CN101418036A - Rapid purification of gamma trichosanthin and crystallization technology thereof - Google Patents

Abstract

The invention relates to a technology for quickly purifying and crystallizing gamma trichosanthin, and discloses a process for extracting novel ribosome inactivating protein-gamma trichosanthin(gamma-trichosanthin) from tuberous root of Trichosanthes kirilowii; and according to the measurement of ESI-MS, the molecular weight of the gamma-trichosanthin is 27,262Da. The gamma-trichosanthin is placed in phosphate buffer solution, citric acid buffer solution or Tris-HCl buffer solution to grow up monocrystal for diffraction of X-ray by taking ammonium sulfate and polyethylene glycol as precipitating agents, and the primary analysis on a crystal structure shows that the gamma-trichosanthin is the type I ribosome inactivating protein, and has actions of resisting tumor and virus, particularly HIV, thereby laying a physical foundation for acquiring medicinal protein or preparing immunotoxin.

Description

The fast purifying of gamma trichosanthin and crystallization technique thereof

Technical field

The present invention relates to biochemical field, particularly relate to a kind of new ribosome inactivating protein--the separation and purification of-gamma trichosanthin, crystal technique and application thereof in the biological chemistry.

Background technology

The ribosome inactivating protein that from higher plant, extracts (Ribosome-Inactivating Protein, be called for short: RIP), RIP is the rrna inactivation that a class can make eukaryotic cells, thereby suppress intracellular protein synthetic cytotoxin, its mechanism of action is to have RNA N-Glycosylase or be polynucleotide adenosine Glycosylase [EC3.2.2.22] activity.Molecular structure based on maturation protein and encoding gene thereof, RIP can be divided into 3 classes, Class1 RIP is the holotype RIPs (holo-RIP) that only contains single RNA N-Glycosylase structural domain (domain), for example, and from the α Trichosanthin (α-Trichosanthin of cucurbitaceous plant snakegourd piece root, be called for short: α-TCS), the α of balsam pear seed, β-Semen Momordicae Charantiae albumen (α, the Semen Luffae albumen a of β-Momorcharin) and sponge gourd seed, b (Luffin a, b).Type 2RIP contains a RNA N-Glycosylase structural domain (A chain) and one to have glycosyl and discern active C end structure territory (B chain), the two is connect by a disulfide linkage, be a kind of RIP (Chimero-RIP) of mosaic type, for example, from the Ricin (Ricin) of euphorbia plant castor-oil plant seed and the abrin (Abrin) of leguminous plants acacia rachii seed.Type 3RIP contains a RNA N-Glycosylase structural domain and directly connects the C end structure territory of a unknown function, also is the RIP (Chimero-RIP) of mosaic type, for example from the JIP60 of barley.The Class1 RIP that from cucurbitaceous plant, separates, for example Trichosanthin, Semen Momordicae Charantiae albumen, Semen Luffae albumen, white bryony albumen etc., all have antitumor, antiviral activity, particularly anti-AIDS cytotoxic activity.About Class1 RIP and existing a lot of patent and the bibliographical informations of type 2RIP.

Snakegourd (Trichosanthes kirilowii Maxim) be Snakegourd Fruit again, the melon building, and the medicine melon is a kind of cucurbitaceous plant snake gourd, snakegourd piece root is exactly the Chinese medicine Snakegourd Root.(Trichosanthin TCS) is a kind of basic protein that separation and purification obtains from the former green material of Chinese medicine Snakegourd Root to Trichosanthin.The chemical nature of Trichosanthin be a kind of molecular weight be 27kDa not sacchariferous single chain polypeptide, belong to 1 type ribosome inactivating protein (Ribosome Inactivating Protein, RIP), have RNA N-glycosidase activity and multiple zymetology and pharmacologically active (people such as Pan Kezhen., Structure-Function Relationship of Trichosanthin, Pure and Appl.Chem.66,57-64,1994; Shaw PC, et al, Resentadvances intrichosanthin, Toxicon, 45,683-689,2005).1989, people such as McGrath find that Trichosanthin can HIV (human immunodeficiency virus) inhibiting (HIV) duplicate (McGrath et al. in the T of acute infection lymphocyte and chronically infected scavenger cell, Proc.Natl.Acad.Sci.USA, 86:2844-2848,1989), soon, the drugs approved by FDA Trichosanthin carries out clinical trial.The induced labor activity of Trichosanthin, antitumor, antiviral, particularly the anti-AIDS cytotoxic activity has caused attention widely.

The Chinese science man at first separation and purification and crystallization Trichosanthin (people such as Wang Yahui, the purifying of Trichosanthin and the preliminary study of character thereof, animal journal, 22,117-143,1976; Fujian Inst. of Matter Structure, Chinese Academy of Sciences etc., the crystal of Trichosanthin is cultivated and the structure cell basic parameter, Science Bulletin, 176-178,1977; People such as Jin Shanwei, the preparation and the physicochemical property thereof of the chemical I crystal trichosanthin of Trichosanthin, chemical journal, 39,917-925,1981; People such as Wang Jiahuai, a kind of new crystal Trichosanthin crystal, Science Bulletin, 943-944,1985).And external, people such as Maraganore ability in 1987 reported first is separated to Trichosanthin (Maraganore et al.J.Biol.Chem.262,116280,1987).Simultaneously, report successively from smallpox powder agglomates root kind and to isolate TAP29 (Lee-Huang et al, Proc.Natl.Acad.Sci.USA 88 6570-6574 1991), draw together building albumen (people such as Jin Shanwei, chemistry wall bulletin 15 160-168 1997), with α β γ-Trichosanthin (Narayanna et al., Plant Science 162 79-85 2002).These work show that these albumen all belong to 1 type ribosome inactivating protein, have the RNA-N glycosidase activity, and antitumor, antiviral, particularly the anti-AIDS cytotoxic activity; The Trichosanthin of previous report mainly is exactly α Trichosanthin (α-Trichosanthin, α-TCS).

Snakegourd piece root crude extract, with the method for acetone fractional precipitation, through the refining Trichosanthin (α Trichosanthin) of lyophilize preparation, refining Trichosanthin is in barbitol buffer solution, with NaNO ₃For precipitation agent grows monoclinic crystal (C2 spacer, a=75.60 dust, b=75.44 dust, c=88.36 dust, Fujian Inst. of Matter Structure, Chinese Academy of Sciences etc. is seen in β=99.50 °, and the crystal of Trichosanthin is cultivated and the structure cell basic parameter, Science Bulletin, 176-178,1977; And P2 ₁Spacer, a=73.4 dust, b=74.7 dust, c=87.9 dust, β=97.7 °); In citrate buffer solution, be precipitation agent with KCl, grow quadrature crystal (P2 ₁2 ₁2 ₁Spacer, the a=38.305 dust, the b=76.225 dust, the c=79.213 dust is seen people such as king family Chinese scholartree, a kind of new crystal Trichosanthin crystal, Science Bulletin, 943-944,1985).The Trichosanthin injection liquid commodity that Kingsoft, Shanghai pharmaceutical factory produces are exactly crystal trichosanthin solution.

People such as Jin Shanwei go the snakegourd piece root of juice from squeezing, after physiological saline extracting, ammonium sulfate precipitation, dialysis, lyophilize, separate through Sephadex G-75, obtain TRICHOBITACIN (Trichobitacin) through the cationite gradient elution again, the molecular weight that mass spectrum (ESI-MS) is measured is 27228Da, and amino acid is formed similar to Trichosanthin with the part order.People such as Lee-Huang from smallpox powder agglomates root through the PBS extracting, dialysis, behind the cationite gradient elution, dialysis also concentrates after Sephadex G-75 separates acquisition TAP29, and it is variant with Trichosanthin on molecular weight (29kDa) and partial amino-acid order.People such as Narayanan are from smallpox powder agglomates root, through the PBS extracting, after the lyophilize, obtain Trichosanthin crude product (lyophilized powder) through the cationite gradient elution, and then go up the Mono-S post and isolate α β gamma trichosanthin on a small quantity, beta trichosanthin protein is at molecular weight (26kDa), and amino acid is formed similar to the α Trichosanthin with the part order; A small amount of isolating gamma trichosanthin is at molecular weight (26kDa), and amino acid is formed upward similar to the α Trichosanthin, but the N terminal amino acid is gone up in proper order and the α Trichosanthin is variant.Chinese patent 2006100849606 confirms to contain in previous Trichosanthin injection liquid of reporting and the Trichosanthin crystal two equipotential albumen: α Trichosanthin and beta trichosanthin protein, but main ingredient is the α Trichosanthin.

Summary of the invention

The objective of the invention is based on above-mentioned prior art basis, from the method and the crystal technique thereof of the primary material preparation gamma trichosanthin of Snakegourd Root.This albumen is in phosphoric acid buffer, citrate buffer solution or Tris-HCl damping fluid, with ammonium sulfate, polyoxyethylene glycol is that precipitation agent grows the hexagonal that can supply X-ray diffraction, through X-ray diffraction with use the synchrotron radiation data analysis, this crystal belongs to the P622 spacer, and its unit cell parameters is the a=180.380 dust, the b=180.380 dust, the c=145.084 dust, γ=120 °.The crystalline structure initial analysis shows that it is the Class1 ribosome inactivating protein, has antitumorly, antiviral, and anti-AIDS toxic action particularly is for obtaining medical protein or the preparation immunotoxin is laid basic substance.

Technical scheme of the present invention is: the former green material peeling of Snakegourd Root is cleaned, chopping, add 5mM, the pH4.5 acetate buffer solution, extracting, homogenate, add 50% Glacial acetic acid and transfer pH4.0, then under 4 ℃ of conditions, the centrifugal 30min of 16000 * g, the supernatant liquor application of sample is to 20mM, on the post of pH4.5 acetate buffer solution equilibrated strong cation-exchanging resin (S-Sepharose Fast Flow or SP-Sepharose Fast Flow), column length requires more than 20cm, and 280nm detects in real time, use 20mM, the pH4.5 acetate buffer solution is washed till baseline, uses 20mM again, and the pH6.5 phosphoric acid buffer is washed till baseline, with the slow gradient elution of 15-20 times of column volume damping fluid, generally can go out 4 peak (see figure 1)s then.Collect the 1st peak, transfer pH4.0 with 50% Glacial acetic acid, add the acetate buffer solution of two volumes, carry out gradient elution with Zeo-karb again, main peak is exactly the gamma trichosanthin (see figure 2).

The gamma trichosanthin of institute of the present invention separation and purification, on SDS-PAGE, present a band, molecular weight is 27kDa, show as a peak at strong cation prepacked column Mono-S post (available from Pharmacia company), on gel-filtration prepacked column Superdex-75 (available from Pharmacia company), show as a peak.Analyze through mass spectrum (ESI-MS), the gamma trichosanthin molecular weight is 27262Da, records α under the same terms, and the molecular weight of beta trichosanthin protein is respectively 27173.0 and 27163.0, and three's molecular weight is close.

The prepared gamma trichosanthin of the present invention adopts vapor diffusion technique, and mother liquid concentration is 20mg/ml, at phosphate buffered saline buffer, in citrate buffer solution or the Tris-HCl damping fluid, with ammonium sulfate, polyoxyethylene glycol is a precipitation agent, just can grow the gamma trichosanthin hexagonal.Through X-ray diffraction analysis or synchrotron radiation diffraction analysis, crystal belongs to the P622 spacer, and its unit cell parameters is the a=180.380 dust, b=180.380 dust, c=145.084 dust, γ=120 °.We have collected the data of a cover 2.2 dusts.With α Trichosanthin (1TCS) is the structure masterplate, made up the structural models of gamma trichosanthin with molecular replacement technique, showing that gamma trichosanthin is the same with the α Trichosanthin has typical ribosome inactivating protein folding (RIP fold) and an active centre, belong to Class1 RIP, the same with the α Trichosanthin have antitumor, antiviral, the function of anti AIDS virus particularly.According to the chemical environment around cloud density figure and the residue, (the N end of writing a Chinese character in simplified form γ-TCS) is DVNFRLLGATSSTYKQFIQN in proper order to determine gamma trichosanthin with the method for X-ray sequencing.

This order and same purification (are write a Chinese character in simplified form 20 amino-acid sequences of N end of Nar ' s γ-TCS) from γ-Trichosanthin of the Narayanna et of snakegourd piece root al. (Plant Science16279-852002), and the α Trichosanthin (is write a Chinese character in simplified form α-TCS), 20 amino-acid sequences of N end of Trichobitacin and TAP29 are compared as follows:

Residue order 156 10 15 20 identical rates

γ of the present invention-TCS DVNF R LLGAT SSTYK QFIQN 100%

Nar’sγ-TCS DVNF?S/R?LLGAT?SATYK?QFIQN 95％

α-TCS DVSF?R LSGAT?SSSYG?VFISN 70％

Trichobitacin DVSF?R LSGAT?SSSYG?VFISN 70％

TAP29 DVSF?R LSGAT?SKKKV?YFISN 55％

γ-TCS of the present invention and Nar ' s γ-TCS, the α Trichosanthin, the identical rate of N end order of Trichobitacin and TAP29 surpasses 55%, and above-mentioned N end comparison shows that in proper order γ-TCS of the present invention is a kind of new RIP that extracts from snakegourd piece root.

The present invention compared with prior art has following positively effect:

1. the present invention adopts 2 step Zeo-karb elution techniques, without acetone or ammonium sulfate precipitation and dialysis, steps such as lyophilize have been avoided many protein-denatured troublesome operation that cause, can go out gamma trichosanthin by sharp separation from the former green material of Trichosanthin.

2. the present invention is preferred for buffer concentration, the pH value of separation and purification, and gradient has improved the efficient of separating gamma Trichosanthin.

3. the prepared gamma trichosanthin of the present invention adopts vapor diffusion technique, and mother liquid concentration is 20mg/ml, at phosphate buffered saline buffer, in citrate buffer solution or the Tris-HCl damping fluid, with ammonium sulfate, polyoxyethylene glycol is a precipitation agent, just can grow the gamma trichosanthin hexagonal.Through X-ray diffraction analysis or synchrotron radiation diffraction analysis, crystal belongs to the P622 spacer, and its unit cell parameters is the a=180.380 dust, b=180.380 dust, c=145.084 dust, γ=120 °.We have collected the data of a cover 2.2 dusts.For this proteic 26S Proteasome Structure and Function research provides important structured data.

Description of drawings

Fig. 1. gamma trichosanthin is elution profile on Sp-Sepharose Fast Flow post: X-coordinate is an elutriant ml number, and ordinate zou is AU, i.e. A ₂₈₀Detected value, dotted line is at 20mM among the figure, under the pH6.5 phosphoric acid buffer condition, the linear gradient line of NaCl concentration, gamma trichosanthin goes out the peak about 0.07M NaCl place, and the α Trichosanthin goes out the peak about 0.15M NaCl place.

Fig. 2. gamma trichosanthin elution profile again on Sp-Sepharose Fast Flow post: X-coordinate is an elutriant ml number, and ordinate zou is AU, i.e. A ₂₈₀Detected value, dotted line is at 10mM among the figure, under the pH7.5 phosphoric acid buffer condition, the linear gradient line of NaCl concentration, gamma trichosanthin goes out the peak about 0.1M NaCl place.

Embodiment

Embodiment 1: the fast separating and purifying gamma trichosanthin

Remove the snakegourd piece root that skin is cleaned, add 100ml pH4.5, the 10mM acetate buffer solution soaks, homogenate secondary on refiner is transferred pH4.0,16000 * g under 4 ℃ of conditions again with 50% Glacial acetic acid then, centrifugal 30 ', collect supernatant liquor, on the Sp-Sepharose FastFlow post of supernatant liquor application of sample to 1.6 * 23cm (this post has been used pH4.5,20mM acetate buffer solution balance), continue to use pH4.5, the 20mM acetate buffer solution is used pH6.5 after washing out an assorted peak, and the 20mM phosphate buffered saline buffer continues to wash post, can wash out an assorted peak again, use 0-0.3M NaCl (at pH6.5, in the 20mM phosphate buffered saline buffer) gradient elution then, collect the 1st peak (see figure 1).

Merge the 1st peak, add 50% Glacial acetic acid and transfer pH4, add the pH4.5 of 2 times of volumes again, the 20mM acetate buffer solution, last sample continues to use pH4.5 to using on the identical acetate buffer solution equilibrated Sp-Sepharose Fact Flow post, and the 20mM acetate buffer solution is washed post, use pH7.5 again, the 10mM phosphoric acid buffer is washed post, uses 0-0.4M NaCl (at pH7.5, in the 10mM phosphoric acid buffer) gradient elution then, collect main peak, this peak is exactly the gamma trichosanthin (see figure 2).

Embodiment 2: strong cation prepacked column Mono-S analyzes:

Gamma trichosanthin elution profile on Mono-S HR5/5 post of the present invention's preparation:

A liquid is 10mM phosphate buffered saline buffer pH7.5, and B liquid is A liquid+1M NaCl, after flow velocity 1ml/min, Mono-S post use A liquid balance, and application of sample, A liquid is washed post 3ml, and 0-10%B liquidus gradient elution 10ml after 100%B liquid is washed 4ml then, uses A liquid balance 5ml again.Gamma trichosanthin reaches 8.85ml at elutriant, and to be that NaCl concentration goes out during for 0.0585M one unimodal.Under the same terms, α, beta trichosanthin protein reaches 10.02ml at elutriant respectively, and 9.05ml is that NaCl concentration is 0.0702M, goes out one unimodal during 0.0605M.

Embodiment 3: the crystal growth of gamma trichosanthin:

The gamma trichosanthin of the present invention preparation is as mother liquor, protein concentration 20mg/ml, pond liquid 0.1M, pH7.5 phosphate buffered saline buffer, 1.8M ammonium sulfate are precipitation agent, and 5%PEG400 is an additive, cultivate protein crystal with sessile drop method, visible crystals after 1 day, left and right sides crystal was grown up in 4 days, through X-ray diffraction analysis or synchrotron radiation diffraction analysis, crystal belongs to the P622 spacer, and its unit cell parameters is the a=180.380 dust, the b=180.380 dust, the c=145.084 dust, γ=120 °.We have collected the data of a cover 2.2 dusts.

Claims (4)

1. a new ribosome inactivating protein-gamma trichosanthin that extracts from snakegourd piece root is characterized in that, molecular weight is 27262Da, and its N terminal amino acid is in proper order: DVNFRLLGATSSTYKQFIQN.

2. according to the technology of preparing of a kind of gamma trichosanthin of claim 1, the peeling of snakegourd piece root is cleaned, chopping adds 5mM, the extracting of pH4.5 acetate buffer solution, homogenate, add 50% Glacial acetic acid and transfer pH4.0, then under 4 ℃ of conditions, the centrifugal 30min of 16000 * g, the supernatant liquor application of sample is to 20mM, on the post of pH4.5 acetate buffer solution equilibrated strong cation-exchanging resin, column length requires more than 20cm, and 280nm detects in real time, use 20mM, the pH4.5 acetate buffer solution is washed till baseline, uses 20mM again, and the pH6.5 phosphoric acid buffer is washed till baseline, with the slow gradient elution of 15-20 times of column volume damping fluid, generally can go out 4 peaks then; Collect the 1st peak, transfer pH4.0 with 50% Glacial acetic acid, the acetate buffer solution that adds two volumes, carry out gradient elution with Zeo-karb again, just can obtain gamma trichosanthin by fast purifying, it is characterized in that: after the damping fluid extracting,, just can obtain gamma trichosanthin through 2 step strong cat ion exchange column gradient elutions.

3. the crystal technique of a gamma trichosanthin is characterized in that, this growing technology is at phosphoric acid buffer, and in citrate buffer solution or the Tris-HCl damping fluid, with ammonium sulfate, polyoxyethylene glycol is that precipitation agent grows the single crystal that can supply X-ray diffraction.

4. according to a kind of gamma trichosanthin of claim 1, it is characterized in that this albumen is antitumor, antiviral as preparation, the particularly application of anti-hiv drug.

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