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Ep 2905332 A1 (19) TZZ Z¥¥ _T (11) EP 2 905 332 A1 (12) EUROPEAN PATENT APPLICATION (43) Date of publication: (51) Int Cl.: 12.08.2015 Bulletin 2015/33 C12N 9/00 (2006.01) C07K 14/00 (2006.01) C07K 19/00 (2006.01) C12N 9/12 (2006.01) (2006.01) (2006.01) (21) Application number: 14198178.7 C12N 9/14 C12N 9/22 C12N 9/50 (2006.01) C12N 9/78 (2006.01) (2006.01) (22) Date of filing: 19.11.2010 C12N 9/96 (84) Designated Contracting States: (72) Inventors: AL AT BE BG CH CY CZ DE DK EE ES FI FR GB •Kahre,Olev GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO 51014 Tartu (EE) PL PT RO RS SE SI SK SM TR •Artma,Kadri Designated Extension States: 51014 Tartu (EE) BA ME • Kahre, Tiina 51014 Tartu (EE) (30) Priority: 19.11.2009 US 262919 P 01.06.2010 US 350457 P (74) Representative: Kremer, Simon Mark et al 18.06.2010 US 356541 P Mewburn Ellis LLP 07.10.2010 US 390857 P City Tower 40 Basinghall Street (62) Document number(s) of the earlier application(s) in London EC2V 5DE (GB) accordance with Art. 76 EPC: 10803492.7 / 2 501 716 Remarks: This application was filed on 16-12-2014 as a (71) Applicant: Solis BioDyne OÜ divisional application to the application mentioned 51014 Tartu (EE) under INID code 62. (54) Compositions for increasing polypeptide stability and activity, and related methods (57) This disclosure provides peptides, polypep- polypeptides. The peptides, polypeptides, fusion tides, fusion polypeptides, compositions, and methods polypeptides, compositions may also enhance the activ- for enhancing or increasing the stability of a polypeptide ity, specificity, and/or fidelity of other polypeptides in a (e.g., Taq polymerase). Such peptides, polypeptides, fu- reaction mixture. The disclosure also provides methods sion polypeptides, or compositions include polypeptides of using such peptides, polypeptides, fusion polypep- linked to a peptide tag that enhances the stability of the tides, compositions. EP 2 905 332 A1 Printed by Jouve, 75001 PARIS (FR) EP 2 905 332 A1 Description CROSS-REFERENCE TO RELATED APPLICATIONS 5 [0001] This application claims the benefit of U.S. Provisional Application No. 61/262,919, filed November 19, 2009; U.S. Provisional Application No. 61/350,457, filed June 1, 2010; U.S. Provisional Application No. 61/356,541, filed June 18, 2010; and U.S. Provisional Application No. 61/390,857, filed October 7, 2010, all of which are incorporated herein by reference in their entirety. 10 BACKGROUND OF THE INVENTION [0002] There is a need in the art for methods and compositions that enhance the stability of proteins. There is particularly a need for compositions that enhance the stability of polymerases such as Taq polymerases so that they can retain enzymatic activity after short-term or long-term exposure to temperatures above freezing. There is also a need in the 15 art for compositions that enhance polymerase fidelity, sensitivity, and yield. SUMMARY OF THE INVENTION [0003] This disclosure provides peptides, polypeptides, fusion polypeptides, compositions, and methods for enabling 20 the retention of activity of an enzyme (e.g., DNA polymerase, RNA polymerase, nuclease, reverse transcriptase, DNA deaminase, RNA deaminase, protease) or a protein (e.g., erythropoietin, human Leukemia Inhibitor Factor (hLIF), gran- ulocyte macrophage colony-stimulating factor (GM-CSF), insulin, vascular endothelial growth factor (VEGF), leptin, bevacizumab) after short-term or long-term exposure to a temperature of from about -20 °C to about 35 °C. In some embodiments, peptides, polypeptides, fusion polypeptides, or compositions provided herein enhance stability of an 25 enzyme or protein at room temperature. In some embodiments, an enzyme or protein provided herein is any nucleic acid binding protein, e.g., a DNA binding protein, a RNA binding protein, a fragment thereof, or any combination thereof. In some embodiments, an enzyme or protein provided herein binds to other proteins, e.g., hormone receptors. [0004] In some embodiments, polypeptides, fusion polypeptides, or compositions provided herein retain activity at a temperature between -20 °C and 50 °C. In some embodiments, polypeptides, fusion polypeptides, or compositions retain 30 enzymatic activity or hormone activity at a temperature between -20 °C and 50 °C. In some embodiments, the enzymatic activity or hormone activity of the polypeptides, fusion polypeptides, or compositions after exposure to a temperature between -20 °C and 50 °C is at least 50% of the enzymatic activity of the polypeptide prior to exposure to said temperature. [0005] In some embodiments, peptide tags provided herein increase stability of the polypeptides, fusion polypeptides, or compositions. In some embodiments, peptide tags stabilize the polypeptides, fusion polypeptides, or compositions. 35 In some embodiments, peptide tags inhibit loss of enzymatic activity of the polypeptides, fusion polypeptides, or com- positions. In some embodiments, peptide tags inhibit degradation of the polypeptides, fusion polypeptides, or composi- tions. In some embodiments, peptide tags increase stability or inhibit loss of enzymatic activity of the polypeptides, fusion polypeptides, or compositions for at least one day. In some embodiments, peptide tags increase stability or inhibit loss of enzymatic activity of the polypeptides, fusion polypeptides, or compositions for at least one week. In some embodi- 40 ments, peptide tags increase stability or inhibit loss of enzymatic activity of the polypeptides, fusion polypeptides, or compositions for at least one month. [0006] In some embodiments, polypeptides, fusion polypeptides, or compositions provided herein demonstrate en- hanced stability, enzymatic activity, or hormone activity when compared to a similar polypeptide that does not comprise a peptide tag provided herein. In some embodiments, polypeptides, fusion polypeptides, or compositions have at least 45 50%, 60%, 70%, 80%, 90%, or 95% of the enzymatic activity or hormone activity of a similar polypeptide that does not comprise a peptide tag provided herein. In some embodiments, polypeptides, fusion polypeptides, or compositions have at least 50%, 60%, 70%, 80%, 90%, or 95% of the enzymatic activity or hormone activity of a similar polypeptide that does not comprise a peptide tag provided herein, after exposure to a temperature between -20 °C and 50 °C. In some embodiments, polypeptides, fusion polypeptides, or compositions have at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50 50%, 60%, 70%, 80%, 90%, 100%, or 200% greater enzymatic activity or hormone activity than the enzymatic activity or hormone activity of a similar polypeptide that does not comprise a peptide tag provided herein, after exposure to a temperature between -20 °C and 50 °C. [0007] In some embodiments, polypeptides, fusion polypeptides, or compositions provided herein comprise a peptide tag that has an amino acid sequence that is at least 70% identical to SEQ ID NO: 1. In some embodiments, polypeptides, 55 fusion polypeptides, or compositions provided herein comprise a peptide tag that has an amino acid sequence that is 50% to 98% identical to SEQ ID NO: 1. In some embodiments, the peptide tag has an amino acid sequence as shown in SEQ ID NO: 1, SEQ ID NO: 13, or SEQ ID NO: 14. In some embodiments, the peptide tag has an amino acid sequence thatis at least 70% identicalto SEQ IDNO: 13 or SEQ IDNO: 14. Insome embodiments, polypeptides, fusion polypeptides, 2 EP 2 905 332 A1 or compositions provided herein comprise an amino acid sequence that is at least 70% identical to SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 11. In some embodiments, polypeptides, fusion polypeptides, or compositions provided herein comprise an amino acid sequence as shown in SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 11. In some embodiments, the fusion polypeptide comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 1, SEQ ID NO: 5 6, SEQ ID NO: 8, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22. In some embodiments, polypeptides, fusion polypeptides, or compositions do not have the amino acid sequence as shown in SEQ ID NO: 2, SEQ ID NO: 6, or SEQ ID NO: 11. [0008] In some embodiments, polypeptides, fusion polypeptides, or compositions provided herein comprise a peptide tag that is encoded by a nucleotide sequence that is at least 70% identical to SEQ ID NO: 3 or SEQ ID NO: 15. In some 10 embodiments, polypeptides, fusion polypeptides, or compositions provided herein comprise a peptide tag that is encoded by a nucleotide sequence as shown in SEQ ID NO:3 or SEQ ID NO: 15. In some embodiments, polypeptides, fusion polypeptides, or compositions provided herein comprise a polypeptide encoded by a nucleotide sequence that is at least 70% identical to SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 12. In some embodiments, polypeptides, fusion polypeptides, or compositions provided herein comprise a polypeptide encoded by a nucleotide sequence as 15 shown in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 12. [0009] In some embodiments, polypeptides, fusion polypeptides, or compositions provided herein comprise a polypep- tide that has an amino acid sequence that is at least 70% identical to SEQ ID NO: 10. In some embodiments, polypeptides, fusion polypeptides, or compositions comprise a polypeptide that has an amino acid sequence as shown in SEQ ID NO: 10. In some embodiments, polypeptides, fusion polypeptides, or compositions comprise a polypeptide comprising a 20 sequence motif that binds to a double stranded DNA. In some embodiments, polypeptides, fusion polypeptides, or compositions comprise a polypeptide that is encoded by a nucleotide sequence that is at least 70% identical to SEQ ID NO: 9.
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