WO2011069280A1

WO2011069280A1 - Antimicrobial pexiganan analogue and preparation process thereof

Info

Publication number: WO2011069280A1
Application number: PCT/CN2009/001423
Authority: WO
Inventors: 吴晓琰; 孙玉琨; 龚铁军
Original assignee: Wu Xiaoyan; Sun Yukun; Gong Tiejun
Priority date: 2009-12-11
Filing date: 2009-12-11
Publication date: 2011-06-16
Also published as: CN102656184A

Abstract

The present invention provides an antimicrobial Pexiganan analogue, the amino acid sequence of which is GIGKFLKKAKKFGKAFVKILKKR. Moreover, the present invention also provides a preparation process of the analogue.

Description

Description. Antimicrobial PEXIGANAN analogue and preparation method thereof

Technical field

The present invention relates to a Pexiganan analog (1-23) which has an antibacterial action and can be used as an antibacterial polypeptide drug. The present invention also provides a method of producing the Pexiganan analog (1-23) by chemical synthesis or genetic engineering.

Background technique

Insects of organisms (animals, plants, insects...) invading the outside microbes produce antibacterial peptides that protect the body from pathogens through natural immunity. More than 800 antimicrobial peptides have been isolated from various organisms in the past 20 years, and more than 200 antimicrobial peptides have been isolated from various frog skins. This peptide, which has a relatively low molecular weight (20-60 amino acids), kills bacteria, fungi, protozoa and certain viruses. It is effective against some resistant microorganisms and is a new broad-spectrum antibacterial drug. "New antibiotics". The antibacterial peptide itself is not toxic, and is amino acid after metabolism, which has been widely noted and studied at home and abroad. As the resistance of various bacterial antibiotics increases, more and more research on antimicrobial peptides has been made, because antimicrobial peptides can also be suppressed against those resistant bacteria. Chemically synthesized Pexiganan consists of 22 amino acids, which are used in clinical trials to achieve good therapeutic results.

The high price of chemical synthetic products limits its wide application. The difficulty in preparing antimicrobial peptides by genetic engineering techniques is that the antibacterial peptides expressed by the fermentation will kill the engineered bacteria, and the fermentation is difficult to carry out. Fermented with Pichia Pastori s yeast, the small peptide secreted into the fermentation broth has a small molecular weight, a small amount, and is difficult to recover. At present, it is expressed by fusion protein, which forms insoluble inclusion bodies and avoids damage to engineering bacteria. However, it is required that the fusion protein carrier has a small molecular weight and a high expression level to increase the yield of the polypeptide of interest. references:

US patene : 7332800

Pexiganan: Antimicrob Agents Chemither (1999) 43, 782-788

Diagn. Microbiol Infect. Dis. (1999) 35, 45-53

Drugs. (1998) 56, 1047-1052

Rev. in Medical Microbiology (2004) 15, 17-25

Drug Communication. (2008) Confirmation Pexiganan genetic engineering preparation:

Biotechnology and applied Biochemistry (2004) 39, 339-345.

Protein Expression and purification (1998) 12, 53-60.

Biochemical and Biophysical. Res.comm (2000) 277, 575-579.

US patent (2001) 6138992

Journal of Bioengineering (2008) 24, 21-26.

The object of the present invention is to overcome the defects of the prior art and to successfully produce by using genetic engineering technology.

Pexiganan analogues (1-23) can reduce costs by up to 500 mg per liter of fermentation broth.

The Pexiganan analog (1-23) of the present invention is characterized in that the amino acid sequence of the Pexiganan analog (1-23) consists of 23 amino acids, and the Pexiganan analog (1-23) has the structural formula:

Gly-I le-Gly-Lys-Phe-Leu-Lys-Lys-Ala-Lys-Lys-Phe-Gly-Lys-Ala-Phe-Val-Lys

-I le-Leu-Lys-Lys-Arg

Among them, the 22 amino acid sequence is Pexiganan, the C terminus is Arg, and the C terminus Arg is -OH or NH2. Synthesis of DNA fragments based on the amino acid sequence of the Pexiganan analog and the corresponding DNA sequence

BamH I X Gly l ie Gly Lys Phe Leu Lys Lys Ala Lys Lys Phe < > GGC ATC GGC AAA TTC CTG AAA AAA GCG AAA AAA TTC

CCG TAG CCG TTT AAG GAC TTT TTT CGC TTT TTT AAG

Gly Lys Ala Phe Val Lys lie Leu Lys Lys Arg

GGC AAA GCG TTC GTT AAA ATC CTG AAA AAA CGT TAG

<==>

CCG TTT CGC AAG CAA TTT TAG GAC TTT TTT GCA ATC

Sal I

Where X=Met 3⁄4=Asp or =Trp or Arg.

In another aspect, the present invention also provides a method of producing the Pexiganan analog (1-23) by genetic engineering or chemical synthesis.

The Pexiganan analog (1-23) of the present invention is an analog of the antibacterial peptide Magainin, which has a strong antibacterial action and can directly kill bacteria, fungi, protozoa and certain viruses without causing drug resistance. The use of the Pexiganan analog (1-23) of the present invention is useful as an antimicrobial polypeptide drug.

The antibacterial peptides such as Pexiganan used so far are chemically synthesized products, which are expensive and hinder their wide application. Genetic engineering using Pichia Pastoris yeast to secrete the expression of antimicrobial peptides to fermentation In the liquid, since the molecular weight is small and the amount of expression is also low, it is very difficult to recover from the fermentation broth. Antibacterial peptide

Pexigarmn analogues (1-23) are expressed in the form of fusion proteins, which are simple to prepare and have industrial production significance. DRAWINGS

Figure 1 is an HPLC detection map of Pexiganan analog (1-23);

Figure 2 is a diagram showing the bactericidal effect of Pexiganan analogues (1-23);

Figure 3 is a diagram showing the synthesis process of a Pexiganan analogue (1-23) synthesized by solid phase synthesis. detailed description

Embodiment 1:

Solid phase synthesis of Pexiganan analogues (1-23).

(1) Chemical structure

Gly-Ile-Gly-Lys-Phe-Leu-Lys-Lys-Ala-Lys-Lys-Phe-Gly-Lys-Ala-Phe-Val-Lys-Ile- Leu-Lys-Lys-Arg

(2) The solid phase resin is Wang Resin, which performs solid phase synthesis. After the synthesis, the obtained peptide resin with side chain protecting group is cleaved, that is, the connection between the polypeptide and the resin is cut off, and the deprotection step is completed in one step. The C-terminus is a free carboxyl group.

(3) Synthesis step

8 kinds of protected groups Fmoc amino acids ~~► Solid phase synthesis - "► Cut the resin, while deprotecting the substrate - HPLC purification - ► freeze-drying to obtain Pexiganan analogues (1-23).

Amino acid with a protecting group Fmoc - Fmoc-L-Ala-OH; Fmoc-L-Lys(Trt)-OH;

Fmoc-L-Arg(pmc)-OH; Fmoc-L-Val-OH;

Fmoc-L-Ile-OH; Fmoc-Gly-OH;

Fmoc-L-Phe-OH; Fmoc-L-Leu-OH

(4) The synthesis process is shown in Figure 3. The first amino acid Arg is linked to the resin (R) from the C-terminus, and then an amino acid linkage is completed from the C-terminus, extending to the N-terminus until the final completion. step. (5) Solid phase peptide synthesizer (Applied Biosystem 433A)

The reagents used were of analytical grade or peptide synthesis grade: N-methylpyrrolidone, chloroform, hexahydrobipyridyl, 2.0 M DIEA NMP, DMAP/DMF, HBTU 100 mmole/0.5M HOBT in F, and the like.

(6) Operation

On the scale of 0.25 mmole, 0.5 g of resin was placed in the reactor of the peptide synthesizer, and 8 kinds of amino acids with protective group Fmoc were bottled in 1 mmole according to the amino acid sequence of Pexiganan analogue (1-23) from C to N. The ends are arranged in a synthesizer, automatically controlled by a computer program (Synth Assiat) at room temperature of 25 ° C, the Fmoc protecting group is removed, activated, and the coupling is completed and then proceeds to the next cycle step.

(7) Pyrolysis and deprotection

Place the Pexiganan analogue (1-23) with a protecting group in a stoppered flask and place the lysis reagent as follows:

Reagent volume (ml)

Water 0.50

Anisole 0.50

Phenol 0.75

Thioglycolic acid 0.20

Trifluoroacetic acid 10.0

Electromagnetic stirring at a constant temperature of 30 ° C for 6 hours, removing the resin by filtration, collecting the supernatant, and washing the resin once with a small amount of trifluoroacetic acid, combining the collected liquid, adding diethyl ether (about 20-30 ml) to precipitate, and filtering with diethyl ether. The precipitate was washed once and immediately placed in a desiccator.

The crude Pexiganan analog (1-23) was dissolved in 0.5% acetic acid at a concentration of 5 mg/ml, and subjected to HPLC. The main peak of Pexiganan analog (1-23) was purified by preparative HPLC using Dalian elestat. The separation and purification conditions were as follows: Gradient separation was carried out by using 0.1% trifluoroacetic acid and 80% acetonitrile containing 0.1% trifluoroacetic acid, and the obtained Pexiganan analog (1-23) was concentrated under reduced pressure to remove acetonitrile, followed by lyophilization to obtain a product.

See Figure 1 for HPLC detection of Pexiganan analog (1-23). Embodiment 2:

The fusion protein is cleaved by Clostripain.

BamH I Arg Gly lie Gly Lys Phe Leu Lys Lys Ala Lys Lys Phe Gly Lys Ala Phe Val ^ ^ CGT GGC ATC GGC AAA TTC CTG AAA AAA GCG AAA AAA TTC GGC AAA GCG TTC GTT

GCA CCG TAG CCG TTT AAG GAC TTT TTT CGC TTT TTT AAG CCG TTT CGC AAG CAA

Lys lie Leu Lys Lys Arg

AAA ATC CTG AAA AAA CGT TAG ^ ►

TTT TAG GAC TTT TTT GCA ATC

Sal I

Gene synthesis and cloning:

Synthesis of DNA fragments based on the amino acid sequence of Pexiganan analogue (1-23) and the corresponding DNA sequence

1 BamH I CGTGGCATCGGCAAATTCCTGAAAAAAGCGAAAAAATT 2

4 Sal I^ CTAGGATCCACGTTTTTTCA

5 TGCCGATGCCACG ^amH

The Arg-containing codon was amplified by a conventional PCR method, cloned into a Lac-containing promoter plasmid to form a fusion protein gene, and an expression plasmid was constructed and transfected into Escherichia coli JM109.

Fermentation:

Pexiganan analogue (1-23) genetically engineered bacteria were inoculated into 300 ml X 2 bottles of LB medium, added with ampicillin (Ampici ll in ) to a final concentration of 50 μg/ml, 37 ° C, and shaken overnight. 150rpm). On the second day, transfer to 20L New Brunswik fermenter. The culture medium is composed of M9 medium, with glucose concentration of 1%, ampicillin (Ampici ll in ) concentration of 50 μg/ml, aeration of 20L/min, dissolved oxygen. Maintained at more than 20%, the antifoaming agent is domestically produced, the pH is maintained at 7~8, and it is adjusted with ammonia water. When the cell concentration reached log curve line, adding of IPTG, at a concentration of 0. 5mM, continue to ferment 4 ~ 6 hours, bacterial cells were collected by centrifugation wet weight 30~ 50g / L _o

Downstream process:

The collected cells were homogenized with pH 8.0 Tri s buffer 20 mM, then lysozyme lg/L was added, incubated at 37 ° C for 1 hour, frozen at 20 ° C overnight, sonicated 3 times, each time 1 minute or so. The precipitate was collected by centrifugation, washed 2 times with 2M Urea, and washed once with water. Separation and purification of inclusion bodies: The precipitate is dissolved in 8 M urea. Sephadex G-100 was chromatographed at A280 nm, and the collected solution was dialyzed overnight, lysed with Clostripain protease, and traced by HPLC. After the enzymatic hydrolysis reaction was completed, ultrafiltration was carried out at a molecular weight of 30,000, followed by purification by HPLC to obtain a Pexiganan analog (1-23). Preparative HPLC purification (see Figure 1). Freeze drying to obtain the product. Embodiment 3:

BrCN cleavage of the fusion protein.

BamH I Met Gly lie Gly Lys Phe Leu Lys Lys Ala Lys Lys Phe Gly Lys Ala Phe Val ►ATG GGC ATC GGC AAA TTC CTG AAA AAA GCG AAA AAA TTC GGC AAA GCG TTC GTT

TAC CCG TAG CCG TTT AAG GAC TTT TTT CGC TTT TTT AAG CCG TTT CGC AAG CAA

Lys He Leu Lys Lys Arg

AAA ATC CTG AAA AAA CGT TAG ~►

TTT TAG GAC TTT TTT GCA ATC

Sal I

Gene synthesis and cloning - synthesis of DNA fragments based on the amino acid sequence of the Pexiganan analogue (1-23) and the corresponding DNA sequence

1 BamH I ATGGGCATCGGCAAATTCCTGAAAAAAGCGAAAAAATT 2

CGGCAAAGCGTTCGTTAAAATCCTGAAAAAACGTGGATCCTAGSal l

►

4 Sal CTAGGATCCACGTTTTTTCA

5 TGCCGATGCCCAT gamH

The Met-containing codon was amplified by a conventional PCR method, cloned into a Lac-containing promoter plasmid to form a fusion protein gene, and an expression plasmid was constructed and transfected into Escherichia coli JM109.

Fermentation:

Pexiganan analogue (1-23) genetically engineered bacteria were inoculated into 300 ml X 2 bottles of LB medium, and added with ampicillin (Ampici ll in) to a final concentration of 50 μg/ml, 37 ° C, and shaken overnight. 150rpm). On the second day, transfer to 20L New Brunswik fermenter. The culture medium is composed of M9 medium, with glucose concentration of 1%, ampicillin (Ampici ll in ) concentration of 50 μg/ml, aeration of 20L/min, dissolved oxygen. Maintained at more than 20%, the antifoaming agent is domestically produced, the pH is maintained at 7~8, and it is adjusted with ammonia water. When the concentration of the bacteria reached the log line, IPTG was added, and the concentration was 0.5 mM. The fermentation was continued for 4 to 6 hours, and the cells were collected by centrifugation to a wet weight of 30 to 50 g/L. Downstream process - The collected cells were homogenized with pH 8.0 Tri s buffer 20 mM, then lysozyme lg/L was added, incubated at 37 ° C for 1 hour, frozen at 20 ° C overnight, sonicated 3 times, each time 1 In about minute, the precipitate was collected by centrifugation, washed 2 times with 2M Urea, and washed once with water. Isolation and purification of inclusion bodies: The precipitate was dissolved in 8 M urea, Sephadex G-100 chromatography was detected at A280 nm, the collected solution was dialyzed overnight, cut through CNBr under 70% HC02H conditions, Na2S03 was added, the reaction was stopped, and 1000 molecular weight ultrafiltration was diluted. Further purification by HPLC gave Pexiganan analog (1-23). Preparative HPLC purification. Freeze drying to obtain the product. Embodiment 4:

Hydroxylamine cleavage of the fusion protein.

BamH 】 Asn Gly lie Gly Lys Phe Leu Lys Lys Ala Lys Lys Phe Gly Lys Ala Phe Val i ► AAC GGC ATC GGC AAA TTC CTG AAA AAA GCG AAA AAA TTC GGC AAA GCG TTC GTT

TTG CCG TAG CCG TTT AAG GAC TTT TTT CGC TTT TTT AAG CCG TTT CGC AAG CAA

Lys lie Leu Lys Lys Arg

AAA ATC CTG AAA AAA CGT TAG ^ ~►

TTT TAG GAC TTT TTT GCA ATC

Sal I

Gene synthesis and cloning:

1 BamH I AACGGCATCGGCAAATTCCTGAAAAAAGCGAAAAAATT 2

CGGCAAAGCGTTCGTTAAAATCCTGAAAAAACGTGGATCCTAGSal I

4 ^Sal CTAGGATCCACGTTTTTTC A

5 TGCCGATGCCGTT ^amH I

The codons containing Asn-Gly were amplified by conventional PCR and cloned into the Lac-containing promoter plasmid to form a fusion protein gene, and the expression plasmid was constructed and transfected into Escherichia coli JM109.

Fermentation:

Pexiganan analog (1-23) genetically engineered bacteria were inoculated into 300 ml X 2 bottles of LB medium, added with ampicillin (Ampici ll in ) to a final concentration of 50 μg/ml, and 37 shaker overnight (150 rpm). On the second day, transfer to 20L New Brunswik fermenter. The culture medium is composed of M9 medium and added with glucose. Degree 1%, ampicillin (Ampici llin) concentration of 50 μ g / ml, ventilation capacity of 20L / min, dissolved oxygen maintained above 20%, antifoaming agent is domestic foaming enemy, pH is maintained at 7~8, with Ammonia adjustment. When the concentration of the bacteria reached the log line, IPTG was added, and the concentration was 0.5 mM. The fermentation was continued for 4 to 6 hours, and the cells were collected by centrifugation to a wet weight of 30 to 50 g/L.

Downstream process - The collected cells were homogenized with pH 8.0 Tri s buffer 20 mM, then lysozyme lg/L was added, incubated at 37 ° C for 1 hour, frozen at 20 ° C overnight, sonicated 3 times, each time 1 In about a minute, the precipitate was collected by centrifugation, and 2M Urea was broken and washed once, and then washed once with water. Isolation and purification of inclusion bodies: The precipitate was dissolved in 8 M urea, Sephadex G-100 chromatography was detected at A280 nm, and the collected solution was dialyzed overnight. The fusion protein of Pexiganan analogue (1-23) was lysed with 2M NH20H/6M guanidine Guanidin·HC1 at pH 9. 0, 45 °C for 8 hours, followed by HPLC, cleavage and ultrafiltration with molecular weight of 1000 The salt was purified by HPLC to give the Pexiganan analog (1-23) product. Preparative HPLC purification. Freeze drying to obtain the product. Embodiment 5:

Cleavage of the fusion egg white residue.

BamH I Trp Gly lie Gly Lys Phe Leu Lys Lys Ala Lys Lys Phe Gly Lys Ala Phe Val ⁴ ^ TGG GGC ATC GGC AAA TTC CTG AAA AAA GCG AAA AAA TTC GGC AAA GCG TTC GTT

ACC CCG TAG CCG TTT AAG GAC TTT TTT CGC TTT TTT AAG CCG TTT CGC AAG CAA

Lys lie Leu Lys Lys Arg

AAA ATC CTG AAA AAA CGT TAG ~►

TTT TAG GAC TTT TTT GCA ATC

Sal I

1 BamH I TGGGGCATCGGCAAATTCCTGAAAAAAGCGAAAAAATT

3

CGGCAAAGCGTTCGTTAAAATCCTGAAAAAACGTGGATCCTAGSal I

4 Sal I CTAGGATCCACGTTTTTTCA

5 TGCCGATGCCCCA gamH^I The Trp-containing codon was amplified by a conventional PCR method, cloned into a Lac-containing promoter plasmid to form a fusion protein gene, and an expression plasmid was constructed and transfected into Escherichia coli JM109.

Fermentation:

Pexiganan analogue (1-23) genetically engineered bacteria were inoculated into 300 ml X 2 bottles of LB medium, and added with ampicillin (Ampici ll in ) to a final concentration of 50 μg/ml, 37 ° C, and shaken overnight. 150rpm). On the second day, transfer to 20L New Brunswik fermenter. The culture medium is composed of M9 medium, with glucose concentration of 1%, ampicillin (Ampici ll in ) concentration of 50 μg/ml, aeration of 20L/min, dissolved oxygen. Maintained at more than 20%, the antifoaming agent is domestically produced, the pH is maintained at 7~8, and it is adjusted with ammonia water. When the concentration of the bacteria reaches the midline of the log curve, add IPTG, the concentration is 0.5 mM, continue the fermentation for 4 to 6 hours, and collect the cells by centrifugation to a wet weight of 30~50g/L.

Downstream process - The collected cells were homogenized with pH 8.0 Tri s buffer 20 mM, then lysozyme lg/L was added, incubated at 37 ° C for 1 hour, frozen at 20 ° C overnight, sonicated 3 times, each time 1 In about minute, the precipitate was collected by centrifugation, washed 2 times with 2M Urea, and washed once with water. Isolation and purification of inclusion bodies: The precipitate was dissolved in 8 M urea, Sephadex G-100 chromatography was detected at A280 nm, and the collected solution was dialyzed overnight. BNPS-Skatole was dissolved in 70% acetic acid (containing 0.1% phenol), and the fusion protein was added at room temperature. The reaction was carried out for 48 hours (or 5 hours at 37 ° C), and the reaction was stopped by adding a 10-fold amount of a reducing agent (such as mercaptoethanol) under nitrogen in the dark. The reaction solution was extracted with ethyl acetate to remove BNPS-Skatole, and the aqueous phase was stored in lyophilized, dissolved and then purified by HPLC to obtain Pexiganan analog (1-23) product. Example 6:

The Pexiganan analog (1-23) of the present invention has an antibacterial action as an antibacterial polypeptide drug, and can directly kill bacteria, fungi, protozoa and certain viruses without causing drug resistance.

See Figure 2, in the figure: The left dish is a Pexiganan analogue (1-23) and the E.Coli resistant bacteria are pre-mixed in a saline solution for half an hour after plating, and the right dish is the premixed physiology of E. coli resistant bacteria. The plate was incubated for half an hour in a saline liquid. The plate was incubated overnight at 37 ° C.

Claims

A Pexiganan analogue (1-23), characterized in that: the amino acid sequence of the Pexiganan analog (1-23) consists of 23 amino acids, and the Pexiganan analog (1-23) has the structural formula:

Gly-Ile-Gly-Lys-Phe-Leu-Lys-Lys-Ala-Lys-Lys-Phe-Qly-Lys-Ala-Phe-Val-Lys-Ile-Leu-Lys-Lys-Argo

2. The Pexiganan analog (1-23) according to claim 1, wherein the 1-22 amino acid sequence is Pexiganan and the C terminus is Arg.

The Pexiganan analogue (1-23) according to claim 1 or 2, wherein the C-terminal Arg is -OH or NH2.

The method for producing a Pexiganan analogue (1-23) according to claim 1, which is produced by a chemical synthesis method, comprising the steps of: synthesizing 8 kinds of protected Fmoc amino acids by solid phase, cutting the resin, and simultaneously deprotecting Purification by base chromatography, HPLC, and lyophilization to obtain Pexiganan analogues (1-23)

The method for producing Pexiganan analog (1-23) according to claim 1, which is produced by genetic engineering.

6. The Pexiganan analogue (1-23) according to claim 1, which is used as an antibacterial polypeptide drug.