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Iiillllllllli'lllll | US005627051A United States Patent [191 [11] Patent Number: 5,627,051 Duman [45] Date of Patent: May 6, 1997

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Iiillllllllli'lllll | US005627051A United States Patent [191 [11] Patent Number: 5,627,051 Duman [45] Date of Patent: May 6, 1997 llilll|||l|l|||||ll||||||i|lllllllllllllllllllllllllllll.nIiillllllllli'lllll | US005627051A United States Patent [191 [11] Patent Number: 5,627,051 Duman [45] Date of Patent: May 6, 1997 [54] NUCLEIC ACID SEQUENCES ENCODING LG. Duman et al.. ‘Thermal hysteresis proteins”. Advances DENDROIDES ANTIFREEZE PROTEINS in Low Temperature Biology, vol. 2. pp. 131-182, (1993). [75] Inventor: John G. Duman. South Bend. Ind. D. Tursman et al., “Cryoprotective eifects of Thermal Hys teresis Protein on Survivorship of Frozen Gut Cells from the [73] Assignee: University of Notre Dame du Lac, Freeze Tolerant Centipede Lithobius for?catus”, Journal of Notre Dame, Ind. Experimental Zoology. vol. 272, pp. 249-257 (1995). D.W. Wu et al., “Activation of antifreeze proteins from [21] Appl. No.: 485,359 larvae of the beetle Dendroides canadensis”, Comp. Physiol. [22] Filed: Jun. 7, 1995 B, vol. 161, pp. 279-283 (1991). [51] Int. Cl.6 ........................... .. C12P 21/02; C07H 21/04 D.W. Wu et al., “Puri?cation and characterization of anti [52] US. Cl. .. 435/69.1; 536/235; 536/2431 freeze proteins from larvae of the beetle Dendroides [58] Field of Search .............................. .. 435/3201, 69.1; canadensis”, Comp. Physiol. B, vol. 161, pp. 271-278 536/235. 24.31 (1991). [56] References Cited Primary Examiner—Dian C. Jacobson PUBLICATIONS Assistant Examiner-Kawai Lau J.G. Duman et al., “Hemolymph proteins involved in insect Attorney, Agent, or Firm—Barnes & Thomburg subzero tolerance: Ice nucleators and antifreeze proteins”, In Insects at Low Temperatures, Chapman and Hall (N .Y.), pp. [57] ABSTRACT 94-127 (1991). The present invention is directed to nucleic acid sequences D.W. Wu et al., “Enhancement of insect antifreeze protein encoding Dendroides canadensis thermal hysteresis pro activity by antibodies”, Biochem. Biophys. Acta 1076, teins. The THPs of Dendroides have signi?cantly greater 416-420 (1991). thermal hysteresis activity than any other known anti-freeze LG. Duman et al., “Adaptations of insects to subzero temperatures”, Quart. Rev. Biol. 66, (4) pp. 387-410 (1991). protein. The thermal hysteresis activity of the puri?ed THPs LG. Duman et al., “Hemolymph Proteins involved in the can be further enhanced by combining the THPs with cold tolerance of terrestrial arthropods: Antifreeze and Ice various “activating” compounds. Nucleator Proteins”. In Water and Life, Springer-Verlag (Berlin). Chapters 17 & 18 pp. 282-315, (1992). 4 Claims, 5 Drawing Sheets US. Patent May 6, 1997 Sheet 1 of 5 5,627,051 3 l 2 - .--l Thermql Hysferesls 2 Pc) . ,.../.... , Protein Concen’rra'rion (mg/ml) 0 4O 80 I20 I60 AFP (mg/ml) US. Patent May 6, 1997 Sheet 2 of 5 5,627,051 O<< <06 PDQ. HE. FOP Poe FOF POF PE **§ *** *** UUU PE “555:4Gun-oz2:.3muucozvwwas35.5%A:?ux-5:2# PUP POP <90 U00 U00 *** axx OOH P<< U<< F<< O<< <<O F<U U00 <UU PUP POP POP OF0 UOF PUP PUP 3mmbum wswcowzoUouzosvvm up?6xk6 mb N: Mg 02 @NN New wow U.S. Patent May 6, 1997 Sheet 4 0f 5 5,627,051 Percent - Survival 40: l -30 -25 ~20 -15 -1O -5 0 Temperature (C) F16 5m 80: Percent 60': Survival 40: 20" 0‘ ' ‘ -3o -25 -2o -15 -1o -5 0 Temperature (C) FIE 5% Percent 60 Survival 40-_ 20" -30 -25 ~20 --15 -10 -5 0 Temperature (C) F170;‘. 54: U-S. Patent May 6, 1997 Sheet 5 of 5 80" Percent 60' Survival 40"‘ 0 -30 -_25 -20 -15 -10 -5 0 Temperature (C) F16 5d 80' Percent ~ _ - Survival 40'. ' 20 -30 -25 -20 -15 -1o -5 0 Temperature (C) F16. 5e 5 ,627,051 1 2 NUCLEIC ACID SEQUENCES ENCODING DENDROIDES ANTIFREEZE PROTEINS TABLE 1 Amino acid compositions (mol %) of representative THPs. Species from a broad phylogenetic range of insects have been reported to produce thermal-hysteresis proteins Amino 1 2 2 (THPs). THPs are believed to play an important role in many acid (H-l) T-4 T-3 3 4 plant and animal species’ ability to survive exposure to Asx 14.3 7.3 5.3 9.5 7.1 subzero temperatures. By de?nition. the equilibrium melting Thr 17.2 i 6.6 2.3 6.0 2.7 Ser 10.3 7.4 11.1 13.0 30.5 and freezing points of water are identical. However. the Glx 5.2 8.9 12.4 11.0 12.3 presence of thermal-hysteresis proteins lowers the non Pro 2.6 5.9 0.0 5.0 0.0 equilibrium freezing point of water without lowering the Gly 6.6 8.3 11.4 15.0 20.0 Ala 8.4 14.3 5.0 8.0 6.8 melting point (equilibrium freezing point). Thus when THPs I/2Cys 15.9 0.0 28.0 6.0 0.0 are added to a solution they produce a diiference between the Val 1.7 11.5 2.3 3.0 3.0 freezing and melting temperatures of the solution. and this Met 0.2 4.8 0.0 0.0 0.0 difference has been termed “thermal-hysteresis”. 116 1.5 7.1 1.0 1.2 1.9 1.611 1.9 0.0 2.2 6.5 3.1 In the absence of THPs a small (about 0.25 mm diameter) Lys 3.4 6.8 15.4 3.1 7.5 ice crystal that is about to melt at the melting point tem Arg 4.8 2.6 0.0 8.0 0.0 perature will normally grow noticeably if the temperature is 'Iyr 3.9 2.3 0.0 1.0 2.0 Phe 0.0 3.9 0.0 2.2 1.1 lowered by 0.0l° to 0.02° C. However. if THPs are present, 20 H15 1.9 1.9 3.1 0.0 2.3 the temperature may be lowered as much as 5° to 6° C. below the melting point (depending upon the speci?c activ l = Dendroides canadensis; 2 = Tenebn'o malitar; 3 = Choristoneura ity and concentration of the proteins present) before notice fumg'ferana; 4 = Oncopeltus fasciatu: able crystal growth occurs. Consequently, because of THPs Several of the insect THPs have signi?cant amounts of ability to lower the freezing point of aqueous solutions. they 25 cysteine. In particular 16 mole % of the amino acid residues are commonly referred to as antifreeze proteins. THPs lower of the THPs of D. canadensis are cysteine, approximately the freezing point of aqueous solutions via a non-colligative half of which are involved in disul?de bridges. Treatment with dithiothreitol, which reduces these disul?de bonds, or mechanism that does not depress the vapor pressure or raise alkylation of free sulfhydryls, results in complete loss of the osmotic pressure of water, as is the case with colligative 30 thermal-hysteresis activity. type antifreezes such as glycerol. The thermal-hysteresis activity present in the Anti-freeze proteins are believed to exert their eifect by hemolymph (the circulatory ?uid of insects) of Dendroides adsorbing onto the surface of potential seed crystals via canadensis insects in mid-winter averages 3°—6° C. with hydrogen bonding. When anti-freeze proteins adsorb onto some individuals having as much as 8°-9° C. This activity the ice surface they interfere with the addition of water 35 is signi?cantly greater than that which is present in the blood molecules and force growth of the crystal into many highly of polar ?sh; the maximal activity achievable with very high curved fronts with high surface free energy. Consequently, concentrations of the ?sh THPs is about 1.7° C. FIG. 1 growth of the crystal requires the temperature to be further compares the activities of puri?ed THPs from two species of lowered to allow crystal growth to proceed ?sh and two species of insects: l=Dendr0ides canadensis Thennal-hysteresis proteins were ?rst discovered, and 40 THP, the most active THP currently known (solid squares); have been best studied, in marine teleost ?shes inhabiting 2=Tenebrio molitor THP (solid circles); 3: activity of the seas where subzero temperatures occur, at least seasonally. most active ?sh THP, i.e. Antarctic eelpout and THP of In these THP-producing Antarctic ?sh an ice crystal present Antarctic nototheniids (open squares); 4: activity of the in the blood serum that melts at -—1.1° C., will fail to grow least active ?sh THP, from cod Gadus morhua (open in size until the temperature is lowered to —2.5° C. (thermal 45 circles). The maximal activity of the most active ?sh and T hysteresis=l.4° C.). Thus the ?sh is protected from freezing molitor THPs are similar, however, the THPs of D. canaden in its ice-laden —1.86° C. seawater environment. sis have a greater speci?c activity and a much greater Over the last 15 years it has been demonstrated that many maximal activity than any other known THP. In addition as insects and other terrestrial arthropods (including certain Will be described later, the Dendroides canadensis THPs can spiders, mites, and centipedes) also produce thermal 50 be activated by the presence of certain other proteins to hysteresis proteins. Low levels of thermal hysteresis activity produce even greater levels of thermal hysteresis activity. are also quite common in overwintering plants and are The present invention is directed to nucleic acid present in certain fungi and bacteria. sequences encoding peptides having antifreeze properties, Thermal-hysteresis proteins (THPs) have been isolated wherein the nucleic acid sequence comprising sequences ?'om four species of insects: Tenebrio molitor, the milkweed 55 from a species selected from the genus Dendroides. The bug, Oncopeltus fasciatus, the spruce budworm, Choristo activity of Dendroides antifreeze proteins is three to six neurafumiferina, and D.
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