CN110407909B

CN110407909B - Polypeptide sequence and preparation method and application of self-assembled material thereof

Info

Publication number: CN110407909B
Application number: CN201811462280.2A
Authority: CN
Inventors: 张志坤; 周英顺
Original assignee: Southwest Medical University
Current assignee: Southwest Medical University
Priority date: 2018-11-23
Filing date: 2018-11-23
Publication date: 2023-06-02
Anticipated expiration: 2038-11-23
Also published as: CN110407909A

Abstract

The invention discloses a synthetic polypeptide, which comprises the following components: (a) The amino acid sequence of the synthetic polypeptide is shown as SEQ ID No.1 and 2, and (b) one or more amino acids are deleted, inserted or substituted in the synthetic polypeptide defined in (a) and the polypeptide has the same biological function as the polypeptide in (a), so that the synthetic polypeptide has self-assembly capacity mediated by hydrogen phosphate, and the self-assembled substance formed has cation affinity activity and can enrich metal ions in aqueous solution. The recombinant strain has the enrichment activity of inorganic phosphate radicals and metal nickel ions after the recombinant induction expression of escherichia coli. The invention is nontoxic and has certain application potential.

Description

Polypeptide sequence and preparation method and application of self-assembled material thereof

Technical field:

the invention belongs to the field of biological materials, and relates to self-assembled polypeptide, a preparation method and application thereof.

The background technology is as follows:

self-assembly refers to a technique whereby the basic building blocks spontaneously form ordered structures. During self-assembly, the basic building blocks spontaneously organize or aggregate into a stable, regular geometric appearance under non-covalent based interactions. Self-assembling polypeptides are the most common and important, and natural gels found in most organisms, as well as many synthetic polymeric gels, are among the self-assembling polypeptides. The self-assembled polypeptide is used as a soft material and has wide application in many aspects, such as contact lens, artificial muscle, cell separation and culture, material extraction, immobilized enzyme, controlled release of medicine and the like. The self-assembled polypeptide material is mainly prepared into two main types, one type is synthetic polymer, and the other type is natural biological material such as polysaccharide, protein and the like. The polypeptide gel is a novel material with better biocompatibility and environmental friendliness, and the diversity of the arrangement of amino acids enables the polypeptide self-assembled molecule to have wide application prospect.

Choline binding sequences refer to proteins encoded by Streptococcus pneumoniae or Streptococcus pneumoniae phage that contain multiple repeats. Proteins containing a choline binding repeat sequence are collectively referred to as choline binding proteins. A variety of binding proteins play a role in the formation of streptococcus pneumoniae biofilms. It has been confirmed that: lytA amidase, lytC lysozyme, lytB aminoglucosidase, cbpA adhesin, pcpA adhesin and PspA (pneumococcal surface protein a) mutants have reduced ability to form biofilms.

The histidine tag refers to 6 consecutive histidines, which may be abbreviated as 6×His. Histidine tags are often used as recombinant protein terminal fusion polypeptide fragments. Histidine is an amino acid with a heterocyclic ring, each group of amino acids contains an imidazole group, the chemical structure carries many extra electrons, there is electrostatic attraction to positively charged chemicals, the tag is uncharged at pH8.0 and is non-immunogenic, and has substantially no effect on secretion, folding, and function of proteins. By virtue of the histidine peptide segment, special interactions with various metal ions can occur, including Ca2+, mg2+, ni2+, co2+ and the like, wherein nickel ions are most widely used. The protein is conveniently purified by metal chelate affinity chromatography.

Phosphorus is a non-renewable limited resource, and excessive discharge can bring about serious water eutrophication. Natural water-soluble phosphorus refers to inorganic phosphorus dissolved in a soil solution and a small amount of phosphorus bound to soluble low relative molecular mass organic compounds. The phosphorus concentration in the urban sewage is about 5-15mg/L, and the phosphorus content in part of sewage is higher. Phosphorus is a non-renewable limited resource, and phosphorus is a limiting factor for the growth of algae in water, so that phosphorus enrichment becomes an important method for solving the problems of phosphorus resource exhaustion and water purification. By utilizing the Enhanced Biological Phosphorus Removal (EBPR) technology, phosphorus in sewage can be enriched in an anaerobic tank or a sludge sedimentation tank by virtue of the action of phosphorus accumulating bacteria (PAOs), higher soluble phosphorus can be obtained, and the high-efficiency and simple removal method of soluble phosphate can better improve the recovery efficiency of phosphorus.

Heavy metal pollution of a water body refers to pollution of the water body caused by pollutants containing heavy metal ions entering the water body. Heavy metal wastewater (containing chromium, cadmium, copper, mercury, nickel, zinc and other heavy metal ions) generated in industrial production processes of mining and metallurgy, mechanical manufacturing, chemical industry, electronics, instruments and the like is one of the industrial wastewater with the most serious pollution to water bodies and the greatest harm to human beings. At present, the discharged wastewater of China contains various heavy metals. The national industrial wastewater discharge in 2014 was 205.3 hundred million tons. The discharge amount of heavy metals such as mercury, cadmium, hexavalent chromium, total chromium, lead and arsenic in the industrial wastewater is respectively 0.7 ton, 16.9 ton, 34.8 ton, 131.8 ton, 71.8 ton and 109.2 ton. The total discharge amount of the industrial wastewater and the discharge amount of heavy metals in the industrial wastewater are huge. Heavy metals, which exist in various chemical states or chemical forms, remain, accumulate and migrate after entering the environment or ecosystem, causing damage. If the concentration of heavy metals discharged with wastewater is small, the heavy metals can accumulate in algae and sediment and are adsorbed by the body surfaces of fish and shellfish, so that food chains are concentrated, and the pollution is caused. Heavy metals in wastewater are not decomposed and destroyed by various common water treatment methods.

The invention comprises the following steps:

in view of the above, the present invention aims to design a trigger type self-assembled amphiphilic polypeptide containing 6×His, wherein a 6×His formed hydrophilic structure exists on the surface of the formed self-assembled particle. The method is characterized in that the trigger factor is hydrogen phosphate, and the formed particles have affinity effect on transition metal ions such as Ni (2+) and the like in the aqueous solution.

The technical scheme adopted by the invention is as follows:

in order to achieve the above object, the present invention provides the following technical solutions: the invention provides a self-assembled polypeptide sequence, a preparation method and application thereof,

1. first, the binding amino acid sequence was simulated by analysis based on the repeated sequence of choline binding protein in Streptococcus pneumoniae. A polypeptide with stronger choline binding capability is designed, and the amino acid sequence of the polypeptide is shown as SEQ ID No.1/2 (1-20 amino acids). And 6 histidines (amino acids 21-26) are attached to the C-terminus. Its complete sequence is found in example 1 for a total of 26 amino acids.

2. The method of solid phase synthesis of polypeptide (synthesis of bivalirudin-2-Cl-Resin) is adopted, the unnatural amino acid is connected with Resin, and then the next natural amino acid is connected. Separating and purifying the synthesized polypeptide by using a high performance liquid chromatograph; mass spectrometry was used for mass detection.

3. The polypeptide powder was weighed and dispersed using sterile deionized water, and the sample was observed to be in a soluble state. Disodium/potassium phosphate (final concentration greater than 0.03M) was added to the solution. Precipitation of white granular material was observed, and flocculent white precipitate was observed at high polypeptide concentration. The resulting white material is the self-assembled polypeptide described herein.

4. After centrifugation of the white precipitate, it was resuspended using sterile deionized water. After a plurality of deionized water washes, the white substance was observed to be insoluble, and the insoluble state was maintained for a long period of time (observation time: 60 days). White particles (about 5 mM) were added to a salt solution of NiSO4 (5 mM) and incubated for 30min. Centrifuging to remove white particles, and detecting the metal ion solubility of the supernatant to find that the concentration of metal ions in the aqueous solution is reduced (see the attached drawing).

5. Cloning the SEQ ID No.2 sequence into pET28a vector, transforming BL21-DE3 strain, and culturing at 37 deg.c for 4 hr via IPTG induction to detect the expression of the polypeptide. Na2HPO4 (10 mmol/L), KH2PO4 (2 mmol/L), niSO4 (5 mg/L) were added to the ordinary LB broth for the culture of the recombinant strain, with the normal pET28a vector strain as a control. The content of soluble phosphate and the content of Ni (2+) ions in the culture solution are obviously reduced.

Compared with the prior art, the invention has the beneficial effects that:

the first, the synthetic polypeptide of the invention is nontoxic, and the cytotoxicity analysis shows that the synthetic polypeptide of the invention has no influence on the growth of 293T cells;

secondly, in the process of preparing self-assembled polypeptide by the synthetic polypeptide, hydrogen phosphate can be recovered for reuse;

thirdly, the self-contained polypeptide prepared by the invention has the activity of adsorbing metal ions;

fourth, the self-contained polypeptide prepared by the invention has higher stability in aqueous solution with pH of 6-8.8.

Fifthly, the self-contained polypeptide prepared by the invention provides a new material for enriching metal ions in a water system and purifying metal pollution of the water system.

Sixth, the invention is polypeptide, if use the biosynthesis method to carry on the large-scale production, the cost is cheaper, easy to use extensively.

Seventh, the polypeptide of the present invention is expressed by E.coli without purification. The recombinant strain expresses the polypeptide to obtain phosphate and Ni (2+) ion enrichment capability.

Drawings

FIGS. 1-1,1-2 show the results of mass spectrometry after purification of a synthetic polypeptide of the invention; 1-1: and (5) synthesizing a polypeptide P26 mass spectrum detection result.

Fig. 1-2: and synthesizing a polypeptide P26 high performance liquid chromatography detection result.

FIGS. 2-1 and 2-2 show the results of circular dichroism and structural analysis of the aqueous solution of the synthetic polypeptide of the present invention; fig. 2-1: and (5) circular dichroism spectrum detection results. Fig. 2-2: and (3) the content of various secondary structures in the polypeptide is estimated according to the circular dichroism spectrum detection result.

FIG. 3 is a photomicrograph of a synthetic polypeptide of the invention after formation of a self-assembled polypeptide; the resulting self-assembled polypeptides were observed with an inverted microscope (400-fold).

FIG. 4 is an electron micrograph of a polypeptide of the invention after formation of the self-assembled polypeptide, as observed by electron microscopy of the self-assembled polypeptide described herein.

FIG. 5 shows the results of the removal test of NI (2+) ions from aqueous solutions for the self-assembled polypeptides of the present invention, for standard and treated samples of NiSO4 at initial concentrations of 1,2,3,4,5mg/L, respectively.

FIG. 6-1,6-2,6-3 is constructed for recombinant SEQ ID No.2 strain, and the functional identification results. Fig. 6-1: the recombinant pET28a vector of SEQ ID No.2 is constructed, and the protein expression detection results (the DNA standard molecular sample of No.1, the plasmid restriction enzyme digestion identification result of No.2, the induced expression result of No. 3, the control induced strain of No. 4 are shown by arrows, and the coding gene fragment of SEQ ID No.2 or the protein electrophoresis band) are shown. Fig. 6-2: and the detection result of the content of soluble phosphate in the recombinant pET28a carrier strain culture system is that the dotted line is a control bacteria sample, and the measurement value is OD690 by adopting a molybdenum-antimony anti-spectrophotometry method. Fig. 6-3: and (3) monitoring the content of Ni2+ ions in a recombinant pET28a carrier strain culture system, wherein a dotted line is a control bacteria sample, and the measurement value is OD450 by a dimethylglyoxime method.

Detailed Description

The invention is further described below with reference to the accompanying drawings.

Polypeptide sequence:

a synthetic polypeptide has an amino acid sequence shown as SEQ ID No.1 and 2; SEQ ID No.2 replaces the first amino acid with the amino acid encoded by the initiation codon for prokaryotic expression.

SEQIDNo.1：TGWVKDNGSWYYLNLSGYMLHHHHHH，

Thr-Gly-Trp-Val-Lys-Asp-Asn-Gly-Ser-Trp-Tyr-Tyr-Leu-Asn-Leu-Ser-Gly-Tyr-Met-Leu-His-His-His-His-His-His

SEQIDNo.2：MGWVKDNGSWYYLNLSGYMLHHHHHH。

Met-Gly-Trp-Val-Lys-Asp-Asn-Gly-Ser-Trp-Tyr-Tyr-Leu-Asn-Leu-Ser-Gly-Tyr-Met-Leu-His-His-His-His-His-His

Embodiment 1: and (3) synthesizing the polypeptide, namely, using 2-Cl-Resin as a carrier (Resin) to synthesize the polypeptide, adopting ethyl glacial ether to carry out suction filtration to obtain a crude peptide product, adopting a C18 and 10 mu m chromatographic column to carry out purification and impurity removal, and sending mass spectrum to confirm that the molecular weight of the product is 3189.40g/mol in accordance with a theoretical value. The freeze-dried pure product is weighed, and the purity of the product is 82.487 percent through analysis and calculation.

Example 2: dissolving polypeptide freeze-dried powder in deionized water to prepare 200 mug/ml solution, wherein the polypeptide is soluble polypeptide, and the polypeptide solution is colorless and transparent. Taking 5ml of the solution for circular dichroism detection. And the percentage of various structures in the secondary structure of the polypeptide is calculated according to the detection result.

The circular dichroism spectrum (CD for short) is the most widely used method for measuring the secondary structure of protein, and is a rapid, simple and accurate method for researching protein conformation in dilute solution.

Example 3: the polypeptides described herein were formulated as 200 μg/ml solutions, which were colorless and transparent. To the solution was added disodium hydrogen phosphate at a final concentration of 0.1M, a white granular substance was seen, and the lowest concentration of disodium hydrogen phosphate capable of forming white granules was 5mM. The solution was pipetted onto a slide in an amount of 50. Mu.l and a cover slip was applied over the drop. The results were observed under an inverted microscope.

Example 4: the polypeptides described herein were formulated as 200 μg/ml solutions, which were colorless and transparent. Disodium hydrogen phosphate was added to the solution at a final concentration of 0.1M. And (3) dripping the sample on the copper sheet, and drying the copper sheet carrying the sample and then adhering the copper sheet carrying the sample on the conductive adhesive tape on the sample holder. And shooting the picture according to the steps.

Example 5: the polypeptides described herein were formulated as a 3mg/ml solution, which was colorless and transparent. Mu.l, 70. Mu.l of the polypeptide were placed in a tube to the solution, respectively. Disodium hydrogen phosphate was added to the tube at a final concentration of 0.1M, and a white precipitate was seen. The white material was harvested separately by centrifugation, resuspended with 100 μl of sterile deionized water, centrifuged and the water removed using a pipette, repeated 4 times to remove residual disodium hydrogen phosphate in the solution. After a plurality of deionized water washes, the white substance was observed to be insoluble, and the insoluble state was maintained for a long period of time (observation time: 60 days).

The above white particles were added to a salt solution (5 mM) of NiSO4 and incubated for 2min, respectively. The white particles were removed by centrifugation and the concentration of Ni (2+) was measured using dimethylglyoxime and found to decrease by about 20% to 35% in the treated solution.

Example 6: cloning the SEQ ID No.2 sequence into pET28a vector by adopting a genetic engineering technology, transforming BL21-DE3 strain, and culturing for 4 hours at 37 ℃ through IPTG induction to detect the expression of the polypeptide (figure 6-1). Na2HPO4 (10 mmol/L), KH2PO4 (2 mmol/L), niSO4 (5 mg/L) were added to the ordinary LB broth for the culture of the recombinant strain, with the normal pET28a vector strain as a control. The soluble phosphate content and Ni (2+) ion content in the culture solution were found to be significantly reduced compared with the control group (FIGS. 6-2, 6-3). PO4 (3-), and Ni (2+) were detected as substances affecting the detection of PO4 (3-), but the difference in the content of PO4 and Ni (2+) was clearly recognized as compared with the control group.

Although embodiments of the present invention have been disclosed above, it is not limited to the details and embodiments shown, it is well suited to various fields of use for which the invention is suited, and further modifications may be readily made by one skilled in the art, and the invention is therefore not to be limited to the particular details and examples shown and described herein, without departing from the general concepts defined by the claims and the equivalents thereof.

Claims

1. A self-assembling polypeptide, characterized in that: the amino acid sequence is shown as SEQ ID No.1, wherein 1-20 amino acids are self-designed sequences and have choline binding activity; amino acids 21-26 are histidine and are capable of electrophilic reaction with metal ions.

2. The method of producing self-assembled particles of self-assembled polypeptides according to claim 1, wherein: the self-assembled polypeptide is subjected to electrophilic reaction in an aqueous solution containing disodium hydrogen phosphate with the concentration of not less than 5mM to form self-assembled particles, and the concentration of the polypeptide in a dispersion medium is more than 30 mug/mL.

3. The method of preparing self-assembled particles according to claim 2, wherein: is formed by the self-assembled polypeptide under the conditions of the temperature of 4-50 ℃ and the pH value of 6-8.8.

4. The method for producing self-assembled particles according to claim 2, wherein the self-assembled particles formed remain stable in a pure water solution after removal of disodium hydrogen phosphate.

5. Use of the self-assembled polypeptide according to any one of claims 1-4 for immobilization and detection of hydrogen phosphate in aqueous solution.

6. Use of a self-assembled polypeptide according to any one of claims 1-4 for adsorption of metal ions in aqueous solutions.

7. The application of the organism expressing the polypeptide in hydrogen phosphate fixation and metal ion adsorption is that the organism is escherichia coli, and the polypeptide is shown as SEQ ID NO. 2.