CZ20002696A3 - Antifreeze protein - Google Patents

Abstract

Describes an antifreeze protein containing at least 40% amino acids S, T or N and showing at least 80% overlap with the following amino acid sequence D-E-Q-P-N-T-I-S-GS-N-N-T-V-R-S-G-S-K-N-V-L-A-G-N-D-N-T-V-I-S-G-D-NN-S-V-S-G-S-N-N-T-V-V-S-G-N-D-N-T-V-T-G-S-N-H-VV-S-G-T-N-H-I-V-T-D-N-N-N-V-S-G-N-D-N-N-V-S-G-SF-H-T-V-S-G-G-H-N-T-V-S-G-S-N-N-T-V-S-G-S-N-H-V-VS-G-S-N-K-V-V-T-D-A and its modified versions that do not affect the inhibition properties for ice recrystallization

Description

Technical area

The invention describes antifreeze proteins and food products that contain AFP.

(AFP) and frozen

State of the art

Antifreeze proteins (AFPs) are used to improve the tolerance of foods to freezing.

The use of AFP for these purposes is well known in the art, particularly suitable are those proteins which exhibit activity to inhibit ice crystal growth (described, for example, in US 5,118,792 ).

WO 90/13571 describes antifreeze peptides produced chemically or by recombinant DNA methods. AFPs are suitable for use in food products. Example 3B describes a method for modifying the shape of ice crystals when a water-ice mixture is frozen in a film in combination with 0.01% (w/w) AFP.

WO 92/22581 describes plant AFPs which can be used to control ice crystal shapes in the preparation of ice cream. This document also describes a method for extracting a polypeptide composition from the intercellular space in plants by infiltrating leaves with an extraction medium without damaging the plants.

Document 94/03617 describes the production of AFPs from yeast and their use in the preparation of ice creams. Document WO 96/11586 describes fish AFPs produced by microbes.

Several references in the literature also describe the isolation and/or use of plant proteins in cryopreservation. Cryopreservation proteins function to protect plant membranes against • · freezing damage. These proteins do not exhibit recrystallization inhibition properties and are therefore not associated with the terms AFP.

Hincha, Journal of Plant Physiology, 1992, 140, 236-240 describes the isolation of proteins for deep-freezing protection of cabbage. Volger, Biochimica et Biophysica Acta, 412 (1975), 335-349 describes the isolation of proteins for deep-freezing protection of spinach. Boothe, Plant Physiol (1995), 108: 759-803 describes the isolation of proteins from Brassica napus. It is believed that the proteins have deep-freezing protective properties rather than AFP. Neven, Plant Molecular Biology 21: 291-305, 1993 describes the characterization of a spinach protein that exhibits deep-freezing protective properties. Salzman, Abstracts and Reviews of the 18 ^th Annual Meeting of the ASEV/Eastern Section in Am. J. Enol. Vitic., Vol. 44, NO. 4, 1993, describes the presence of polypeptides that are stable at boiling point in the buds of the plant vitis. Although these proteins are analogs of fish antifreeze peptides, they are proteins that exhibit deep freezing protection, but they are not AFPs. Lin, Biochemical and Biophysical research Communication, Vol. 183, No. 3, 1992, p. 1103-1108 and Lin, Plant Physiology (1992) 99, 519-525 describe a deep freezing protection polypeptide with a molecular weight of 15 OOO from Arabidopsis Hakaira. Houde, The Plant Journal (1995) 8(4), 583-593 describes cryoprotective proteins from wheat.

However, AFPs have not been applied in commonly available foods to date. One reason is the high cost and complicated method of obtaining AFPs. Another reason is that AFPs cannot be incorporated into standard mixtures because they tend to destabilize during processing, especially during the pasteurization step. This destabilization is believed to be caused by

3· · ♦ · · · ···· • · ·· ····· ·· · • · · · ··

PCT/EP97/03634 discloses that AFPs can be isolated from natural sources, such as grass acclimatized to low temperatures, by a novel, relatively simple method. This method first leads to the identification of AFPs, which can be conveniently added to the mixture for the preparation of frozen products prior to pasteurization.

This procedure for obtaining AFP from grass includes the steps:

a) isolation of juice containing AFP from grass,

b) treatment of grass or juice containing AFP at a temperature of at least 60 °C,

c) removal of insoluble fractions.

The step described in paragraph C) typically occurs after the steps described in paragraphs a) and b). The step described in paragraphs a) and b) may be performed in any desired order. For example, step a) is followed by step b) (in which case the AFP-rich juice is heated), or step b) is followed by step a) (in which case the natural source is heated), or steps a) and b) occur simultaneously.

This process has a number of advantages. First, it is not necessary to prevent the grass from being damaged by this process, as is the case with the process described in WO 92/22581. This immediately significantly increases the commercial applicability of the process compared to, for example, the process described in WO 92/22581, since high costs for specific processing are not necessary. Also, using high temperatures, it is possible to extract from a large group of peptides present in grass AFPs that are very active in inhibiting ice recrystallization. Third, contrary to what was expected, using high temperatures does not denature all protein materials, but only some proteins seem to denature, while the remaining AFPs of grasses show increased stability to temperature. This allows the inclusion of isolated AFPs in compositions that need to be

exposed to high temperatures, for example during pasteurization. This is particularly surprising since the AFP of WO 92/22581 does not appear to be stable upon heating.

The process described above comprises, in the step described in paragraph b), heating the AFP-rich grass or juice to a temperature of more than 60°C. It is preferred that the temperature is in the range of 60 to 110°C, more preferably in the range of 80 to 105°C. The heating step is carried out after the isolation of the protein-rich juice (step described in paragraph a)) or before the isolation of said juice. Any suitable method of heating the juice, heating in an added extraction medium, heating with steam, etc. can be used.

If an extraction medium is used, it may preferably be used in small volumes to avoid the necessary dilution of the AFP fraction. Any suitable extraction medium may be used, although the use of water is particularly preferred. If necessary, additives may be added to the water before it is used as the extraction medium. It is most preferred to use water that is free of additives.

It has been found that a highly active AFP can be obtained from grass in this way. According to the invention, the name grass includes members of the family Gramineae, which includes, for example, perennial grasses such as Lolium perenne, Parapholis strigosa, Nardus strica, Catapodium loliaceum and Lolium multiflorum, Poa trivialis, Poa pratensis and cereals such as winter rye and winter barley. The amino acid sequence of AFP has been determined.

The essence of the invention

Surprisingly, it has been found that highly active AFPs obtained from plants are characterized by a high amount of the amino acids: serine (S), threonine (T) and asparagine (N). It has further been found that preferred AFPs according to the invention are characterized in that at least 40% of the amino acids in the protein are selected from the group consisting of S, T and N. It is preferred that the AFPs are obtained from grasses, in particular from the Gramineae family.

• ·

The preferred molecular weight of the AFP according to the invention is 8,000 to 16,000, more preferably 10,000 to 14,000, this molecular weight being determined from the gene sequence or by mass spectroscopy of the unmodified form, for example in the form which is not glycosylated.

The invention further relates to an AFP obtainable from grass, said AFP having the amino acid sequence (from the N-terminus):

_d _ _e _q-PNTISGSNNTVRSGSKNVLAG-NDNT-Vj-SGDNNSVSGSNNTVVSGNDNT-VTGSNHV _v _S_G-TNHIVTDNNNNVSGNDNNVS--GSFHTV _s _ _g _ _g _H-NTVSGSNNTVSGSNHVVSGSN-KVV-TD-A

The invention also includes proteins having a sequence with a high degree of similarity to the above-described sequence. The invention further describes all RI active proteins having an amino acid sequence showing at least 80% overlap with the above-described sequence. More preferably, at least 90% overlap, most preferably more than 95% overlap, for example, amino acid sequences that are either the same as or differ by only two amino acids from the above-described sequence. Such isoforms of the above-described protein of the invention.

The invention further describes modified versions of the proteins, wherein said modifications do not materially affect the ice recrystallization inhibitory properties. Such a form is, for example, their glycosylated version.

The AFP-rich juice can be separated from the grass by any suitable method, for example, pressing, filtration, homogenization, subject to the above-described β

• · ·· 99 · ♦ • · · · · · · · · · · · · · · • 9 9 · 9 9 99··

9 9 9 9 9 999 · 9 9 9 9 • 9 ·· ···· ···· 9999 99 ·· ·· ·· extraction etc. It is preferred that the grass is chopped into small pieces or made into a mash before the protein-rich fraction is collected. The maceration can be carried out in any suitable manner, for example in a blender. The skilled person is able to prepare the grass in such a way that the step of capturing the protein-rich juice can easily take place.

After collecting said juice and heating (in the desired order), the protein fraction of the resulting sample containing AFP can then be processed in a suitable manner to remove the insoluble fraction and leave only the liquid fraction rich in AFP. For example, the insoluble fraction can be removed by filtration, precipitation, etc. The liquid rich in AFP can then advantageously be processed to increase its concentration or to isolate the AFP, thereby bringing it into a form suitable for further use. Examples of suitable processing are drying, whereby a powder or paste is obtained, upon concentration, a concentrate of AFP is obtained, further chromatography, whereby AFP is separated from the extraction medium, etc. It is easy for a person skilled in the art to determine suitable methods and conditions for proper isolation.

A nucleic acid sequence encoding the AFP described above has also been determined. The invention further describes a nucleic acid sequence capable of encoding the AFP of the invention.

It is preferred that said nucleic acid has the sequence:

GAT GAA CAG CCG AAT ACG ATT TCT GGG AGC AAC AAT ACT GTC AGA TCC GGG AGC AAA AAT GTT CTT GCT GGG AAT GAC AAC ACC GTC ATA TCT GGG GAC AAC AAT AGT GTG TCT GGG AGC AAC AAC ACT GTC GTA AGT GGG AAT GAC AAT ACC GTA ACC GGC AGC AAC CAT GTC GTA TCA GGG ACA AAC CAT ATC GTT ACA GAC AAC AAC AAT AAC GTA TCC GGG AAC GAT AAT AAT GTA TCC GGG AGC TTT CAT ACC GTA TCC GGG GGG CAC AAT ACT GTG TCC GGG AGC AAC AAT ACC GTA TCT GGG AGC AAC

CAC GTT GTA TCT GGA AGC AAC AAA GTC GTG ACA GAC GCT TAA ·· ·· ·· • ♦ · · · ········ · ·

The invention further provides alleles or other nucleic acid sequences capable of encoding the AFPs described above. These are, for example, nucleic acid sequences where in the above sequence one or more codons are replaced by their synonyms (i.e. codons encoding the same amino acid).

The invention further describes vectors that contain a nucleic acid sequence capable of encoding an AFP according to the invention.

Based on the above information, it is also possible to genetically modify other natural sources so that they produce AFP.

It has also been found that the AFP of the above sequence exhibits improved ice recrystallization inhibitory properties. A suitable assay for determining recrystallization inhibitory properties is described in the examples. It involves rapid freezing to -40°C followed by storage at -60°C. Preferably, the AFPs which are subjected to the assay after heat treatment are in the form of ice crystal particles which are less than 5 pm larger in size than the ice crystal size in a sample to which the same AFP has been added but the sample has not been heat treated. Preferably, the difference is less than 3 pm, most preferably, the difference is less than 1 pm.

Preferably, those AFPs are selected which exhibit substantial ice recrystallization inhibitory properties. A suitable assay for determining recrystallization inhibitory properties is described in the Examples. It is preferred that the AFPs of the invention provide ice particles after an ice recrystallization inhibitory assay as described in the Examples, of 15 pm or less in size. More preferably, the particle size is from 5 to 15 pm.

The AFP according to the invention can be advantageously used in food products, with food products which are frozen or intended to be frozen being preferred. The use of AFP in products which are particularly preferred is • · • 9 9 9 9 9 9 9 4

9999 9999 99 99 99 99 which are heated before freezing, for example by pasteurization, blanching or sterilization. It is particularly preferred for use in frozen confectionery products.

Examples of such products are: frozen confectionery mixes, such as ice cream mixes, which are pasteurized before freezing. Such mixes are usually stored at ambient temperature. Suitable product forms are, for example, a powder mix, which is packaged, for example, in a sachet or pouch. Said mix is capable of forming the basis of a frozen food product, for example after the addition of water and other ingredients and after possible aeration.

Another example of a suitable mixture could be a liquid mixture (which can be aerated) which can be frozen after adding other ingredients and aeration.

An obvious advantage of the above mixtures is that the presence of AFP allows the mixture to be frozen under conditions where mixing is not necessary, for example in a store or in a home freezer, without the formation of unacceptable ice crystal shapes and a structure that differs from that of products obtained by quick freezing.

It is very convenient to pack this mixture in containers (e.g. boxes, bags, plastic containers, etc.). In the case of a single portion, the package size is generally 10 to 1000 g. In the case of multiple portions, the package size is up to 500 kg. In the general case, the package size is 10 g to 5 OOOg.

As mentioned above, the preferred products where AFP is used are frozen confectionary products such as ice cream. The amount of AFP is preferably 0.00001 to 0.5% (w/w) of the final product. If dry mixes or concentrates are used, the AFP concentration must be higher to ensure that the amount of the final product is within the above range.

For purposes of the invention, the term "frozen confectionary product" includes frozen confections such as ice cream, frozen yogurt, frozen sorbet, sorbet, milk ice cream and egg toto • to

·· ·· ·· to · · to ·· to • to · · toto to • ·>· * · to· · • · · · · · · · · · · to· frozen cream, water ices and frozen fruit purees. In some cases, its use in fermented food products is less preferred.

It is preferred that the amount of solids in the frozen confectionery products (e.g. sugar, fat, flavors) is greater than 4% (w/w). For example, to be greater than 30% (w/w), an amount of from 40 to 70% (w/w) is more preferred.

The frozen confectionery products of the invention can be produced by any method suitable for the production of frozen confectionery products. It is particularly preferred that all ingredients are thoroughly mixed before pasteurization and freezing. The freezing process preferably includes hardening, for example, at a temperature of -30°C or lower.

Examples of embodiments of the invention

Example 1:

AFP isolation, where the grass is first heated, then the juice with a high AFP content is isolated and AFP is isolated from it.

In January, tissue from various grasses (Poa trivialis, Lolium perenne, Holcus lanatus, Bromus sterilis) was cut (the average temperature in this month is 3.5 °C to ensure proper cold acclimation of the plants). The grass tissue was quickly transported to the laboratory for further processing. It was washed with water to remove impurities.

A 500 g portion of cut grass was placed in a 659 watt microwave oven and heated at full power for 5 minutes, with the temperature rising to 85 to 100° C. The cut grass was then cooled to ambient temperature.

In another case, the grass pieces were mixed with 500 g of boiling water and the mixture was heated again to a temperature of 100 °C, followed by heating for 10 minutes with constant stirring and then cooling to a temperature of 60 °C.

φφ φφ φφ φφ φ® φφ φφφφ φφφφ φ··· φ φ φφφφ φφφφ

Π Φ ΦΦΦΦ Φ·Φ Φ φ φ φ Φ

J- '-Ζ · · ·Φ ΦΦΦΦΦ φφφφ φφφφ φφ φφ φφ φφ

After heating, the AFP-rich juice was separated from the chopped grass by filtration. The mass was continuously stirred for 5 minutes in the presence of an equal volume of water and pressed through three layers of gauze.

The supernatant may be freeze-dried to remove water and then stored. Alternatively, the supernatant may be frozen for storage.

Example 2:

A liquid mixture suitable for making ice cream was prepared by mixing:

ingredients % (wt.) skimmed milk powder 10.00 sucrose 13, 00 maltodextrin (MD40) 4.00 carob gum 0.14 butter oil 8.00 monoglyceride (palmitate) 0.30 vanillin 0.01 AFP (described in Example I*) 0.01 or none (control) water balance

*note: AFP is added as a concentrated AFP solution in a small amount of water, the percentage refers to the amount of AFP.

The ingredients were mixed at ambient temperature, followed by pasteurization for 60 seconds at 89°C. The mixture was aseptically filled into 500 ml bags. The bags were sealed and stored at ambient temperature.

The mixture is used to prepare ice cream by whipping with a suitable home blender, followed by freezing in a home freezer without the need for mixing.

After two months of storage according to the invention, the mixture had a noticeably better structure than the control sample.

• · • ftft • · · • · ft ftft. ft ftft ·

Example 3:

The ice recrystallization inhibitory properties of AFP can be determined as follows.

The AFP-containing product sample was adjusted to a sucrose content of 30% (w/w) (if the initial sample content was higher than 30%, then this was done by dilution; if the initial sucrose content was lower than 30% (w/w), then sucrose was added to this content).

A 3 μΐ drop of sample was placed on a 22 mm coverslip. The drop was covered with a 16 mm coverslip and a 200 g weight was placed on the sample to ensure uniform thickness. The edges of the coverslips were sealed with nail polish.

The slide was placed under a Linkham THM 600 temperature-controlled microscope. The microscope stage was rapidly cooled (50 °C per minute) to -40 °C, forming a large population of small crystals. The microscope stage temperature was then rapidly increased (50 °C per minute) to -6 °C and held at this temperature.

The ice phase was observed at -6 °C using a Leica Aristoplan microscope. To enhance the contrast of the ice crystals, polarized light was used in conjunction with a lambda plate. The ice phase stage (ice crystal size) was recorded with 35 mm photomicrographs at time T=0 and T= 1 hour.

In general, this test can be applied to any suitable composition comprising AFP and water. In general, the amount of AFP in such a test composition is not very critical and can, for example, range from 0.0001 to 0.5% (w/w), more preferably from 0.0005 to 0.1% (w/w), most preferably from 0.001 to 0.05% (w/w), for example 0.01% (w/w).

Any suitable composition containing AFP and water may be used in the assay. However, it is generally not necessary to isolate AFP in pure form. In practical use ·* • ti • ••ti titititi ti· • titi ti • ti ti ti

It is sufficient to prepare a liquid extract or juice from the natural material, and this “extract or juice” can then be tested.

This method can then be applied, for example, to AFP-containing extracts obtained in Example I, with or without a concentration step.

The recrystallization inhibition properties of several samples were measured. The AFP-containing juice obtained after extraction and heating according to Example I was measured for its recrystallization properties as described above.

An extract of grass harvested in January, which had not been heat treated, was obtained from Silsoe (UK). The extract was centrifuged for one hour to remove soil and insoluble impurities (centrifuge: Sorvall RC3C, rotor: H6000A, temperature: + 5°C, speed 5,000 rpm, 7,268 xg).

A sample of the extract was freeze-dried to determine the total solids content. This value was found to be 11.48 mg/ml. The dry extract was then rehydrated with 30% sucrose solution to its original solids concentration. Several solutions were prepared by diluting the extract with 30% sucrose solution.

The ice crystal recrystallization inhibitory activity was measured using the assay described above.

At T=0 and T=1, the recrystallization inhibition tests showed the average size of the ice crystals measured using a Zeiss TGA 10 analyzer. The ice crystal size (length) was determined by tracing the crystals. The maximum length for each individual ice crystal in the ice cream batch was transferred to a spreadsheet where the data was analyzed and the mean and standard deviation were determined.

The results obtained are presented in the table below:

• · • ft ·· ·· • · · « · · · • · · · · · · • ····· · · · • · · · · ·

sample total solids (mg/ml) ice crystal size (μπι) ice crystal growth in one hour at -6°C (μιη) T=0 T=1 hour at a temperature of -6 °C undiluted 11.48 5.2 7.3 2.1 50% extract 5.74 5, 5 7.6 2.1 25% extract 2.87 6, 3 8.9 2.6 12.5% extract 1,435 6, 6 13.1 6.5 6.25% extract 0.7175 8.1 14.7 6, 6 3.125% extract 0.359 7.4 17.0 9.6 1.5625% extract 0.179 9.0 20.3 11.3

These results show the differences in final crystal size and the change in crystal size over one hour at 6°C for different dilutions of the grass extract. It can be seen that the amount of solids in the grass extract can vary over a wide range while still obtaining good recrystallization inhibition properties. Those concentrations are preferably selected where their use results in an ice crystal size of 15 micrometers or less after one hour.

A similar test was performed with the grass extract subjected to heat treatment (10 minutes at 100°C). No significant reduction in the recrystallization inhibitory properties could be observed.

Additionally, the grass extracts of Example I were tested using the same recrystallization inhibition assay. The following results were obtained:

heat treatment crystal size T=0 T=1 60 °C for 1 hour 9, 6 11.1 boil for 10 minutes WHAT 11.3

These results show that even after warming the cold-acclimated grass extract, its ability to inhibit ice crystal growth was maintained.

Example 4:

Perennial ryegrass (Lolium perenne) seeds were obtained from Barenbrug (UK) Ltd. These seeds were sown in 22.5 cm pots with Levingston No. 2 compost and after fertilization, which usually occurred between 4 and 7 days after planting. The plants were grown in the greenhouse for a further 7 to 10 days until they reached a size of approximately 15 to 20 cm. The plants were then transferred to a cold room for cold acclimation. Acclimation took place for 30 days at +4 °C with 8-16 hours of light/dark.

Leaves of a cold-acclimated plant were cut and homogenized in 50 mM Tris, 10 mM EDTA, pH adjusted with concentrated HCl. Homogenization was performed for 2 x 30 seconds in a Waring blender, with a buffer to leaf ratio of 2.5:1 (w/w).

The homogenate was then placed in a boiling water bath for minutes and allowed to drain through layers of gauze. Collected • · · · • · · ·

000 xg on an 8 x • ♦ · · • · · 9 • · · · • · · · · · · · The supernatant was further centrifuged at 50 ml Sorvall centrifuge.

Recrystallization properties of ice described in Example 3.

The heat-stable extract (10 to 50 ml) was concentrated approximately tenfold on an Amicon using a PM10 membrane. 1 ml aliquots were desalted on a Fast Desalting Column in an FPLC separation system (Pharmacia). The column was operated at a flow rate of 1 ml/min in 50 mM Tris/Cl, 10 mM EDTA, pH 8.5. Fraction collection (1 ml volume) was started when the O.D. at 280 nm began to increase. The R1 active fractions were pooled to a volume of 3 ml.

Samples with R1 activity were loaded onto a Mono Q column (HR5 x 5) equilibrated with the same Tris buffer pH 8.5. The effluent was collected as a single fraction and the column was immediately eluted with a linear gradient of 0 to 0.25 M NaCl over 25 minutes at a flow rate of 1 ml/min. All activity was found to remain unbound and eluted in the flow-through fraction.

The pH of the flow-through fraction was carefully adjusted to pH 9.5 using one microliter drops of 4M NaOH and the fraction was loaded onto a Mono Q column which was re-equilibrated with Tris buffer pH 9.5. The flow-through fraction was collected as a single fraction and the column was then immediately eluted with a linear NaCl gradient from 0 to 0.25 M over 25 minutes. The flow rate was 1 ml/min. 1 ml fractions were collected and the Rl assay was performed. It was found that all of the Rl activity remained bound to the column and no activity was detected in the flow-through fraction. The activity eluted as a single peak between 0.08 and 0.14 M NaCl.

The active fractions obtained from the Mono Q column were pooled and concentrated to a volume of 50 microliters on a PM10 device.

Centricon and applied to a Sephadex 75 column in a Smart system (Pharmacia). The column was equilibrated with 50 mM buffer and tested as follows: • ·

Tris/Cl pH 9.5 and the flow rate was 50 μΐ/min. After draining a volume of 1 μΐ from the column to remove the column headspace, 50 ml fractions were collected. The active fractions were tested on SDS PAGE.

Samples were then run on 10% Nu Page gels from Novex. Samples and gel buffer were prepared according to the manufacturer's instructions, and the gels were stained using a Novex silver staining kit. The active fractions contained a single stained protein band with an apparent molecular weight of 29,000.

Protein samples were also separated by SDS PAGE followed by electroblotting onto a PVDF membrane, which is based on the Matsudaira method in 10 mM Caps buffer, pH 11.0, and 10% methanol. The membrane was wetted with methanol and then equilibrated with Caps buffer for 10 minutes. Blotting was performed for 16 hours at a constant voltage of 20 volts. After blotting, proteins were visualized by staining the membrane with 0.2% Coomassie brilliant blue in 50% methanol, 1% acetic acid for 1 minute. The membrane was then destaining with 50% methanol until protein bands were clearly visible. The membrane was washed with Milia Q, air-dried, and stored at -20°C.

The N-terminus of the 29,000 band was sequenced using conventional methods that provide the amino acid sequence.

A protein with a molecular weight of 29,000, whose RI activity is considered to be tolerant to boiling temperature, was immobilized on a PVDF membrane after separation by SDS PAGE. The N-terminus of this protein was sequenced and 15 of the first 17 residues were successfully sequenced. A protein with a molecular weight of 27,000 isolated from Poa pratensis was also sequenced at its N-terminus (Table 1) and was shown to have the same sequence, suggesting that it is part of the same protein family. This suggests that there is a single class of AFP responsible for the boiling temperature tolerance of RI activity in Gramineae.

Table No. 1

Sequence alignment of the N-terminal RI active protein with a molecular weight of 29,000, originating from Lolium perenne, and a protein with a molecular weight of 27,000 from Poa pretensis.

Lolium -DEQPNTISGXNNTVRXG

Poa -AETPNTISGTNN

Example 5:

Initial

1:1-phenol increased approximately Amicon chambers extraction was performed with a chloroform mixture, followed by extraction with chloroform alone. Phenol denatures proteins, exposing hydrophobic amino acid residues that allow the protein to pass into the organic phase. More hydrophilic nucleic acids and carbohydrates form part of the aqueous phase. Proteins with a substantial degree of glycosylation form part of the phase interface. Chloroform acts as a protein resolving agent more effectively when two different organic solvents are used and a final extraction with chloroform removes any remaining traces of phenol.

A heat-stable extract was prepared from the leaves of Lolium perenne plants and its concentration was tenfold using ultrafiltration with a membrane that separates molecules with a molecular weight of 10,000. The resulting concentrate (volume 0.05 ml) was applied to a Superdex 75 PC 3.2/30 gel filtration column, which operates on the basis of the Smart microseparation system (Pharmacia). The column was eluted with 50 mM Tris/Cl buffer pH 9.5 and a flow rate of 0.05 ml/min and fractions of 0.05 ml were collected. Individual fractions were tested for ice recrystallization inhibitory activity and the most active fractions were pooled together. These were then applied to a Mono Q PC 1.6/5 column, which was equilibrated with 50 mM Tris/Cl buffer pH 9.5, also operating on the basis of the Smart system. The flow rate was 0.1 ml/min. After loading and washing, the column • 9 99 • ♦ 9 ·

9 9 ·

9 9 9

9 9 9

9 a · eluted with a linear sodium chloride gradient with a concentration gradient of 0 to 0.4 M and 0.1 ml fractions were collected. The fractions were assayed and the activity eluted as a single peak at approximately 0.1 M sodium chloride concentration.

The active fractions were vortexed with an equal volume of phenol:chloroform:isoamyl alcohol (1:1(24:1)) for 1 minute. The aqueous (upper) and organic (lower) phases were collected separately without a visible phase interface. The two phases were then precipitated by adding ten volumes of cold acetone and stored at -20°C for 16 hours. The resulting pellet was resuspended in 50 mM Tris/Cl buffer and samples were assayed for RI activity and subjected to SDS PAGE.

Assay of resuspended fractions shows that the RI activity is entirely in the aqueous phase. The organic phase contained no RI activity even after the fraction was denatured/renatured with 6M guanidine hydrochloride. This is an unexpected result, as the protein should migrate to the organic phase or the glycoprotein should remain at the phase interface. However, analysis by SDS PAGE showed that although most of the protein is in the organic phase, there is a single protein band with an apparent molecular weight of 29,000 in the aqueous active fraction, but no such band is visible in the organic phase. The band stained a different color than the other proteins and was very diffuse.

A similar phenol:chloroform extraction was performed with a boiling-tolerant extract prepared from cold-acclimated leaves of meadow grass (Poa pratensis var Barcelona). The extract also preferentially partitioned RI activity into the aqueous phase and a protein with a molecular weight of 27,000 appeared on SDS PAGE.

Sequencing of the N-termini of the proteins confirmed that they are identical to the AFPs described in Example 4.

• ·

99 9

Example 6:

A degenerate oligonucleotide primer (Lol 1) was designed and synthesized from the N-terminal sequence of the protein (ASP-GLU-GLN-PROASN-THR-ILE) as GAYGARCARCCIAAYACIAT, where Y=C+T, R=A+G and I is inosine.

First-strand cDNA was prepared from 5 pg of RNA from Lolium perenne leaves that had been cold-acclimated for 30 days using Superscript reverse transcriptase (Stratagene) and the oligonucleotide primer OG1 (GAGAGAGGATCCTCGAG (T) 15) according to the manufacturer's instructions. 1% of the first-strand cDNA was used as a template together with primers Loll and OG1 in the PCR reaction. The reactions were then carried out in a thermal cycler using Tag DNA polymerase (Gibco BRL) for 30 cycles (1 min at 94 °C, 1 min at 55 °C and 1 min at 72 °C) after initiating a denaturation step for 2 min at 14 °C.

A PCR product of approximately 600 bp was amplified, isolated from a 1% agarose gel, and cloned into the pTAg vector (R and D systems) according to the manufacturer's instructions. The cloned PCR product was sequenced on a Perkin Elmer automated DNA sequencer (Applied Biosystems) using primers T3 and T7. This product contained an open reading frame that is essentially similar to the sequence:

GAT GAA CAG TCC GGG AGC

TCT GGG GAC AGT GGG AAT GGG ACA AAC AAC GAT AAT CAC AAT ACT CAC GTT GTA

CCG AAT AAA AAT AAC AAT GAC AAT CAT ATC AAT GTA GTG TCC TCT GGA

ACG ATT TCT GTT CTT GCT AGT GTG TCT ACC GTA ACC GTT ACA GAC TCC GGG AGC GGG AGC AAC AGC AAC AAA

GGG AGC

GGG AAT GGG AGC GGC AGC AAC AAC TTT CAT AAT ACC GTC GTG

AAC AAT ACT GTC AGA GAC AAC ACC GTC ATA AAC AAC ACT GTC GTA AAC CAT GTC GTA TCA AAT AAC GTA TCC GGG ACC GTA TCC GGG GGG GTA TCT GGG AGC AAC ACA GAC GCT TAA

·· to· » to· · • · and the deduced amino acid sequence is essentially similar to the sequence:

_d _ _e _q_P_N-TISGSNNTVRSGSKNVLAGND-NT-Vt-sGDNNSVSGSNNTVVSGNDNT-VTGSNHV _v _ _S _ _G -TNHIVTDNNNNVSGNDNNVSG-SFHT-Vs-gghntvsgsnntvsgsnkvvs-gsnkvv-tD-A

The open reading frame encodes a pronein with a molecular weight of approximately 12,000, which is less than its apparent molecular weight on SDS PAGE (29,000). It was also observed that the coding sequence also contains six glycosylation sites (ASN-XSER/THR) and a substantial number of SER and THR residues that may also be glycosylated. This evidence suggests that the native protein isolated from Lolium is highly glycosylated. When the protein is enzymatically deglycosylated, it still retains its ice recrystallization inhibitory activity, suggesting that glycosylation is not essential for the protein to be active.

Example 7:

To demonstrate that the cDNA from Lolium perenne described in Example 6 encodes AFP, the coding region was expressed. A strain of Pichia pastoris, a methylotrophic yeast, was generated that contains the Lolium AFP cDNA.

The Lolium cDNA was cloned into the pPIC9 vector with the α-factor signal sequence to ensure cell secretion and glycosylation. All enzymes were obtained from Boehringer Mannheim and used according to the manufacturer's instructions. Construction of expression vectors, transformation and growth of Pichia yeast were performed as described in the Invitrogen Pichia Expression Kit (Version B) Manual.

The Lolium plant cDNA was cloned into the pPIC9 vector as a PCR amplification fragment with compatible restriction ends suitable for ligation into the pPIC9 vector. This was done in • · ftftftft · ♦ · · · · · · • · ftftftft ····

2i · · · · **· · · · :

ftft·· ftftftft ftft ·· »· ·· using cDNA from the Lolium plant as a template and primers GTAT CT T CGAGAAAAGAGAT G AGC AGC CGAAAC AGAT T and

TTAATTCGCGGCCGCCTGTAGGAAAAGTATGGTATATC, which introduce an XhoI restriction site at the 5' end of the amplified fragment and a NotI restriction site at its 3' end, thus ensuring that the Lolium plant cDNA is in frame with the secretion signal of the open reading frame. The reactions were performed in a thermal cycler using Taq DNA polymerase and the reading enzyme Pfu (Boehringer Mannheim) in 30 cycles (1 minute at 94 °C, 1 minute at 55 °C and 1 minute at 72 °C). All subsequent PCR reactions were performed under the same conditions, but without the Pfu enzyme. The XhoI/NotI cDNA fragment was cloned into the pPIC 9 vector digested with the restriction enzymes XhoI/NotI and competent E. coli cells (strain XL1-blue) were transformed with the vector. After transformation, cells were plated on LB plates with ampicillin at a concentration of 50 μρ/μιΐ and grown at 37 °C for 16 hours. Then, 20 ampicillin-resistant transformants were isolated and analyzed for their incorporation into Lolium cDNA by PCR using primers 5Ά0Χ1 and 3Ά0Χ1, which were synthesized as described in the Invitrogen Kit Manual.

Plasmid DNA was isolated from four positive transformants by miniprep method and the DNA was linearized by restriction enzyme digestion with SalI for transformation into Pichia cells, which were cultured and washed as described in the Invitrogen manual. The linearized DNA was electroporated into Pichia cells using the BioRad GenePulser. After addition of ice-cold 1 M sorbitol, the cells were plated on MD plates and grown at 30°C for 24 hours until visible colonies formed. Sixteen transformants were then isolated on fresh MM and MD plates and transformants that grew on MD plates and on MM where they grew more slowly were selected. These transformants were then analyzed by PCR using the 5' and 3' primers A0X1 and 8 positive transformants were tested for expression.

Colonies were inoculated into BMGY medium and grown at 30°C for 24 hours, then the cells were centrifuged and transferred to BMMY medium to induce expression and then further incubated for 24 hours at 30°C. Methanol was added twice more to a concentration of 0.5% after 24 and 48 hours to maintain induction. The cells were then removed by centrifugation and an aliquot of the medium was adjusted to a sucrose concentration of 30% and tested for ice recrystallization inhibitory activity. The medium contained substantial R1 activity in all transformants, whereas medium produced in the same manner from control Pichia cells not containing the inserted Lolium cDNA showed no activity.

The above example shows that the protein isolated from the plant Lolium perenne and the corresponding cDNA represent the R1 active AFP.

Example 8:

E. coli XL-1 was used in all cloning steps. Cultures were grown on 2YT medium with ampicillin or kanamycin as the selection agent. All oligonucleotide primers were synthesized on a Perkin Elmer 381A DNA synthesizer using a phosphoramidite group as recommended by the manufacturer. Culture plates were prepared by plating 2 ml of Nicotiana bethiama cell suspension onto Petri dishes containing 25 ml of MS salts, B5 vitamins (1 mg/l nicotinic acid, 1 mg/l pyridoxine, 10 mg/l thiamine, 100 mg/l inisitol) + 1 mg/l 2-4D, 0.2 mg/l BAP + 0.8% agar. The cells were mixed by swirling to cover the entire agar and then covered with a sterile filter paper disc.

• ft • ft ftft ft ftft · • ftft · • ftft ft • ftft · • · ftft • · • ftftft ftftftft

Selection medium: MS basal medium + 3% sucrose, 0.2 mg/l IAA, 1mg/l BAP, 0.9% agar + 500 mg/l cefotaxime + 100 mg/l kanamycin.

The Loll AFP-encoding sequences and the PRla apoplastic targeting sequence were PCR amplified with 5' and 3' oligonucleotide primers designed to create appropriate restriction sites that allow them to be ligated at the BamHI restriction site and the ligated product was then inserted into the plasmid vector pUC opened with the restriction enzymes NcoI and EcoRI (Messing 1983). The Loll AFP was inserted separately into three pUC vectors behind different promoter sequences (granule-bound starch synthase, cauliflower ring virus, cauliflower mosaic virus double 35S). These were then further cloned into a plant transformation binary vector and introduced into tobacco plants using Agrobacterium-mediated transformation.

The PCR reaction mixture contained 5 μΐ 10x Taq reaction buffer, 1 μΐ dNTP, 1 μΐ 5' and 3' primers (50 pmol/μΐ), 1 μΐ Loll template sequence, 0.5 μΐ Taq polymerase and 0.05 μΐ Pfu enzyme. The tubes with the mixture were covered with mineral oil and the amplification was carried out in 30 cycles of 1 minute at 94 °C, 1 minute at 55 °C and 1 minute at 72 °C after an initial denaturation step, 2 minutes at 94 °C.

Digestion of the PCR amplification product was performed with appropriate restriction enzymes in the recommended buffer (Boehringer Mannheim) for 16 hours at 37°C. The digested mixtures were separated on a 1% agarose gel and the relevant bands were excised and purified using a Qiagen gel DNA isolation kit according to the manufacturer's instructions.

The insert and plasmid vector were ligated with T4 ligase using 10x concentrated buffer recommended by Stratagene for 16 hours at 16°C. Competent E. coli cells were transformed using 2 μΐ of the ligation reaction, plated and grown at 37°C overnight. Transformants were identified by PCR using specific 5'a S^rimers. Plasmid DNA was then isolated as described in Maniatis Manual, Volume 1, and the correct alignment was confirmed by restriction analysis.

The HindIII/EcoRI inserts were excised and ligated as before into a general plant transformation vector (GPTV) digested with the restriction enzymes HindIII/EcoRI. Competent E. coli cells were transformed and transformants were identified by PCR. The correctness of the subsequently isolated plasmid DNA was confirmed by sequencing.

Three different GPTV vectors were transformed separately into Agrobacterium tumefaciens (strain LBA 4404) using the freeze/thaw method as described in the Plant Molecular Biology manual PMAN A3/7.

Seeds of the plant Nicotiana tabacum (variety Petit Havana) were surface sterilized for 10 minutes with a solution of 10% sodium hypochlorite and, after three repeated washings with distilled water, were transferred to the basic culture medium according to Murashige and Skoog with 3% sucrose and 0.9% agar. Agrobacterium cells containing the appropriate plasmid were cultured overnight on Lennox medium (5 g/l NaCl, 10 g/l yeast extract, 10 g/l bactotryptone and 15 g/l agar), then the cells were obtained by centrifugation for 10 minutes at 3,000 x g and resuspended in basic MS medium with 3% sucrose. Leaf discs of the tobacco plant, cut with a sterile cork cutter, were infected by incubation with a culture of the microorganism Agrobacterium for 10 minutes. They were then allowed to dry on sterile filter paper and placed face down on tobacco plant cell culture plates.

Infected tobacco discs were incubated at 26°C for 2 days at 2000 lux and then transferred to selection medium. There they were incubated at 3000 lux for 26°C and 16 hours, followed by an eight-hour dark cycle. They were transferred to fresh medium every two weeks. When new shoots appeared at the edge of the disc, they were transferred to MS basal medium with 3% sucrose, 500 mg/l cefotaxime and 100 mg/l kanamycin for rooting.

After rooting and growing to a size of 3 to 4 leaves, leaves from several lines of each construct plus several untransformed control plants were removed for RI assay. The leaf tissue was crushed into an Eppendorf tube and the released juice was obtained by centrifugation. The juice was adjusted with 30% sucrose (w/v) before assay.

Several plant lines from each construct were shown to have substantial ice recrystallization inhibitory activity. Extracts from each control plant showed no activity. This clearly indicates that Loll AFP was transformed into tobacco plant cells that do not have their own AFP. This protein is successfully expressed as an active protein and the plant does not need to be cold acclimatized.

Plants selected for further cultivation were placed in pots with a 50% perlite/50% peat mixture and grown to inflorescence at 26°C with a 16-hour light/22°C with 8-hour dark. Flowers were covered to ensure self-pollination and seeds were collected and stored at +4°C.

Example 9:

To demonstrate that the AFPs of the invention do not require glycosylation for their activity, the coding region was expressed in a prokaryotic system. An E. coli strain was generated containing the Lolium AFP cDNA.

The Lolium plant cDNA was cloned into pET32 plasmids from Novagen, which contains thioredoxin suitable for expression of the target gene as a fusion protein. All enzymes used were from Boehringer Mannheim and were used according to the manufacturer's recommendations. The construction of expression plasmids, transformation and growth of E. coli cells were carried out as described in the Novagen pET system manual and the Promega Applications guide.

The Lolium cDNA was cloned into the plasmid pET-32 as a fragment generated by PCR amplification with compatible restriction ends suitable for ligation into the multiple cloning site of the plasmid pET-32. The fragment was generated using the Lolium cDNA as a template and a 5' oligonucleotide primer (TTTGCGCTCGAGTTAAGCGTCTGTCACGACTTTG) and a 3' oligonucleotide primer (TTTGCGCCATGGATGAACAGCCGAATACGATTTC). In this way, a NcoI restriction site was introduced at the 5' end and an XhoI site at the 3' end of the amplified fragment. The reactions were carried out in a thermal cycler using Taq DNA polymerase for 25 cycles (denaturation at 94°C, annealing at 35°C to 60°C, expression at 72°C). All subsequent PCR reactions were carried out under the same conditions. The NcoI/XhoI PCR amplified fragment was then cloned into the NcoI/XhoI restriction enzyme-digested pET-32 plasmid and transformed into competent non-expressing E. coli cells (strain XLl-Blue). After transformation, the cells were plated on LB medium with ampicillin and cultured at 37°C for 16 hours. Ten ampicillin-resistant transformants were isolated and analyzed for incorporation of the Lolium cDNA by PCR using upstream and downstream pET primers from Novagen.

Using the small volume method, plasmid DNA from three positive transformants was isolated and digested with the restriction enzymes XhoI and NcoI for transformation into competent expression cells of E. coli BL21(DE3) as described in the pET system manual. Transformed cells were plated on LB medium with ampicillin and incubated at 37°C for 24 hours along with a control prepared from untransformed E. coli cells.

BL21(DE3).

• Φ φφ φφ φφ φ φ φφφφ φ • φφφφ φ * · φ φφφφφφ φφ φ • * φ · φφφφ φφφφ φφ φφ φφ φ φ

Three positive transformed colonies were selected and subcultured in LB culture medium with ampicillin and incubated at 37°C for 1 hour to reach an O.D. (λ=600 nm) of 0.6 and then inoculated with a large volume of medium and mixed at 37°C. When the O.D. reached 0.5, the cells were induced with IPTG to express for 3 hours as described in the pET system manual.

The expressed proteins were isolated from the cytoplasm of E. coli cells as described in the pET system manual. The cells were harvested by centrifugation, resuspended in HEPES buffer (25 mM HEPES, 10 mM MgCl2, 100 mM NaCl, 1 mM DTT, pH7.5) and sonicated for 10 seconds. The cytoplasmic protein fraction was centrifuged and the supernatant was collected. An aliquot of the supernatant was adjusted with 30% sucrose and tested for ice recrystallization inhibitory activity as described in Example 3. The cytoplasmic protein extract showed substantial RI activity, whereas the cytoplasmic protein extract produced in the same manner from control E. coli cells carrying the integrated Lolium cDNA showed no activity.

The fusion protein was purified on a metal affinity column and thioredoxin was removed by thrombin digestion. The identity of the cleaved AFP was confirmed by N-terminal sequencing and this cleaved protein was shown to exhibit substantial ice recrystallization inhibitory activity.

This fact described above demonstrates that the non-glycosylated antifreeze protein of the plant Lolium perenne expressed in the microorganism E. coli retains recrystallization inhibitory activity, whether in the form of a fusion product with thioredoxin or after its cleavage by thrombin.

Claims (6)

Claims Uro 1. An antifreeze protein which is characterized in that it comprises at least from the group serine, β-eonin and asparagine and has at least 80% overlap with the following amino acid sequence: D-E-Q-P-N-T-I-S-G-S-N-N-T-V-R-S-G-S-K-N-V-L-A-S-N-D-NT-V-I-SG-D-N-N-S-VS-GS-N-N-T-V-VS-G-N-D-N-T-V-T-GS-N-H-V-VS-G-T-N-H-I-V-T-D-N-N-N-N-VS-G-N-D-N-N-V-SG-S-F-H-T-PS-G-G-H-N-T-VS-GS-N-N-T-VS-GS-N-H-V-VS-GS-N-K-V-V-T-D-A as well as modified versions of the protein, said arrangement 'matenalne affect' recrystallisation inhibition properties of ice. The anti-freeze protein of claim 1, which is obtainable from grass. The anti-freeze protein of claim 1, wherein the overlap is at least 95%. The anti-freeze protein of claim 1, wherein the overlap is 100%. The anti-freeze protein of claim 1, wherein the protein has been glycosylated. A nucleic acid sequence capable of encoding the antifreeze protein of claim 1.