamination Certificate
1999-04-14
2001-08-14
Carlson, Karen Cochrane (Department: 1653)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
C530S350000, C530S324000, C530S379000, C435S006120, C435S069100, C435S173300, C435S320100, C435S410000, C536S023100, C536S023600, C426S548000, C800S281000
Reexamination Certificate
active
06274707
ABSTRACT:
CROSS REFERENCE TO RELATED APPLICATIONS
Not applicable.
BACKGROUND OF THE INVENTION
The present invention relates to sweet proteins. Brazzein protein that has been modified to improve its taste profile and to permit sweetness perception at significantly lower concentrations is disclosed.
The most widely used natural sweetener, sugar (sucrose), has significant problems associated with its use (especially causing weight gain by users). Many other sweeteners either have undesirable side effects or are deficient in certain respects. For example, aspartame loses its sweetness when exposed to elevated temperatures for long periods. This renders aspartame unsuitable for use in most baking applications. Moreover, most existing artificial sweeteners have temporal sweetness profiles which do not adequately match that of sugar. For example, their sweetness may die out sooner or leave an undesirable after taste, and/or may be perceived sooner than sugar. It may therefore be desirable to mix an existing artificial sweetener with one or more other sweeteners having different temporal profiles (so as to create a mixed sweetener that more closely matches the overall temporal sweetness profile of sugar).
Only relatively few sweet proteins (as distinguished from sweet carbohydrates) have been found in nature. One protein that appears to be particularly promising is Brazzein (SEQ ID NO: 1). See also U.S. Pat. Nos. 5,346,998 and 5,527,555, PCT publication WO 94/19467, and J. Caldwell et al., Solution Structure Of The Thermostable Sweet-tasting Protein Brazzein, 5 Nature Structural Biology 427-431 (1998). This protein is particularly desirable because it is stable when subjected to the level of heat typically present during baking of foods. The disclosure of these publications and of all other publications referred to herein are incorporated by reference as if fully set forth herein.
While naturally occurring Brazzein has certain desirable characteristics, there are some concerns regarding its temporal profile and the amount of the sweetener needed for threshold perception. Moreover, recombinant expression systems for Brazzein have to date been relatively inefficient. Prior research had not been able to identify any consensus sequence or structure in Brazzein responsible for sweetness or the sweetness profile. Thus, efforts to date to improve Brazzein's sweetness characteristics were not successful.
As such, it can be seen that the need exists for an improved protein sweetener.
BRIEF SUMMARY OF THE INVENTION
In one aspect the invention provides a peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14.
In another aspect, the invention provides a peptide having the SEQ ID NO: 1 amino acid sequence, albeit with one additional Ala either inserted therein, or inserted therein in replacement for an amino acid other than Ala in SEQ ID NO: 1, or positioned at a terminus of SEQ ID NO: 1.
In yet another aspect, the invention provides a peptide having the SEQ ID NO: 1 amino acid sequence, albeit with one additional Arg either inserted therein, or inserted therein in replacement for an amino acid other than Arg in SEQ ID NO: 1, or positioned at a terminus of SEQ ID NO: 1.
Other forms of the invention include providing nucleotide sequences for expressing such peptides. For example, a SEQ ID NOS: 15 and 17 are sequences capable of expressing SEQ ID NO: 3 and SEQ ID NO: 9 respectively.
Surprisingly, it has been learned that the replacement of a single amino acid in SEQ ID NO: 1 with either Ala or Arg, or the addition of either, can create peptides with desirable sweetness characteristics. For example, the addition of a single Ala at the beginning of the SEQ ID NO: 1 sequence to create SEQ ID NO: 3, or the replacement of one His with Ala, leads to a substantially lower detectable threshold of sweetness.
The sweet proteins of the present invention will be useful to sweeten consumable foods and beverages. For example, a small amount of the peptide can be mixed in a carrier such as lactose and poured into a beverage such as ice tea in order to sweeten it.
Further, production of genes coding for these peptides and their insertion into production vectors will allow large quantities to be created at low cost. Further, the genes can be inserted directly into a plant genome (and even possibly an animal genome) so that the fruit, vegetables, and/or edible meats, milk or the like may be sweeter.
The objects of the present invention therefore include providing:
(a) improved protein sweeteners that are stable when exposed for long periods of time to elevated temperatures;
(b) improved protein sweeteners which can be detected by humans at concentrations lower than concentrations usually required for Brazzein to be detected; and
(c) genes coding for such protein sweeteners. These and still other objects and advantages of the present invention will be apparent from the description which follows. The following description is merely of the preferred embodiments. Thus, the claims should be looked to in order to understand the full scope of the invention.
REFERENCES:
patent: 5221624 (1993-06-01), Blair et al.
patent: 5346998 (1994-09-01), Hellekant et al.
patent: 5527555 (1996-06-01), Hellekant et al.
patent: 94/19467 (1994-09-01), None
patent: WO 95/31547 (1995-11-01), None
Alignments, Ming et al. FEBS Letters, vol. 355, pp. 106-108, 1994.*
Ming et al., FEBS Letters, vol. 355, pp. 106-108, 1994.*
Alignments, Hellenkant et al., 1995.
Assadi-Porter Fariba M.
Markley John L.
Carlson Karen Cochrane
Quarles & Brady LLP
Robinson Hope A.
Wisconsin Alumni Research Foundation
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