JP2019014663A - Peptide synthesis method - Google Patents

Abstract

To provide a stable and inexpensive peptide synthesis method.SOLUTION: Escherichia coli BL21-pETHis-ywfE and Escherichia coli BL21-pETHis-ack are prepared respectively, these strains are cultured separately, and recombinant amino acid ligase and acetic acid kinase are obtained. A phosphate group of acetyl phosphate is transferred to ADP under the action of acid kinase to turn into ATP, and the ATP is used to synthesize peptides.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a peptide synthesis method.

  Conventionally, synthesis of L alanyl L glutamine has been performed by chemical synthesis or chemical enzyme synthesis. In the process of chemical synthesis, the same applies to Ala-Ala-Gln D-Ala-Glu Ala-Glu or the like in which a toxic ligand such as carbonyl chloride or D-2 chloropropionic acid is used in the process of synthesis.

  Further, in chemical enzyme synthesis, Ala Gln is synthesized from alanine methyl ester and Gln by an enzyme isolated from Empedobacterium brevis or Sphingobacterium siyangensis. However, the synthesis of alanine methyl ester is difficult. The same applies to the synthesis of Ala-Ala and Ala-Ala-Gln. In addition, as a biological method, there is a method for synthesizing a peptide by using genetically modified E. coli or the like (Patent Document 1).

JP2012-44909

  However, chemical synthesis is expensive. Furthermore, although there is a method for synthesizing a peptide using Escherichia coli or the like that has undergone genetic recombination, ATP is consumed at the time of synthesis, and the production costs increase, making it unsuitable for industrial production (Patent Document 1).

  In order to solve the above-described problems, an object of the present invention is to provide a peptide synthesis method that is inexpensive and can be industrially mass-produced.

  In the present invention, E. coli BL21-pETHis-ywfE and E. coli BL21-pETHis-ack are prepared, respectively, and these strains are cultured separately to obtain recombinant amino acid ligase YwfE and acetate kinase Ack. Furthermore, the phosphate group of acetyl phosphate generates ATP from ADP under the action of acetate kinase, and the ATP is used to synthesize a peptide.

The present invention
A first recombinant E. coli having an acetate kinase gene;
Culturing a second recombinant E. coli having an amino acid ligase gene;
Producing ATP using acetate kinase from the first recombinant E. coli;
A peptide synthesis method comprising a step of synthesizing a peptide using a second recombinant E. coli-derived amino acid ligase using the ATP.

  The present invention may also be a peptide synthesis method in which the peptide is L alanyl L glutamine.

  The first recombinant E. coli expresses acetate kinase (acetate kinase), which synthesizes ATP from ADP using acetic acid in the substrate. The amino acid ligase expressed by the second recombinant E. coli synthesizes a peptide from the amino acids in the substrate using ATP. Since Escherichia coli can grow in one culture solution because the growth environment is almost the same, compared to using different strains, the cost of equipment, medium, etc. and space costs can be saved. Furthermore, since acetic acid is cheaper and more stably available than ATP, a peptide synthesis method that is inexpensive and can be industrially mass-produced can be provided.

It is a conceptual diagram explaining the vector concerning embodiment of this invention. It is a conceptual diagram explaining the vector concerning embodiment of this invention.

[Features of the present invention]
The Bacillus spp amino acid ligase having the ywfE gene is an ATP-dependent dipeptide ligase and is used for the synthesis of L-Alanyl-anticapsin. In addition, it is considered to have an activity of binding Ala-Gln depending on ATP. However, this enzyme method requires a lot of ATP as a substrate, and therefore costs high.

  In order to solve this problem, the applicant has used Acetate kinase of Clostridium acetobutylicum to generate ATP from ADP using acetic acid in the substrate.

  Acetic acid is cheaper and easier to obtain than ATP. Mass production of Ala-Gln can be stably performed at low cost by a technique using a plurality of enzymes including recombinant amino acid ligase and acetate kinase.

[Property of plasmid]
The pET-His vector is an E. coli expression vector having ampicillin resistance. The promoter is T7, the label is N-His, the enzyme recognition site is thrombin, and the plasmid size is 2.9 kb.

  The pET-His vector can be transformed into E. coli with many copies (37 ° C., LB). The N-terminal 6 His and T7 promoters are present in the pET-His vector. The N-terminus of the highly expressed product has 6 Hiss that facilitate the purification of the target protein using chelate sepharose affinity chromatography. pET-His has four restriction sites: BamHI, EcoR I, Nhe I and Hind III.

  In this patent, a recombinant expression vector for the amino acid ligase gene pET-His-ywfE and a recombinant expression vector for the acetate kinase gene pET-His-ack are constructed, respectively. The amino acid ligase gene is derived from Bacillus subtilis (ATCC 15245), and the acetate kinase gene is derived from Clostridium acetobutylicum (ATCC 4259).

  Clostridium acetobutylicum (ATCC 824) is a commonly used butanol-producing bacterium. Using the KEGG database (http://www.kegg.jp/kegg-bin/show_pathway?cac00620), the applicant converted acetyl-CoA to acetyl phosphate by acetyltransferase and acetate kinase in the metabolic pathway of butanol. I found out that

  Acetyl phosphate can be further converted to acetic acid by acetate kinase. ATP produced by the above reaction can be used for cell growth under anaerobic conditions. We therefore attempted to use acetate kinase from Clostridium acetobutylicum to achieve ATP recycling.

[Method of gene recombination]
In the gene ligation method, 100 μl of E. coli BL21 (DE3) was subjected to heat shock for 30 minutes in an ice bath and 60 seconds at 37 ° C. The mixture was added to SOC medium and incubated for 1 hour. Cells were centrifuged and cultured in LB solid medium with ampicillin (Ampr) resistance for 12 hours at 37 ° C. to screen for positive transformants.

[Properties and culture of recombinant strains]
E. coli BL21 (DE3) is used to efficiently express genes cloned into expression vectors containing the phage T7 promoter (eg, pET series). T7 phage RNA polymerase is located in the lambda phage DE3 region and is integrated on the chromosome of E. coli BL21. This strain is suitable for the expression of non-toxic proteins.

  Two recombinant expression vectors are each transformed into E. coli BL21 (ACCC11171) to obtain engineered E. coli pET-His-ywfE and E. coli pET-His-ack.

  The recombinant strain was inoculated from a glycerol tube into 100 mL LB liquid medium containing 100 μg / mL ampicillin at an inoculum of 0.1% (v / v) and cultured at 37 ° C. and 200 rpm for 12 hours. The culture solution was transferred to 400 mL of LB medium containing 100 μg / mL of ampicillin at an inoculum of 1% (v / v) and cultured at 37 ° C. and 200 rpm. When the cell concentration OD600 nm reached 0.6, IPTG having a final concentration of 0.1 mmol / L was added, and the recombinant strain was cultured at 28 ° C. for 10 hours to express the protein.

[Purification method]
Enzyme purification: After incubation, cells were harvested by centrifugation at 4 ° C. and 6000 rpm. The cells were then washed three times with pH 7.4 PBS buffer, resuspended in pH 7.4 PBS buffer, sonicated at 4 ° C. for 20 minutes, and centrifuged at 10,000 rpm to obtain a supernatant.

  The supernatant was passed through Ni + -NTA resin at a flow rate of 1 mL / min, eluted with a 50 mM imidazole solution at a flow rate of 1 mL / min, and eluted with a 250 mM imidazole solution at a flow rate of 1 mL / min to obtain a target protein.

  The eluted target protein was dialyzed and desalted with PBS buffer at pH 7.4 at 4 ° C. The dialysate was changed every 2 hours. After repeating 4 times, the obtained target protein was obtained.

When ATP is used directly in the Ala-Gln enzymatic reaction, the amount of ATP is the same amount (mole) as other substrates such as Ala and Gln. However, the present invention does not use ATP directly. ATP is formed by acetate kinase using acetyl phosphate and ADP. Detailed enzyme reaction data and some comparative data are shown in the table below.

It is known that an amino acid ligase derived from Bacillus subtilis can synthesize 44 types of α-dipeptides by using ATP. As shown in Table 2, by using the technique of the present invention, some of the dipeptides can be synthesized.
In addition, as for the black circle of Table 2, the corresponding dipeptide was confirmed by HPLC analysis. Open circles were suggested by the formation of ADP and phosphoric acid, HPLC peaks, etc., but could not be confirmed because standard dipeptides were not available.

  As described in this embodiment, recombinant Escherichia coli BL21-pETHis-ywfE and Escherichia coli BL21-pETHis-ack were prepared, and these strains were separately cultured to obtain recombinant amino acid ligase YwfE and acetate kinase Ack. obtain. Furthermore, the phosphate group of acetyl phosphate generates ATP from ADP under the action of acetate kinase, and amino acid ligase synthesizes a peptide such as L-alanyl L-glutamine using the generated ATP. In this way, the efficiency of peptide synthesis can be increased by using acetic acid without adding expensive ATP.

  In addition, the recombinant Escherichia coli BL21-pETHis-ywfE and Escherichia coli BL21-pETHis-ack have almost the same growth environment. Because almost the same type of medium can be used, costs such as money and space can be saved. Furthermore, since acetic acid is cheaper and more stably available than ATP, a peptide synthesis method that is inexpensive and can be industrially mass-produced can be provided.

Claims (2)

A first recombinant E. coli having an acetate kinase gene;
Culturing a second recombinant E. coli having an amino acid ligase gene;
Producing ATP using acetate kinase from the first recombinant E. coli;
A peptide synthesis method comprising a step in which an amino acid ligase derived from a second recombinant Escherichia coli synthesizes a peptide using the ATP. The peptide synthesis method according to claim 1,
A peptide synthesis method wherein the peptide is L alanyl L glutamine.