CN102168057B

CN102168057B - Engineering bacteria expressing active peptides and method of preparing mixed polypeptide

Info

Publication number: CN102168057B
Application number: CN201010546991A
Authority: CN
Inventors: 李云亮; 马海乐; 任晓锋; 黄六容; 曲文娟
Original assignee: Jiangsu University
Current assignee: Jiangsu University
Priority date: 2010-11-17
Filing date: 2010-11-17
Publication date: 2012-08-29
Anticipated expiration: 2030-11-17
Also published as: CN102168057A

Abstract

The present invention provides escherichia coli engineering bacteria expressing active peptides and a method of preparing mixed polypeptide CEI-12 and CEI-7 with the escherichia coli engineering bacteria, and relates to the bioengineering field. The preservation number of the engineering bacteria is Escherichia Coli BL21-MF3A3 CCTCC No: 2010204. By using smaller His label segment on pET-30a to subject CEI-12 polypeptide and CEI-7 polypeptide to fusion expression and alternative cascade three times, the method increases the ratio of target polypeptide in recombination protein, greatly increases the expression efficiency, and reduces the separating difficulty of small peptides.

Description

Express the engineering bacteria of many bioactive peptides and prepare the method for mixed polypeptide

Technical field

The present invention relates to bioengineering field,, utilize the engineering bacteria of its expression vector establishment and utilize it to prepare the method for said many bioactive peptides more specifically to making up a kind of many bioactive peptide series connection DNA.

Background technology

Biologically active peptides is that natural amino acid is formed the general name with the different peptide classes of the linearity from the dipeptides to the complicacy of arrangement mode formation, ring structure with difference in the protein, is to come from proteinic multi-functional compounds.Bioactive peptide has multiple body metabolism and physiological regulation function; Be prone to digest and assimilate; Effects such as promoting immunity, hormone regulation, antibiotic, antiviral, hypotensive, reducing blood-fat is arranged, and edible safety is high, is the most popular research topic of current international food circle and the functional factor that has development prospect.

In treatment essential hypertension drug research; From natural food protein, isolated at present biologically active peptides with antihypertensive function; This type of bioactive peptide then is through suppressing blood plasma and vascular endothelial cell hypertensinase (Angiotensin I-Converting Enzyme; ACE) activity reaches the purpose that brings high blood pressure down.Because of its blood pressure lowering effect is obvious, safe without toxic side effect, become the focus of bioactive peptide research.Now successively from multiple animal-plant material and tankage; As viper venom, sardines, cheese, soybean meal, fermenting bean milk, corn refuse lac, former albumen (referring to Huang Jiayin. wait the people; Food and fermentation industries, 32:81-86 (2006)) etc. isolated multiple active polypeptide with antihypertensive function.

At present from the cow's milk protein enzymolysis product, found multiple polypeptide with antihypertensive function, wherein studying many is peptide C EI-12 (Phe-Phe-Val-Ala-Pro-Phe-Glu-Val-Phe-Gly-Lys), peptide C EI-5 (Phe-Phe-Val-Ala-

Pro), peptide C EI-7 (Ala-Leu-Pro-Met-His-Ile-Arg), peptide C EI-β 7 (Ala-Leu-Pro-Met-His-Ile-Arg) etc. (referring to Dong Kaifa. wait the people; Chinese food journal, 4:86-90 (2004)) has good blood pressure lowering effect.

Because the cow's milk protein enzymolysis product is a polypeptide mixture; Of a great variety; Activity difference is very big, and the productive rate that really has highly active polypeptide is also not really high, is the important operation of active enrichment to this separation and purification; But facts have proved to reach very difficult to the effective separation and purification of high reactivity polypeptide, cost increases significantly.Therefore enzymolysis product is difficult to become the raw material of preparation high reactivity Altace Ramipril, has limited the deep development of this industry.

The fast development of biotechnology realizes that for overcoming the problems referred to above the efficient production of bioactive peptide provides possibility.Utilize engineering bacteria efficiently express the single-activity peptide the application of antibacterial peptide, tumor suppression peptide etc. (referring to: learn military affairs recklessly. wait the people, Chinese biological chemistry and molecular biosciences journal, 18 (3): 287-292 (2002); Ma Hongxing. wait the people, international genetics magazine, 30:169-172 (2007).), obtained success.In to newborn source property blood pressure lowering peptide CEI-12 research; There is the investigator to use bigger fusion rotein (molecular weight is about 29 kDa) not placed in-line 12 the amino acid CEI-12 sequences of amalgamation and expression (molecular weight is about 1.3 kDa); Expression efficiency is extremely low (referring to Lv, people such as G.S., J.Dairy Sci.; 86:1927-1931 (2003)); Simultaneously, comprised multiple high reactivity polypeptide composition because the cow's milk protein enzymolysis product is a polypeptide mixture, only utilize engineering bacterium expression wherein the single-activity peptide possibly not reach good blood pressure lowering effect.Therefore research and establishment utilizes two or more series connection blood pressure lowering peptide gene of less fusion rotein label amalgamation and expression, and in the hope of improving blood pressure lowering peptide expression efficiency and hypotensive activity, it is necessary just to seem.The research that utilizes engineering bacterium expression breast source property active antihypertensive peptide at present seldom, the research that utilizes engineering bacteria to express two or more blood pressure lowering peptide does not simultaneously more appear in the newspapers.

Summary of the invention

The present invention is with the example that is prepared as of two kinds of newborn source property blood pressure lowering peptides, explain many bioactive peptides the DNA that alternately connects construction and expression and make up the method for engineering bacteria.

First aspect of the present invention provides the recombination bacillus coli engineering bacteria that can efficiently express two kinds of newborn source property blood pressure lowering peptide CEI-12 and CEI-7.This bacterial classification is the Chinese typical culture collection center preservation in Chinese Wuhan on August 19th, 2010, and deposit number is intestinal bacteria Escherichai coliBL21-MF3A3 CCTCC NO:M 2010204.

Second aspect of the present invention provides the method for utilizing described intestinal bacteria to prepare mixed polypeptide CEI-12 and CEI-7; Carry out according to following step: (1) is in the LB liquid nutrient medium that is inoculated in of 1% inoculum size with above-mentioned e. coli bl21-MF3A3 by volume earlier; Wherein substratum is formed as follows: Tryptones 10g/L; Yeast extract 5g/L, NaCl 10g/L, pH 7.0; 37 ℃, 200rmin ^-1It is 1.5 o'clock that the constant temperature shaking table is cultured to OD600, adds isopropyl-to final concentration 1mmolL ^{– 1}, induce 12 h for 28 ℃, 5000r min ^-1Centrifugal collection thalline freezes thalline molten 3 times repeatedly, carries out ultrasonication at PBS buffered soln, 8000r min ^-1The centrifuging and taking deposition is dissolved in upper prop buffered soln, obtains recombinant protein through activatory Ni-column purification; (2) with above-mentioned fully dissolving in the 6M urea of preparing through cryodesiccated recombinant protein; The 0.1% enteropeptidase solution that adds two volumes pH8.0,37 ℃ of temperature are bathed 8h, remove His mark fusion tag; Make the tandem polypeptide of fusion tag; Resulting tandem polypeptide solution is added 0.1% (w/v) trypsinase that monoploid amasss pH8.0, and 37 ℃ of temperature are bathed 8h; Dialysis tubing through molecular weight cut-off 7000Da is removed enteropeptidase and trypsinase, sees through liquid obtains purifying after concentrated with desalination polypeptide.

Advantage of the present invention is: utilize pET-30a to go up less His label segment amalgamation and expression alternately connect 3 CEI-12 and CEI-7 polypeptide, increased the shared ratio of desired polypeptides in the recombinant protein, increase expression efficiency greatly and reduced the separating difficulty of little peptide.

Description of drawings

Fig. 1 has shown e. coli bl21-MF3A3 bacterium colony PCR electrophorogram, explains that the plasmid of engineering bacillus has inserted the big or small 120bp left and right sides goal gene that is; Wherein annotate: 1, pET-30a ⁺Goal gene, 2, the pET-30a empty carrier, 3, Marker.

Fig. 2 has shown e. coli bl21-MF3A3 expression product SDS-PAGE electrophorogram, the engineering bacillus successful expression has been described size be the target protein about 12kDa; Wherein annotate: 1, protein Marker; 2, BL21-MF3A3 (not adding IPTG induces); 3, BL21-MF3A3 (adding IPTG induces), 4, BL21-MF3A3 (adding IPTG induces) ultrasonication supernatant, 5, BL21-MF3A3 (adding IPTG induces) ultrasonication deposition.

Fig. 3 has shown original hypertensive rat filling a certain amount of newborn source property blood pressure lowering peptide CEI-12 of stomach and CEI-7 mixed peptide blood pressure figure, has explained that the CEI-12 and the CEI-7 mixed peptide of the present invention's preparation has good blood pressure lowering effect.

Embodiment

Employed in the present invention term only if other explanation is arranged, generally has the implication of those of ordinary skills' common sense.

Below in conjunction with concrete embodiment, and comparable data is described the present invention in further detail.Should be understood that these embodiment just in order to demonstrate the invention, but not limit scope of the present invention by any way.

In following embodiment, various processes and the method do not described in detail are ordinary methods as known in the art.The source of agents useful for same, trade(brand)name and be necessary to list its moity person indicate when occurring first that all used thereafter identical reagent is like no specified otherwise, and is all identical with the content of indicating first.

From description taken in conjunction accompanying drawing given below, will become clear with other purposes and characteristic above of the present invention.

Embodiment 1:Two kinds of newborn source property blood pressure lowering peptide CEI-12 and CEI-7 series connection DNA design are with synthetic

A, two kinds of newborn source property blood pressure lowering peptide CEI-12 and CEI-7 tandem polypeptide sequences Design

Newborn source property blood pressure lowering peptide CEI-12 (SEQ ID NO3) and the CEI-7 (SEQ ID NO4) that the present invention relates to correspond respectively to the residue sequence of cow's milk alpha-casein 23-34 and the 177-183 residue sequence of cow's milk beta-casein.The N section amino-acid residue of CEI-12 and CEI-7 is respectively Lys and Arg; Be all tryptic restriction enzyme site; The aminoacid sequence of CEI-12 and CEI-7 is alternately connected 3 times, add Asp-Asp-Asp-Asp-Lys sequence in the N of gained aminoacid sequence section, sequence is shown in SEQ ID NO2.

B, two kinds of newborn source property blood pressure lowering peptide CEI-12 and CEI-7 tandem polypeptide gene design

With gained aminoacid sequence (SEQ ID NO2) according to intestinal bacteria ( Escherichia coli) BL-21 bacterial strain codon-bias change into nucleotide sequence and 3 ' end add the TAATAATAA sequence with BamH I restriction endonuclease recognition sequence GGATCC adds at 5 ' end KpnI restriction endonuclease recognition sequence GGTACC, the gained nucleotide sequence is shown in SEQ ID NO1.

Embodiment 2:Expression vector pUC18-CEI-12, the structure of 7 structure and e. coli bl21-MF3A3 engineering bacteria

The nucleotide sequence SEQ ID NO1 that a, embodiment 1 are designed is cloned into pUC18 (available from Novagen company) by synthetic the obtaining of Shanghai rising sun hat biotech development ltd, and restriction enzyme site does KpnI with BamThe H I, with this recombinant vectors called after pUC18-CEI-12,7, change over to intestinal bacteria ( Escherichia coli) DH5 α (available from Novagen company).

B, will contain pUC18-CEI-12,7 bacillus coli DH 5 alpha extracts plasmid, carries out KpnI with BamH I (available from TaKaRa company) double digestion reclaims the fragment about 200bp.

C, expression vector pET-30a (available from Novagen company) is carried out KpnI with BamH I (available from TaKaRa company) double digestion reclaims big fragment.

D, step b gained had a sticky end recovery fragment and step c in the big fragment that reclaims link to each other; Change DH5 α bacterial strain over to; Utilize conventional bacterium colony PCR to obtain clone's; Primer is 5'-ACGACTCACTATAGGGGAATTGTGA-3' and 5'-CGGATATAGTTCCTCCTTTCAGCA-3' (it is synthetic that worker's biotechnology ltd is given birth in Shanghai), and the result is as shown in Figure 1.

E, clone's that steps d is obtained are delivered to Shanghai and are given birth to the order-checking of worker's biotechnology ltd, sequencing result with design consistent.

F, will check order clone's correct and extract plasmid, called after pET30a-CEI-12,7, change over to intestinal bacteria ( Escherichia coli) BL-21 (available from Novagen company), with the transformant called after " e. coli bl21-MF3A3 " for preparing like this.This bacterial classification is the Chinese typical culture collection center preservation in Chinese Wuhan on August 19th, 2010, and deposit number does Escherichai coliBL21-MF3A3 CCTCC NO:M 2010204.

Embodiment 3:The expression and the detection of two kinds of newborn source property blood pressure lowering peptide CEI-12 and CEI-7 tandem polypeptide

E. coli bl21-MF3A3 that instance 2 is obtained is that 1% inoculum size is inoculated in LB liquid nutrient medium (the Tryptones 10g/L that 50mL contains kantlex Kan (50 μ g/mL) by volume; Yeast extract 5g/L; NaCl 10g/L, pH7.0) in, 37 ℃, 200rmin ^-1It is 1.5 o'clock that the constant temperature shaking table is cultured to OD600, adds isopropyl-(IPTG) to final concentration 1mmolL ^{– 1}, induce 12 h for 28 ℃, 5000r min ^-1Centrifugal collection thalline freezes thalline repeatedly and to dissolve 3 times, PBS buffered soln (pH is 7.0) carry out ultrasonication (300W, 2s/2s, 2min), 8000r min ^-1The centrifuging and taking deposition is dissolved in 8mL upper prop buffered soln, obtains the recombinant protein of His mark through activatory Ni-post (available from Novagen company) purifying, carries out the SDS-PAGE electrophoresis, and is as shown in Figure 2.Electrophoresis is illustrated in intestinal bacteria and induces the back target protein mainly to be present in the deposition of intestinal bacteria ultrasonication liquid after centrifugal, and molecular weight size be about 12kDa, and is big or small identical with the designed molecules amount.

Embodiment 4:Two kinds of newborn source property blood pressure lowering peptide CEI-12 and CEI-7 tandem polypeptide enzyme are cut purifying

With fully dissolving in the 6M urea of embodiment 2 said method preparations through cryodesiccated recombinant protein; The 0.1% enteropeptidase solution (available from Novagen company) that adds two volumes pH8.0; 37 ℃ of temperature are bathed 8h; Carry out activatory Ni-post (available from Novagen company)) purifying removal His mark fusion tag; Make the tandem polypeptide of fusion tag, 0.1% (w/v) trypsin that resulting tandem polypeptide solution is added the long-pending pH8.0 of monoploid is available from Sigma company), 37 ℃ of temperature are bathed 8h; Dialysis tubing (reaching instrument Science and Technology Ltd. available from Shanghai Ou Wei) through molecular weight cut-off 7000Da is removed enteropeptidase and trypsinase, sees through liquid and after concentrating, obtains the polypeptide of purifying with CELLUFINE GH-25 post (available from CHISSO company) desalination, and the expression amount that detects CEI-12 and CEI-7 mixed polypeptide through the Xylene Brilliant Cyanine G method is 0.2gL ^{– 1}

Embodiment 5:Mixed polypeptide CEI-12 and CEI-7 are active to be detected

Active used mixed polypeptide CEI-12 of detection and CEI-7 prepare with embodiment 4 methods according to embodiment 3.

A. mixed polypeptide CEI-12 and CEI-7 external activity detect

External activity detection employing Vermeirssen etc. are improved to be the spectrophotometry of angiotensin stand-in with FAPGG, like table 1, records the IC of mixed polypeptide CEI-12 and CEI-7 ₅₀Value is 0.012g/L, and activity is higher than CEI-12 (IC ₅₀Value is 0.099g/L) and CEI-7 (IC ₅₀Value is for 0.036g/L) wherein separately any one (referring to people such as Richard J. F., J Nutr, 134 (4): 980-988. (2004)).

Table 1 ACE suppresses active measurement operation parameter

	Blank well (μ L)	Sample well (μ L)
			ACE（0.1U/mL）	10	10
FAPGG（mmol/L） ¹	50	50
			The matrix damping fluid ²	40	0
Mixed polypeptide (CEI-12 and CEI-7)	0	40

Annotate: 1.FAPGG (1.0mmol/L): get 3.994mg FAPGG and add the matrix damping fluid, be settled to 10mL, dissolving mixes, and puts 4 ℃ of lucifuges and places; 2. matrix damping fluid: HEPES 1.910g, NaCl 1.755g after the bi-distilled water dissolving, is transferred to pH8.3 with NaOH, restock water is to 100mL, put 4 ℃ subsequent use.

B. mixed polypeptide CEI-12 and CEI-7 activity in vivo detect

Experiment material is: 20 of SPF level male essential hypertension model Wistar rats in 10 age in week, body weight 180 ~ 240 g purchase in Shanghai Slac Experimental Animal Co., Ltd..Experimental result is as shown in Figure 3.Experimental result shows that at 4h to 6h, diastole is pressed with and declines to a great extent after the various dose administration, and wherein middle dose groups is maximum at the 4h drop-out value of blood pressure, reaches 32mmHg, the blood pressure lowering effect that this dosage is good to original hypertensive rat.

SEQUENCE LISTING

< 110>Jiangsu University

< 120>express the engineering bacteria of many bioactive peptides and prepare the method for mixed polypeptide

<130>

<160> 4

<170> PatentIn version 3.3

<210> 1

<211> 207

<212> DNA

< 213>artificial sequence

<400> 1

GGTACCGACG ACGACGACAA GTTCTTTGTG GCGCCGTTTC CGGAAGTGTT TGGCAAAGCG 60

CTGCCGATGC ATATTCGCTT CTTTGTGGCG CCGTTTCCGG AAGTGTTTGG CAAAGCGCTG 120

CCGATGCATA TTCGCTTCTT TGTGGCGCCG TTTCCGGAAG TGTTTGGCAA AGCGCTGCCG 180

ATGCATATTC GCTAATAATA AGGATCC 207

<210> 2

<211> 62

<212> PRT

<213>

<400> 2

Asp Asp Asp Asp Lys Phe Phe Val Ala Pro Phe Pro Glu Val Phe Gly

1 5 10 15

Lys Ala Leu Pro Met His Ile Arg Phe Phe Val Ala Pro Phe Pro Glu

20 25 30

Val Phe Gly Lys Ala Leu Pro Met His Ile Arg Phe Phe Val Ala Pro

35 40 45

Phe Pro Glu Val Phe Gly Lys Ala Leu Pro Met His Ile Arg

50 55 60

<210> 3

<211> 12

<212> PRT

<213>

<400> 3

Phe Phe Val Ala Pro Phe Pro Glu Val Phe Gly Lys

1 5 10

<210> 4

<211> 7

<212> PRT

<213>

<400> 4

Ala Leu Pro Met His Ile Arg

1 5

Claims

1. express the colibacillus engineering of many bioactive peptides Escherichai coliBL21-MF3A3, said engineering bacteria are preserved in Chinese typical culture collection center, and deposit number is CCTCC NO:M 2010204, and preservation date is on August 19th, 2010.

2. utilize the described engineering bacteria of claim 1 to prepare the method for mixed polypeptide CEI-12 and CEI-7; Carry out according to following step: (1) is that 1% inoculum size is inoculated in the LB liquid nutrient medium with above-mentioned intestinal bacteria earlier by volume; Wherein the LB substratum is formed as follows: Tryptones 10g/L; Yeast extract 5g/L, NaCl 10g/L, pH 7.0; 37 ℃, 200rmin ^-1It is 1.5 o'clock that the constant temperature shaking table is cultured to OD600, adds isopropyl-to final concentration 1mmolL ^{– 1}, induce 12 h, 5000rmin for 28 ℃ ^-1Centrifugal collection thalline freezes thalline molten 3 times repeatedly, carries out ultrasonication, 8000rmin at PBS buffered soln ^-1The centrifuging and taking deposition is dissolved in upper prop buffered soln, obtains recombinant protein through activatory Ni-column purification; (2) with above-mentioned fully dissolving in the 6M urea of preparing through cryodesiccated recombinant protein; The 0.1% enteropeptidase solution that adds two volumes pH8.0; 37 ℃ of temperature are bathed 8h, remove His mark fusion tag, make the tandem polypeptide of fusion tag; The quality and the volume ratio that resulting tandem polypeptide solution are added the long-pending pH8.0 of monoploid are 0.1% trypsinase; 37 ℃ of temperature are bathed 8h, remove enteropeptidase and trypsinase through the dialysis tubing of molecular weight cut-off 7000Da, see through liquid obtains purifying after concentrated with desalination polypeptide.