CN107916243A

CN107916243A - For handling microbial cell and albumen, the corresponding processing method and kit of heavy metal pollution

Info

Publication number: CN107916243A
Application number: CN201610884462.3A
Authority: CN
Inventors: 娄春波; 杜沛; 魏伟佳; 王瑞沙; 姜浩; 张浩千
Original assignee: Institute of Microbiology of CAS
Current assignee: Institute of Microbiology of CAS
Priority date: 2016-10-10
Filing date: 2016-10-10
Publication date: 2018-04-17

Abstract

The present invention relates to for handle heavy metal pollution microbial cell and albumen, utilize the microbial cell and albumen processing heavy metal pollution method and kit.

Description

For handling the microbial cell and albumen, corresponding processing method of heavy metal pollution And kit

Technical field

The present invention relates to pollution process field, in particular to for handle heavy metal pollution microbial cell and Albumen, utilize the microbial cell and the method and kit of albumen processing heavy metal pollution.

Background technology

The activity such as the mining of the mankind, smelting, fertilising generates plurality of heavy metal pollution in natural water.In water body The heavy metals such as Hg, Cd, Pb not only all have toxic action to most of biology, but also may be enriched with via food chain, final right Human health damages.At present, the manner of cleaning up of the heavy metal element in water body is broadly divided into based on chemistry and biology Method.Wherein, the method for cleaning based on chemistry includes chemical precipitation, chemisorbed, redox, electrolysis, ion exchange, film point From etc., the above method is small, of high cost and easily cause secondary pollution problems there are processing flux to varying degrees.

Heavy metal adsorption based on biology is the method that is generally used in recent years, can realize environmentally friendly, is not produced The target of raw secondary pollution.Microorganism has the advantages that speed of growth is fast, good environmental adaptability, can efficient purification of heavy metal The relatively low waste water of concentration.Research both domestic and external shows, a variety of micro- lifes including bacterium, actinomyces, mould, yeast and algae Thing all has the ability to be enriched with heavy metal ion micro in aqueous solution.For example, the adsorbable Co of brown alga²⁺, aspergillus oryzae is adsorbable Zn²⁺, the adsorbable Cd of yeast²⁺, the adsorbable Pb of bacillus licheniformis²⁺, the adsorbable Cu of micrococcus luteus²⁺Deng.

The various microbial population of nature evolves a variety of albumen that can be adsorbed, detoxified to heavy metal.For example, Being widely present in MerR albumen in the common bacterias such as pseudomonas aeruginosa, bacillus megaterium, staphylococcus aureus can be with Hg in special adsorbent solution²⁺Ion., can meanwhile other albumen in the gene cluster where MerR also have function of detoxification With by Hg²⁺HgO is converted into, substantially reduces its toxicity.In addition, CadR, PbrR, CueR and ZntR are able to absorption Cd² ⁺、Pb²⁺、Cu²⁺And Zn²⁺.Therefore, Heavy-Metal-Contaminated Environments purification is carried out using the microorganism with heavy metal adsorption Genetic elements With great potentiality.

The content of the invention

In a first aspect, be used to handle the microbial cell of heavy metal pollution the present invention provides a kind of, wherein, it is described micro- Biological cell expresses Heavy Metal Binding Proteins and biotin-binding protein；Wherein, the heavy metal is selected from a following huge sum of money One or more in category：Cadmium, mercury, lead and arsenic.

In second aspect, the present invention provides a kind of fusion protein for being used to handle heavy metal pollution, wherein, the fusion Albumen includes heavy metal binding structural domain and biotin binding structural domain；Wherein, the heavy metal is selected from a following huge sum of money One or more in category：Cadmium, mercury, lead and arsenic.

In the third aspect, the present invention provides a kind of kit for being used to handle heavy metal pollution, the kit includes The fusion protein described in microbial cell or second aspect described in first aspect, and biotinylated solid phase material；Wherein, The heavy metal is the one or more in following heavy metal：Cadmium, mercury, lead and arsenic.

In fourth aspect, the present invention provides a kind of method for handling heavy metal pollution in sample, the method bag Include：

(a) microbial cell described in first aspect or the fusion protein described in second aspect are provided；

(b) biotinylated solid phase material is provided；

(c) the biotinylated solid phase that the microbial cell or fusion protein and step (b) provided step (a) provides Material is mixed with sample, so that the heavy metal ion in sample is captured, and

(d) the biotinylated solid phase material of step (b) is recycled；

Wherein, the heavy metal is the one or more in following heavy metal：Cadmium, mercury, lead and arsenic.

Brief description of the drawings

Fig. 1 is principle schematic diagram of the present invention.(A) the heavy metal processing method based on cell.Cell can expression and localization in The Heavy Metal Binding Proteins of membrane surface and the biotin-binding protein for being positioned at membrane surface, so that the cell It can be combined respectively with heavy metal and biotin (so that with biotinylated solid phase material).When there are during heavy metal, this is thin Born of the same parents can capture heavy metal to biotinylated solid phase material.(B) the heavy metal processing method based on albumen.Fusion protein bag Containing two domains, heavy metal binding structural domain and biotin binding structural domain, so as to respectively with heavy metal and life Thing element (so that with biotinylated solid phase material) combines.When there are during heavy metal, which can capture heavy metal To biotinylated solid phase material.

Fig. 2 is according to embodiment 2, and biotin-binding protein is in the expression of microbial cell surface and mutual with biotin Effect.(A) structure of a variety of biotin-binding proteins with cross-film and film localization domain.(B-D) it is respectively to utilize enzyme mark Each biotin-binding protein that the surface expression (A) that instrument is read, flow cytometry and fluorescence microscopy obtain is shown it is big Enterobacteria and with fluorescence biotin (Biotin-Atto88) combination result.(B) square frame is represented compared with mSA2, table The ability not specifically bound up to the Streptavidin of the wild type in cell surface with biotin.(C) square frame expression and eMA Compared with SCD-E2, mSA2 has the specific binding most strong with biotin.Error bar is the standard deviation of independent experiment three times. (D) IPTG+ represents induced expression biotin-binding protein, and IPTG- represents not induce.N is represented without biotin integrated structure Domain.

Fig. 3 shows what the Escherichia coli that different biotin-binding proteins are expressed in embodiment 2 were combined with biotinylation magnetic bead Scanning electron microscope as a result, cell surface do not express biotin-binding protein Escherichia coli (N) be difficult to be attached to it is biotinylated Magnetic bead.Biotinylated magnetic is largely attached in the Escherichia coli (mSA2) of cell surface expression biotin-binding protein mSA2 Pearl.Biotinylated magnetic bead is attached on a small quantity in the Escherichia coli (Streptavidin) of cell surface expression Streptavidin. IPTG+ represents induced expression mSA2 or Streptavidin, and IPTG- represents not induce.

Fig. 4 is according to embodiment 2, in the surface of E. coli expression protein-bonded construct of mercury ion (A-C) and right The absorption (D) of mercury ion in sample.(A) structure diagram of MerR, shows DNA binding structural domains, mercury ion binding structural domain And catenation sequence.(B) structure of MerR is simulated, designs the protein-bonded mercury ion bound fraction MBP (Hg) of mercury ion, the part With two mercury ion binding structural domains.(C) the protein-bonded overall structure diagram of mercury ion, shows cross-film and film positioning knot The connection mode of structure domain Lpp-OmpA and MBP (Hg).(D) according to the absorption to mercury ion of AFS DETERMINATION. Error bar is the standard deviation of independent experiment three times.

Fig. 5 is according to embodiment 3, design (A) and the SDS-PAGE verification (B) of SA-MBP (Hg) fusion protein.(A) SA-MBP (Hg) fusion protein includes the biotin binding structural domain (core of the Streptavidin as biotin binding structural domain Functional domain), the MBP (Hg) as heavy metal binding structural domain and for the histidine-tagged of purifying, the structure of the MBP (Hg) For as shown in Figure 4 B.

Fig. 6 is the SDS-PAGE results to be interacted according to the SA-MBP (Hg) that embodiment 3 obtains with magnetic bead.

Fig. 7 is that the mercury in sample is carried out according to the SA-MBP (Hg) that the embodiment 3 of AFS DETERMINATION obtains The effect of removing.Error bar is the standard deviation of independent experiment three times.

Fig. 8 is according to embodiment 4, the design (A-C) and SA-MBP (Cd) and magnetic bead phase of SA-MBP (Cd) fusion protein The SDS-PAGE results (D) of interaction.(A) structure diagram of CadR, shows DNA binding structural domains, cadmium ion integrated structure Domain and random sequence.(B) structure of CadR is simulated, design cadmium ion binding structural domain MBP (Cd) includes the funtion part of CadR (cadmium ion binding structural domain and random sequence).(C) overall structure diagram of SA-MBP (Cd) fusion protein, it includes conduct The biotin binding structural domain (Core Feature domain) of the Streptavidin of biotin binding structural domain, as heavy metal integrated structure The MBP (Cd) in domain and for the histidine-tagged of purifying.

Fig. 9 is that the SA-MBP (Cd) that the embodiment 4 measured according to inductivity coupled plasma mass spectrometry (ICP-MS) obtains is right The effect that cadmium in sample is purged.

Figure 10 is the secreting type SA-MBP (Hg) (A-C) and SDS-PAGE results designed according to embodiment 5.(A) strepto- parent With plain (its Core Feature domain is 38-163 amino acids) and the structure diagram of secreting type SA-MBP (Hg).It is used more Kind secreting signal peptide (SP) is such as shown in (C).MBP regions are identical with the MBP (Hg) of embodiment 2.(B) pBE-S-lacZ plasmids are illustrated Figure, shows the insertion point of secreting type SA-MBP (Hg).(D) the SDS-PAGE results of secreting type SA-MBP (Hg).1-8 points of sample Wei not the SA-MBP (Hg) with the secreting signal peptide 1-8 of (C).

Embodiment

The present invention can combine the albumen realization of cadmium (Cd), mercury (Hg), lead (Pb) and arsenic (As) with much money known to utilizing The capture of category, and by the protein-bonded interaction of biotin-biotin, by the heavy metal ion enrichment of capture to solid phase material Material, so as to be purged to the heavy metal in sample, is effectively treated pollution.

Present invention employs the cooperation that two ways realizes Heavy Metal Binding Proteins (MBP) and biotin-binding protein： Both albumen are expressed at the same time in same microbial cell, or Heavy Metal Binding Proteins are operably coupled to biotin knot Hop protein and be used as fusion protein.Wherein, since various heavy associated proteins can be expressed in same microbial cell, so that Realize the capture to contents of many kinds of heavy metal ion in sample, the shadow without considering the fusion protein function when domain is excessive Ring, the mode captured using microbial cell has more flexibility.However, the mode of fusion protein is not related to sample (example Such as environmental sample) in launch transgenic microorganism, environmental friendliness and applicability higher.

Terms used herein " being operably connected " has implication well known in the art, refers to two domains or fragment Between feature connection.In general, being operably connected means that nucleotide sequence to be connected is adjacent, and right It is necessary, adjacent and in same reading frame for being connected of two protein encoding regions.

Ground is not intended to be limited by theory, although in the present invention, Heavy Metal Binding Proteins and biotin-binding protein are even Connection, and by biotin-conjugated to the solid phase surface for collecting, may be alternatively provided as Heavy Metal Binding Proteins is coupled with biotin, and Biotin-binding protein is conjugated to the solid phase surface for collection.For example, it can be incited somebody to action by methods known in the art and kit Heavy Metal Binding Proteins biotinylation (such as Chen etc., 2Nat.Methods 99 (2005)), and be conjugated with using commercially available The solid phase material (such as Streptavidin agarose resin) of biotin-binding protein completes the present invention.Alternatively, using biology Plain sandwich method (such as Davis etc., Proc.Natl.Acad.Sci.USA, 103：8155-60 (2006)), i.e. solid phase surface with Heavy Metal Binding Proteins are conjugated with biotin, by the biotin-binding protein (such as Streptavidin) that in addition adds by two Person is coupled.

Microbial cell in the present invention is not particularly limited, and it is thin to may be, for example, bacterial cell, actinomycetes cells, mould Born of the same parents, yeast cells and alga cells.The bacterium can be especially Escherichia coli (Escherichia coli), bacillus subtilis Bacterium (Bacillus subtilis), pseudomonas aeruginosa (Pseudomonas aeruginosa), pseudomonas putida (Pseudomonas putida), corynebacterium glutamicum (Corynebacterium glutamicum), but not limited to this.

Various heavy associated proteins (MBP) known in the art, the heavy metal binding structural domain and function such as table of the MBP Shown in 1.Above-mentioned Heavy Metal Binding Proteins can be as the Heavy Metal Binding Proteins of the present invention.It is pointed out that cited by table 1 Heavy Metal Binding Proteins it is most of be transcription factor.Lead to since the present invention is not related to conduct heavy metal binding signal to downstream Road, can be used only the heavy metal binding structural domain of above-mentioned Heavy Metal Binding Proteins, and without using DNA binding structural domains.This area The heavy metal binding structural domain of known above-mentioned Heavy Metal Binding Proteins.The Heavy Metal Binding Proteins or sheet of microbial cell of the present invention The heavy metal binding structural domain of invention fusion protein can be known in the art MBP, or the heavy metal integrated structure of these MBP Domain.In addition, it is possible to use with above-mentioned albumen with more than 80% uniformity, preferably 90%, 95%, more than 99% uniformity and tool There is the albumen of heavy metal binding function.Heavy Metal Binding Proteins as the present invention include but not limited to these cited by table 1.

Table 1：MBP and its heavy metal target known in the art

Terminology used in the present invention " biotin-binding protein " refer to Non-covalent binding to biotin or its analog, spread out The albumen of biology, also referred to as Avidin sample albumen (avidin-like protein).In general, biotin-binding protein includes life Thing element binding structural domain.Term as used herein " biotin binding structural domain " refers to the polypeptide for being bound to biotin.Although Complete biotin-binding protein can be used as biotin binding structural domain；But it can also be used only in albumen and be combined with biotin Part.A variety of biotin-binding proteins and its biotin binding structural domain known in the art are as shown in table 2.

Table 2：Biotin-binding protein known in the art

Dimerization is difficult to when being fixed on cell surface in view of the biotin-binding protein of tetravalence or bivalent form Or tetramerization, when using the biotin-binding protein of bivalent form, by the biotin-binding proteins of two bivalent forms (such as Rhizavidin it is) operably associated, dimer (that is, the expression list having the function of with reference to biotin is expressed in the form of single chain polypeptide Chain homodimer).When using the biotin-binding protein of tetravalent form, by the biotin combination egg of two or four tetravalent form (such as Streptavidin or ovum Avidin) is operably associated in vain, and being expressed in the form of single chain polypeptide has the function of with reference to biotin Single-stranded dimer or the tetramer.In one embodiment, single-stranded Streptavidin dimer SCD-E2 can be used.Also can use The biotin-binding protein of monomeric form, such as eMA, mSA (or mSA2).In addition, above-mentioned biotin combination egg can also be used only White biotin binding structural domain, or use and above-mentioned biotin-binding protein or biotin binding structural domain have 80% with Upper uniformity, preferably 90%, 95%, more than 99% uniformity and the albumen with biotin binding function.Similarly, using During functional domain (that is, the biotin binding structural domain) of the biotin-binding protein of bivalent form, by the biology of two bivalent forms The biotin binding structural domain of plain associated proteins (such as rhizavidin) is operably associated, and being expressed in the form of single chain polypeptide has With reference to the dimer of biotin function.In functional domain (that is, the biotin combination knot of the biotin-binding protein using tetravalent form Structure domain) when, by the biotin integrated structure of the biotin-binding protein (such as Streptavidin) of two or four tetravalent form Domain is operably associated, and the dimer or the tetramer having the function of with reference to biotin are expressed in the form of single chain polypeptide.

In some embodiments, Heavy Metal Binding Proteins and/or biotin-binding protein can further include cross-film and Film localization domain, so that Heavy Metal Binding Proteins and/or biotin-binding protein are illustrated in cell outer surface.Know this area N-terminal or C-terminal the addition cross-film and film localization domain in albumen, the method positioned to albumen are crossed by Xiaotong.For example, with withered For careless bacillus as in the embodiment of microbial cell, cross-film and film localization domain can be CotB (SEQ ID NO：15) [15].In the embodiment using Escherichia coli as microbial cell, cross-film and film localization domain can melt for Lpp-OmpA Hop protein (SEQ ID NO：Or INP (SEQ ID NO 16)：17)[16].Cross-film and film localization domain can be connected directly to Heavy Metal Binding Proteins and/or biotin-binding protein, or operationally connected cross-film and film localization domain by connecting peptide It is connected to Heavy Metal Binding Proteins and/or biotin-binding protein.Peptide is connected as flexibility, not comprising any active structure domain, preferably It is made of glycine and/or serine.In fact it is extremely limited to connect influence of the length of peptide to fusion protein, and can be any Length, those skilled in the art can make appropriate selection.For example, connection peptide amino acid sequence can be GGGGSGGGGS, GVD, GVDG, GI or TR.

In some embodiments, Heavy Metal Binding Proteins and/or biotin-binding protein can further include convenient egg The labelled peptide purified in vain.This area is known on the premise of protein function is not influenced, and mark is added by the N-terminal or C-terminal in albumen Peptide is signed, protein purification can be facilitated.The labelled peptide be such as, but not limited to hexahistine label, c-Myc labels, Halo labels, Flag labels etc..Above-mentioned label is known in the art, such as described in WO2012/155007.

Terminology used in the present invention " heavy metal binding structural domain " refers to the polypeptide that there is heavy metal to combine activity, such as For the MBP listed by table 1 or its heavy metal binding structural domain.In addition, it is possible to use with above-mentioned albumen or polypeptide have 80% with Upper uniformity, preferably 90%, 95%, more than 99% uniformity and albumen or polypeptide with heavy metal binding function.It is used as this The heavy metal binding structural domain of invention includes but not limited to these cited by table 1.

Terminology used in the present invention " biotin binding structural domain " refers to the polypeptide with biotin binding activity, such as The total length of biotin-binding protein or its part with biotin binding function.The present invention biotin binding structural domain can be Such as, but not limited to following biotin-binding protein or its biotin binding structural domain：EMA, mSA, mSA2, rhizavidin, chain Mould Avidin and ovum Avidin, is preferably mSA2.Since fusion protein can move freely in detection architecture, without by single-stranded Form causes the advance multimerization of Avidin sample albumen.It is also possible, however, to use the Streptavidin dimer of single stranded form, single-stranded shape The ovum Avidin dimer of formula, the rhizavidin dimers of single stranded form, the Streptavidin tetramer, single-stranded of single stranded form The ovum Avidin tetramer of form.

For heavy metal binding structural domain is operably coupled to biotin binding structural domain, as long as not destroying two Heavy metal binding structural domain, can be connected directly to the N-terminal or C-terminal of biotin binding structural domain by the respective function of domain, or Heavy metal binding structural domain is connected to the N-terminal or C-terminal of biotin binding structural domain using connection peptide.When there are multiple heavy metals During binding structural domain, on the premise of function is not influenced, the company of each heavy metal binding structural domain and biotin binding structural domain The mode of connecing is not particularly limited.Peptide is connected as flexibility, not comprising any active structure domain, preferably by glycine and/or serine Composition.The length of connection peptide hardly has an impact the function of fusion protein, therefore can be random length.For example, connection peptide Amino acid sequence can be GGGGSGGGGS, GVD, GVDG, GI or TR.

In some embodiments, fusion protein can further include secreting signal peptide, so that fusion protein be secreted out of Outside microbial cell, avoid being likely to form inclusion body and influencing to fold.This area known on the premise of protein function is not influenced, Secreting signal peptide is added by the N-terminal in albumen, protein secretion is gone out to the method for cell.For example, make with bacillus subtilis For in the embodiment of expressive host, secreting signal peptide can be such as, but not limited to from AmyA, AmyE, NprE, AprE and The signal peptide of SacB.

In some embodiments, fusion protein can further include the labelled peptide for facilitating fusion protein purification.This area Know on the premise of protein function is not influenced, labelled peptide is added by the N-terminal or C-terminal in albumen, protein purification can be facilitated.Institute State labelled peptide and be such as, but not limited to hexahistine label, c-Myc labels, Halo labels, Flag labels etc..Above-mentioned label is this Known to field, such as described in WO2012/155007.

Biotinylated solid phase material as described herein can be any solid phase material for being covalently attached biotin.This area institute Know, function dough, and the biotin using function dough and the table with amino or carboxyl can be carried out to biotin Face carries out esterification.Such as esterification occurs instead using n-hydroxysuccinimide biotin and the surface with amino or carboxyl Should, biotinylation modification [17] is carried out to solid phase surface.It can also be used with polyethyleneglycol modified biotin to solid phase surface Modified so that the surface hydrophilicity after modification is stronger.The solid phase material is such as, but not limited to glass, agarose, polyphenyl Ethene and other high molecular materials.In this regard, being carried with free amino or carboxyl or its surface can be by ammonia Any solid phase material of base or the free group of carboxyl modified (such as amino) can serve as the solid phase material of the present invention.Or Person, since protein fragments generally comprise free amino and/or carboxyl, the solid phase material can be protein modified material.

In some embodiments, directly can separate biotinylated solid phase material with processed sample (such as it is straight Pick up out), to recycle captured heavy metal ion.In this regard, biotinylated solid phase material can be sheet, bulk Or filamentous form.It is more than 1mm in solid phase material in the embodiment of sheet, it can have²Surface area.It is in solid phase material In block embodiment, it, which can have, is more than 1mm³Volume.In solid phase material in Filamentous embodiment, it can have Length more than 1cm.

In some embodiments, can be by way of centrifugation by biotinylated solid phase material and processed sample point From to recycle captured heavy metal ion.In this regard, biotinylated solid phase material can be the form of particle, example Such as, biotinylated solid phase material can be 5 μm of particles to about 1mm of size.

In other embodiments, biotinylated solid phase material and processed sample can be divided by Magneto separate From to recycle captured heavy metal ion.In this embodiment, biotinylated solid phase material can be biotinylated glass Glass or high molecular material (such as agarose or polystyrene) magnetic bead.Glass, agarose or polystyrene magnetic beads are commercially available , such as castor nanometer (glass magnetic bead, article No. 70201-5；Agarose magnetic bead, article No. 70203-5), ThermoFisher is (poly- Styrene magnetic bead, article No. 14307D).Preferably, the magnetic bead has 1 μm to 100 μm, preferably 10 μm to 80 μm of size.

Ground is not intended to be limited by theory, due to the parent needed for the hydrophobicity and biotin and magnetic bead JA(junction ambient) of biotin It is water-based, when carrying out biotinylation to magnetic bead, add biotin and cause the hydrophobicity of local environment to make it possible to magnetic bead Aggregation, so as to reduce biotinylated efficiency.In a preferred embodiment, biotin and magnetic during biotinylation are carried out to magnetic bead The mass ratio of pearl is (0.5-1):1 (m/m), preferably 3:4 (m/m), the ionic strength of biotinylation system buffer solution is 0.5-3M, It is preferred that 1M, surfactant is preferably Tween-20 or Triton X-100.

(b) biotinylated solid phase material is provided；

(d) the biotinylated solid phase material of step (b) is recycled；

In the present invention, pending sample can be any source, especially from the sample of environment.It is described to come from environment Sample for example obtained from or from following environment sample：Water (including waste water), pond, river, reservoir, swimming pool, soil, Food processing and/or packing factory, agriculture ground, aquatic farm (including water planting food farm), medicine manufacturing works, fauna Facility, or any combination of above-mentioned ambient source.Sample can be solid, liquid or solid-liquid mixed form.

The method of the present invention can utilize the sample of taking-up to implement, and sampling ground, realization pair are put back to after heavy metal pollution processing The showering of environment.Alternatively, in the directly embodiment of recycling or Magneto separate, can be by step (a) and the material of (b) offer Mixed with sample in-situ, original position recycling, so as to carry out in-situ immobilization to environment.In two ways, to the heavy metal being recovered Disposal/recycling it is all independent with the recycling step of the present invention, therefore the method for the present invention can easily with it is follow-up a variety of Heavy metal method of disposal is docked.

Microbial cell, fusion protein and the biotinylated solid phase material and recovery method of the present invention such as this paper is other Described in part.

The embodiment of each side described herein can be by the paragraph explanation numbered as follows：

1. a kind of microbial cell for being used to handle heavy metal pollution, wherein, the microbial cell expresses heavy metal knot Hop protein and biotin-binding protein；Wherein, the heavy metal is the one or more in following heavy metal：Cadmium, Mercury, lead and arsenic.

2. the microbial cell as described in paragraph 1, wherein, the microorganism is selected from what is be made of following Institute of Micro-biology Group：Bacterium, actinomyces, mould, yeast and algae.

3. the microbial cell as described in paragraph 2, wherein, the bacterium is selected from Escherichia coli, bacillus subtilis, copper Green pseudomonad, pseudomonas putida and corynebacterium glutamicum.

4. such as the microbial cell any one of paragraph 1-3, wherein, the Heavy Metal Binding Proteins are selected from by such as One or more in the group that lower albumen is formed：Cadmium-binding protein, mercury-binding proteins, lead associated proteins and arsenic combination egg In vain.

5. the microbial cell as described in paragraph 4, wherein, the Heavy Metal Binding Proteins in following albumen one Kind is a variety of：MerR, Crs5, CadR, CmtR, PbrR, PbrR691 and ArsR.

6. the microbial cell as described in paragraph 4, wherein, the Heavy Metal Binding Proteins are selected from following one or more The heavy metal binding structural domain of albumen：MerR, Crs5, CadR, CmtR, PbrR, PbrR691 and ArsR.

7. such as the microbial cell any one of paragraph 1-6, wherein, the biotin-binding protein is selected from as follows Albumen：EMA, mSA, mSA2, the Streptavidin dimer of single stranded form, the ovum Avidin dimer of single stranded form, single-stranded shape The rhizavidin dimers of formula, the Streptavidin tetramer of single stranded form, the ovum Avidin tetramer of single stranded form；It is preferred that Ground, the biotin-binding protein are mSA2.

8. such as the microbial cell any one of paragraph 1-6, wherein, the biotin-binding protein is selected from：It is single The Streptavidin biotin binding structural domain dimer of chain form, the ovum Avidin biotin binding structural domain two of single stranded form Aggressiveness, the rhizavidin biotin binding structural domains dimer of single stranded form, the Streptavidin biotin knot of single stranded form Close the domain tetramer, the ovum Avidin biotin binding structural domain tetramer of single stranded form；Preferably, the biotin combines Albumen is the biotin binding structural domain of mSA2.

9. such as the microbial cell any one of paragraph 1-8, wherein, Heavy Metal Binding Proteins and/or described Biotin-binding protein further includes cross-film and film localization domain；Preferably, the heavy metal is combined by connecting peptide Albumen and/or the biotin-binding protein are connected with cross-film and film localization domain.

10. such as the microbial cell any one of paragraph 1-9, wherein, Heavy Metal Binding Proteins and/or described Biotin-binding protein further includes labelled peptide, and the labelled peptide is located at the Heavy Metal Binding Proteins and/or the biology The protein-bonded N-terminal of element or C-terminal；Preferably, the labelled peptide is one kind in the group being made of following labelled peptide It is or a variety of：Hexahistine label, c-Myc labels, Halo labels, Flag labels.

11. a kind of fusion protein for being used to handle heavy metal pollution, wherein, the fusion protein includes heavy metal and combines knot Structure domain and biotin binding structural domain；Wherein, the heavy metal is the one or more in following heavy metal：Cadmium, Mercury, lead and arsenic.

12. the fusion protein as described in paragraph 11, wherein, the heavy metal binding structural domain is selected from by following heavy metal One or more in the group that associated proteins are formed：Cadmium-binding protein, mercury-binding proteins, lead associated proteins and arsenic combination egg In vain.

13. the fusion protein as described in paragraph 12, wherein, the heavy metal binding structural domain is in following albumen It is one or more：MerR, Crs5, CadR, CmtR, PbrR, PbrR691 and ArsR.

14. the fusion protein as described in paragraph 12, wherein, the heavy metal binding structural domain is selected from following a kind of or more The heavy metal binding structural domain of kind albumen：MerR, Crs5, CadR, CmtR, PbrR, PbrR691 and ArsR.

15. such as the fusion protein any one of paragraph 11-14, wherein, the biotin binding structural domain is selected from Following albumen or its biotin binding structural domain：EMA, mSA, mSA2, Streptavidin, ovum Avidin and rhizavidin； Preferably, the biotin-binding protein is Streptavidin or the biotin binding structural domain of Streptavidin.

16. such as the fusion protein any one of paragraph 11-14, wherein, the biotin binding structural domain is selected from： The Streptavidin dimer of single stranded form, the ovum Avidin dimer of single stranded form, the rhizavidin dimerization of single stranded form Body, the Streptavidin tetramer of single stranded form, the ovum Avidin tetramer of single stranded form；The Streptavidin life of single stranded form Thing element binding structural domain dimer, the ovum Avidin biotin binding structural domain dimer of single stranded form, single stranded form Rhizavidin biotin binding structural domains dimer, single stranded form the Streptavidin biotin binding structural domain tetramer, The ovum Avidin biotin binding structural domain tetramer structure domain of single stranded form.

17. such as the fusion protein any one of paragraph 11-16, the fusion protein is further included the huge sum of money Belong to the connection peptide that binding structural domain is connected to the biotin binding structural domain.

18. such as the fusion protein any one of paragraph 11-17, the fusion protein further includes secretion signal Peptide；Preferably, the secreting signal peptide is located at the N-terminal of the fusion protein.

19. such as the fusion protein any one of paragraph 11-18, the fusion protein further includes labelled peptide, institute State N-terminal or C-terminal that labelled peptide is located at the fusion protein；Preferably, the labelled peptide is selected from by following labelled peptide institute group Into group in one or more：Hexahistine label, c-Myc labels, Halo labels, Flag labels.

20. a kind of kit for being used to handle heavy metal pollution, the kit are included such as any one of paragraph 1-10 institutes The microbial cell or the fusion protein as any one of paragraph 11-19 stated, and biotinylated solid phase material；Its In, the heavy metal is the one or more in following heavy metal：Cadmium, mercury, lead and arsenic.

21. the kit as described in paragraph 20, wherein, the solid phase material carries amino or carboxyl or band for surface Having can be by amino or the group of carboxyl modified (such as hydroxyl), so as to be covalently attached with biotin to form biotinylation Solid phase material material；Preferably, the solid phase material has albumen for glass, agarose, polystyrene or surface modification Material.

22. the kit as described in paragraph 21, wherein, the biotinylated solid phase material is flaky material, it is preferable that The flaky material, which has, is more than 1mm²Surface area.

23. the kit as described in paragraph 21, wherein, the biotinylated solid phase material is bulk material, it is preferable that The bulk material, which has, is more than 1mm³Volume.

24. the kit as described in paragraph 21, wherein, the biotinylated solid phase material is filamentary material, it is preferable that The filamentary material has the length more than 1cm.

25. the kit as described in paragraph 21, wherein, the biotinylated solid phase material is particle form, it is preferable that The particle has 5 μm of sizes to 1mm.

26. the kit as described in paragraph 21, wherein, the biotinylated solid phase material is magnetic bead form, it is preferable that The magnetic bead is glass magnetic bead, agarose magnetic bead or polystyrene magnetic beads, more preferably agarose magnetic bead；Preferably, the magnetic Pearl has 1 μm to 100 μm, preferably 10 μm to 80 μm of size.

27. the kit as described in paragraph 26, wherein, when carrying out biotinylation to the magnetic bead, biotin and the magnetic The mass ratio of pearl is (0.5-1):1 (m/m), preferably 3:4(m/m)；Preferably, the ionic strength of biotinylation system buffer solution is 0.5-3M, preferably 1M；Preferably, biotinylation system includes surfactant, and the surfactant is preferably Tween-20 Or Triton X-100.

28. a kind of method for handling heavy metal pollution in sample, the described method includes：

(a) microbial cell as any one of paragraph 1-10 is provided or as any one of paragraph 11-19 Fusion protein；

(b) biotinylated solid phase material is provided；

(c) biotin that the microbial cell or the fusion protein and step (b) provided step (a) provides The solid phase material of change is mixed with sample, so that the heavy metal ion in sample is captured, and

(d) the biotinylated solid phase material of step (b) is recycled；

29. the method as described in paragraph 28, wherein, the solid phase material in step (b) carries amino or carboxylic for surface Base or with can be by amino or the group of carboxyl modified (such as hydroxyl), so as to be covalently attached with biotin with shape Into the material of biotinylated solid phase material；Preferably, the solid phase material is repaiied for glass, agarose, polystyrene or surface It is decorated with the material of albumen.

30. the method as described in paragraph 29, wherein, it is recovered as directly recycling described in step (d).

31. the method as described in paragraph 30, wherein, the biotinylated solid phase material in step (b) is sheet material Material, it is preferable that the flaky material, which has, is more than 1mm²Surface area.

32. the method as described in paragraph 30, wherein, the biotinylated solid phase material in step (b) is block-shaped material Material, it is preferable that the bulk material, which has, is more than 1mm³Volume.

33. the method as described in paragraph 30, wherein, the biotinylated solid phase material in step (b) is Filamentous material Material, it is preferable that the filamentary material has the length more than 1cm.

34. the method as described in paragraph 29, wherein, it is recovered as being recovered by centrifugation described in step (d), and step (b) the biotinylated solid phase material in is particle form, it is preferable that the particle has 5 μm of sizes to 1mm.

35. the method as described in paragraph 29, wherein, Magneto separate is recovered as described in step (d), and in step (b) The biotinylated solid phase material is magnetic bead form, it is preferable that the magnetic bead is glass magnetic bead, agarose magnetic bead or polyphenyl second Alkene magnetic bead, more preferably agarose magnetic bead；Preferably, the magnetic bead has 1 μm to 100 μm, preferably 10 μm to 80 μm of size.

36. the method as described in paragraph 35, wherein, when carrying out biotinylation to the magnetic bead, biotin and the magnetic bead Mass ratio be (0.5-1):1 (m/m), preferably 3:4(m/m)；Preferably, the ionic strength of biotinylation system buffer solution is 0.5-3M, preferably 1M；Preferably, biotinylation system includes surfactant, and the surfactant is preferably Tween-20 Or Triton X-100.

37. such as the method any one of paragraph 28-36, wherein, the sample is the sample from environment, particularly Obtained from or from following environment sample：Water (including waste water), pond, river, reservoir, swimming pool, soil, food processing And/or packing factory, agriculture ground, aquatic farm (including water planting food farm), medicine manufacturing works, fauna facility, or Any combination of above-mentioned ambient source；The sample is solid, liquid or solid-liquid mixed form.

Embodiment

Embodiment 1 prepares biotinylated magnetic bead

1. reagent

The magnetic bead that the present embodiment uses is respectively glass magnetic bead (2 μm of diameter, castor nanometer, article No. 70201-5) and fine jade Lipolysaccharide magnetic bead (30 μm to 150 μm of diameter, castor nanometer, article No. 70203-5).

Biotin liquid storage：Biotin is dissolved in dimethyl sulfoxide (DMSO) (DMSO), final concentration of 50mg/ml.

MES (2- (N- morpholinoes) ethyl sulfonic acid) buffer solution：0.1M MES are dissolved in water, and pH to 5.0 is adjusted using NaOH.

1 × PBS buffer：

NaCl：7.9g (Beijing Chemical Plant, purity >=99.5%, pH：5.0~8.0)；KCl：0.2g (Beijing Chemical Plant, it is pure Spend >=99.5%, pH：5.0~8.0)；

KH₂PO₄：0.24g (Beijing Chemical Plant, purity >=99.5%, pH：4.2~4.5)；

K₂HPO₄：1.8g (Beijing Chemical Plant, purity >=99.0%, pH：8.9~9.4),

It is dissolved in 800ml distilled water, finally plus distilled water is settled to 1L, and (pure, Chinese medicines group chemical reagent is analyzed with HCl Co., Ltd) pH value of solution is adjusted to 7.4.

High salt PBS-T solution：1M NaCl are added in 1 × PBS buffer, and add 0.1%Tween-20 or TritonX- 100。

High salt TBS-T solution：In the Tris-HCl buffer solutions of 10mM add 1M NaCl, and add 0.1%Tween-20 or Triton X-100。

Unless otherwise indicated, reagent used in the present embodiment is purchased from SIGMA.

2. method

(a) 0.15ml DMSO, 0.15ml biotin liquid storages are added into the 2.7ml MES buffer solutions of precooling, acutely shake Swing to mixed liquor and clarify, obtain containing 10%DMSO, the MES solution that biotin concentration is 2.5mg/ml (~10mM).

(b) 30mg 1- (3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides (EDC) are added to 3ml steps (a) in the solution obtained, the solution that EDC concentration is 1wt% is obtained.

(c) magnetic bead for taking 1ml concentration to be 10mg/ml, is removed supernatant after being adsorbed using magnetic frame, uses the MES of precooling Solution cleans 3 times.

(d) magnetic bead is resuspended with the solution of 3ml steps (b), (violent concussion in every 20 minutes subtracts when concussion incubation 2 is small on ice Few magnetic bead aggregation).

(e) using magnetic frame absorption magnetic bead after the completion of being incubated, supernatant is removed, is cleaned 3 times using high salt PBS-T solution, And magnetic bead is resuspended in the solution, final volume 1ml, 4 DEG C of preservations.

The final concentration of 10mg/ml of the magnetic bead obtained.

Embodiment 2 prepares the microbial cell that can remove mercury ion pollution

1. reagent and culture medium

Luria-Bertani (LB) fluid nutrient medium：

Peptone (Fisher Scientific) 10g/L；

NaCl(Fisher Scientific) 10g/L；

Dusty yeast (Fisher Scientific) 5g/L.

121 DEG C of autoclaving 20min.

LB solid mediums：

It is identical with the formula of LB fluid nutrient mediums, add 1.5% agar powder.

Chloramphenicol (Acros)：It is dissolved in LB culture mediums, 20 μ g/ml of final concentration.

Ampicillin (Acros)：It is dissolved in LB culture mediums, 25 μ g/ml of final concentration.

Isopropyl-β-D-1- thiogalactosides (IPTG) (USB Corporation) stock concentrations 1M, it is final concentration of 100μM。

1M Tris-HCl buffer solutions：

Tris base：121.14g(American Bioanalytical#AB14042)；It is dissolved in 800ml distilled water, Finally plus distilled water is settled to 1L, with HCl (analyze pure, Sinopharm Chemical Reagent Co., Ltd.) adjust the pH value of solution to 8.0, the liquid storage of 100 times of concentration is made.Liquid storage is diluted 100 times during use.

LB liquid and solid medium are used in plasmid construction, strain culturing and maintenance, induction.

Restriction enzyme, T4 polynucleotide kinases and T4DNA ligases are purchased from New England Biolabs (Frankfurt, Germany).Primer and Single-stranded DNA fragments (oligonucleotides) are purchased from BGI (Beijing genome institute).UseSpectrophotometer ND-2000 (Peqlab, Erlangen, Germany) detects DNA concentration.

2. structure is used for plasmid and bacterial strain in cell surface expression biotin-binding protein

Whole plasmid constructions are used as clone strain by the use of e.colistraindh5α (TransGen Biotech).Using big Enterobacteria DH5 α bacterial strains (TransGen Biotech) and BL21 (DE3) bacterial strain (TransGen Biotech) are used as experimental bacteria Strain.

Build RGP carriers (SEQ ID NO：18) DNA for sequencing is assembled.PTac startups are integrated with RGP carriers The LacI and AmpR that son, p15A replicons and composing type are overexpressed；In addition, two Bsal sites are located at the side of lacZ α fragments The wing, so as to remove recognition site from plasmid after digestion.By Golden Gate cloning process, LacZ α fragments are replaced with Biotin-binding protein interested, so as to build RGP series plasmids.

The plasmid of structure 4 kinds of different biotin-binding proteins (Fig. 2A) of expression, specifically, carries by clone of RGP carriers Body, uses 4 kinds of fusion proteins in Golden Gate cloning process difference assembling figure 2A.Wherein, Streptavidin (tetravalence parent With plain sample albumen) sequence be SEQ ID NO：19.The sequence of eMA (monovalent Avidin sample albumen) is SEQ ID NO：20. The sequence of mSA2 (monovalent Avidin sample albumen) is SEQ ID NO：21.SCD-E2 is the Streptavidin dimerization of single stranded form Body, sequence are SEQ ID NO：22.Respectively these biotin-binding proteins N-terminal add inner membrance localization domain Lpp and across Film peptide OmpA (Lpp-OmpA).The sequence of Lpp-OmpA is SEQ ID NO：23, so as to be located in cell surface.-T-R- For-Thr-Arg- catenation sequences.

Plasmid is converted to expression strain Escherichia coli BL21 (DE3), screening positive clone.

3. the interaction of the microbial cell and biotin of surface expression biotin-binding protein

The seed liquor being incubated overnight is diluted 100 times using LB, (37 DEG C, 225rpm) is to OD when culture 3 is small₆₀₀~0.6, Add 0.1mM IPTG inducible proteins expression, using the same terms be further cultured for 4 it is small when.1.5ml cultures are centrifuged (4000rpm, 2min) and cell is collected, abandon supernatant.Cell is resuspended using the PBS buffer of 1ml, takes 200 μ l to add to 96 orifice plates Enter the Biotin-atto488 (0.5mM, Sigma, article No. 30574-1MG-F) of 4 μ l, be incubated 1h (37 DEG C, 1000rpm)；Then Use PBS cleaning cell 3 times.

Pass through the plain interaction with constructed microbial cell of three kinds of method test organisms：

Flow cytometer (BD^TMLSR II)/microplate reader (BioTek, SynergyMx Multi-Mode Microplate Reader)：Cell is diluted 100 times, detection cell carries green glimmering under 488nm excitation wavelengths, 525nm Detection wavelengths Light.Fluorescence microscope (Zeiss AXIO IMAGER A2)：Cell is fixed on the agarose coated on glass slide, uses fluorescence The observation of microscope FITC passages is taken pictures, excitation wavelength 490nm, Detection wavelength 520nm.

Microplate reader and flow cytomery show that the wild type Streptavidin of cell surface expression is not special with biotin The opposite sex combines, this is because Streptavidin could be combined after needing tetramerization with biotin, and merges egg with Lpp-OmpA The Streptavidin that white mode expresses in cell surface is difficult to freely form the tetramer.Two kinds of monomer Avidins eMA, mSA2 And the combination of the dimer Avidin SCD-E2 and biotin of single chain polypeptide are stronger, the combination effect of mSA2 is preferably (Fig. 2 B-2C). The fluorescence micrograph of Fig. 2 D shows, the cell of surface expression mSA2 and biotin-Atto488 specific bonds are simultaneously presented stronger Fluorescence.

4. the interaction of the microbial cell of surface expression biotin-binding protein and biotinylation magnetic bead

The seed liquor being incubated overnight is diluted 100 times using LB, when culture 3 is small (37 DEG C, 225rpm) to OD600~0.6, Add 0.1mM IPTG inducible proteins expression, using the same terms be further cultured for 4 it is small when.1.5ml cultures are centrifuged (4000rpm, 2min) and cell is collected, abandon supernatant.Cell is resuspended using the PBS buffer of 1ml, takes 200 μ l to add to 96 orifice plates Enter the biotinylated magnetic bead (concentration of 20 μ l embodiments 1 preparation：10mg/ml), it is incubated 1h (37 DEG C, 1000rpm)；Use magnetic force Frame adsorbs magnetic bead, removes supernatant, and cleans magnetic bead 3 times using buffer solution, prepares scanning electron microscope (SEM) sample [18] and observation is taken pictures.

SEM image such as Fig. 3 shows, the cell of surface expression mSA2 can effectively be adsorbed in magnetic bead surfaces, and surface table Up to monomer streptavidin cell due to being unable to tetramerization, it is difficult to be effectively adsorbed in magnetic bead surfaces.

5. structure is used for plasmid and bacterial strain in cell surface expression Heavy Metal Binding Proteins

Whole plasmid constructions are used as clone strain by the use of e.colistraindh5α (TransGen Biotech).Using big Enterobacteria DH5 α bacterial strains (TransGen Biotech) and BL21 (DE3) bacterial strain (TransGen Biotech) are as experiment Bacterial strain.

Build pSB1C3-pT7 (SEQ ID NO：24) carrier is used for the DNA assemblings of sequencing.PSB1C3 plasmids are Biobrick standard plasmids, are integrated with pMB1 replicons and CmR resistant genes.PSB1C3-pT7 is assembled on the basis of pSB1C3 T7 promoters, also, it is similar with RGP carriers, LacZ α fragment of its flank with two BsaI sites is used to use Golden Gate cloning process assembles pSB1C3-pT7 series plasmids.

Design can adsorb mercury ion and in Lpp-OmpA-MBP (Hg) fusion protein of cell surface positioning.According to wild The structure of type MerR, design Heavy Metal Binding Proteins MBP (Hg).The sequence and structure of wild type MerR such as SEQ ID NO：1 He Shown in Fig. 4 A.The allosteric of the transcription factor is caused due to not being related to Hg in the present invention, without using DNA binding structural domains.This Heavy metal bound fraction used in embodiment as shown in Figure 4 B, intercept MerR albumen mercury ion binding structural domain and with its phase Two block coupled in series are obtained MBP (Hg) (SEQ ID NO by catenation sequence even as mercury ion binding modules：25).Separately Outside, cell outer surface can be positioned at by the albumen in the N-terminal addition Lpp-OmpA of MBP (Hg).- G-I- is-Gly- Ile- catenation sequences ,-G-V-D- are-Gly-Val-Asp- catenation sequences (Fig. 4 C).

Lpp-OmpA-MBP (Hg) fusion protein is inserted into pSB1C3-pT7 carriers using Golden Gate cloning process In.Plasmid is converted to expression strain Escherichia coli BL21 (DE3), screening positive clone.

6. absorption of the microbial cell of surface expression MBP (Hg) to mercury ion

The seed liquor being incubated overnight is diluted 100 times using LB, (37 DEG C, 225rpm) is to OD when culture 3 is small₆₀₀~0.6, Add 0.1mM IPTG inducible proteins expression, using the same terms be further cultured for 4 it is small when.1.5ml cultures are centrifuged (4000rpm, 2min) and cell is collected, abandon supernatant.Cell is resuspended using the high salt TBS-T buffer solutions of 500 μ l, distinguishes thereto Nitric acid mercury standard solution is added, makes final concentration of 10 μM and 100 μM of mercury ion, 37 DEG C of oscillation incubation 1h (1000rpm)；Centrifugation (4000rpm, 2min) removes cell, and mercury concentration is detected after supernatant is diluted 100 times.

Concentration of heavy metal ion measures work and is completed by Beijing Xin Aohuanbiao companies, uses AFS DETERMINATION mercury Ion concentration, the concentration of cadmium and lead ion is measured using ICP-MS methods.

As shown in Figure 4 D, when initial ion concentration of mercury is 10 μM or 100 μM, the cell of surface expression MBP (Hg) With the mercury ion in adsorbent solution, the remaining ion concentration of mercury in solution is greatly reduced.

7. structure expresses the cell of MBP (Hg) and biotin-binding protein at the same time

Respectively by the above-mentioned pSB1C3-pT7 plasmids with Lpp-OmpA-MBP (Hg) and with Lpp-OmpA-mSA2's For RGP plasmids cotransformation to e. coli bl21 (DE3) bacterial strain, obtain has heavy metal adsorption and and biotinylation at the same time The microbial cell of magnetic bead binding ability.

Embodiment 3 prepares the fusion protein S A-MBP (Hg) that can remove mercury ion pollution

1. reagent and culture medium

Culture medium used in embodiment 3 and buffer solution are as described in 2 Part I of embodiment.

Protein extraction and renaturation are following [19] with reagent.

The structure of 2.SA-MBP (Hg) fusion protein

Whole plasmid constructions are used as clone strain by the use of e.colistraindh5α (TransGen Biotech).Using big Enterobacteria BL21 (DE3) bacterial strain (TransGen Biotech) is used as experimental strain.

As shown in Figure 5A, by Streptavidin (SEQ ID NO：20) C-terminal and MBP (the Hg) (figures described in embodiment 2 4B, SEQ ID NO：25) N-terminal connects structure SA-MBP (Hg) fusion protein, its molecular weight is about 26kD.Connecting peptide is Flexibility, not comprising any active structure domain, its sequence is GVDG.By SA-MBP (Hg) be inserted into pET28a (+) carrier (Novagen, Article No. 69864-3) multiple cloning sites NcoI restriction enzyme sites.

By the seed liquor being incubated overnight using LB culture mediums dilute 100 times to cumulative volume be 1L, cultivate 3 it is small when (37 DEG C, 225rpm) to OD600~0.6, add the IPTG inducible proteins expression of 0.1mM, using the same terms be further cultured for 6 it is small when.Centrifugation, Supernatant is abandoned, collects bacterium.Suspended with 1 × PBS of 30ml or so, and ultrasonic degradation (ultrasonic 6s, interval 12s, 99 times, 300W or so) extremely Liquid is clarified；10000rpm centrifuges 10min, abandons supernatant, and precipitation is resuspended with lavation buffer solution, continues 10000rpm centrifugation 10min, Abandon supernatant；It is repeated once, is resuspended with buffer solution is resuspended, 10000rpm centrifugations 10min.Sediment weight is weighed, according to precipitation quality Add and redissolve buffer solution (guanidine hydrochloride or urea) dissolving, it is 30mg/ml to make precipitating concentration.Kept for 4 DEG C to be stirred overnight.12000rpm 10min is centrifuged, supernatant is added into bag filter, is dialyzed overnight for 4 DEG C in refolding buffers.12000rpm centrifugations 10min is removed Precipitation in bag filter, collects supernatant, uses aperture that pipe (Millipore, article No. is concentrated by ultrafiltration for the albumen of 10kD UFC901096 it is) protein storage buffer solution by solvent replacement.Using BCA methods measure protein concentration, add 50% glycerine, -80 DEG C Preserve.As shown in Figure 5 B, sample 4 is the SA-MBP (Hg) after renaturation, and stripe size is correct, and foreign protein amount is seldom, no longer needs His-tag is used to carry out ni-sepharose purification.Compared with sample 2, the albumen in sample 4 is only accounted for less than 20% (range estimation), illustrates SA- The major part of MBP (Hg) albumen cannot correct renaturation, appear in precipitation in expection meet.In albumen and sample 3 in sample 2 Protein classes it is similar, illustrate to be redissolved in the albumen of buffer solution most of albumen in refolding cannot correct renaturation, Sediment fraction is stayed in, refolding buffers have the ability of specificity renaturation SA and SA-MBP (Hg)；

Calculate that, using this method, 1L bacterium solutions about can be with output renaturation according to the protein concentration (4.4mg/ml) after concentration The about 3mg of SA-MBP afterwards.

The combination of 3.SA-MBP (Hg) fusion proteins and magnetic bead

High salt PBS-Ts of the SA-MBP (Hg) of purifying containing 1M NaCl is diluted 20 times to 0.1mg/ml.By 400 μ l The 40 μ l agaroses magnetic beads (average diameter is 65 μm) that albumen is prepared with embodiment 1 mix, and 1 is incubated in 37 DEG C, 1000rpm concussions Hour.Magnetic bead is adsorbed using magnetic frame, takes out supernatant, subsequent high salt PBS-T cleanings magnetic bead three times, adds 10 μ l loading buffers Liquid, boils 5min, takes supernatant.SDS-PAGE, coomassie brilliant blue staining are carried out to supernatant.With the control without biotin Agarose magnetic bead implements identical step.

As shown in fig. 6, after biotinylated magnetic bead mixes incubation with SA-MBP (Hg), it is no longer able to detect in supernatant Albumen, this shows that SA-MBP (Hg) can be combined with biotinylated magnetic bead, its Avidin sample part has bioactivity.And magnetic Pearl boil after supernatant in can detect albumen, and mix incubation with SA-MBP (Hg) without the control magnetic bead of biotin Afterwards, the albumen similar with addition can be still detected in supernatant, shows that SA-MBP (Hg) cannot be by not biotinylated Magnetic bead combines.In addition, SA-MBP (Hg) albumen cannot be combined explanation with control magnetic bead, it is existed in the form of inclusion body, and It is to need biotin-binding protein-biotin specific reaction to combine.

Absorption of 4.SA-MBP (Hg) fusion proteins to mercury ion

The SA-MBP (Hg) of purifying is added into high salt TBS-T buffer solutions (final concentration of 0.1mg/ml), adds nitre The mixing of sour mercury standard solution, cumulative volume are 500 μ l, final concentration of 1 μM and 10 μM of mercury ion, 37 DEG C of oscillation incubation 1h.It is diluted to 10ml, is concentrated by ultrafiltration pipe (Millipore, article No. UFC901096) using the albumen that aperture is 10kD and filters SA-MBP (Hg) egg In vain, take filter liquor to be diluted to 50ml, ion concentration of mercury is detected using method same as Example 2.As shown in fig. 7, initial When ion concentration of mercury is 1 μM or 10 μM, SA-MBP (Hg) albumen can make the residue in solution with the mercury ion in adsorbent solution Ion concentration of mercury is greatly reduced.

Embodiment 4 prepares the fusion protein S A-MBP (Cd) that can remove cadmium ion pollution

Reagent and culture medium are same as Example 3 used in embodiment 4.

The structure of 1.SA-MBP (Cd) fusion protein

According to structure (the SEQ ID NO of wild type CadR：3, Fig. 8 A), MBP (Cd) egg of Adsorption of Cadmium is capable of in design (SEQ ID NO in vain：26, Fig. 8 B).MBP (Cd) albumen interception CadR albumen cadmium ion binding structural domain and it is coupled with Machine sequence is as cadmium ion binding modules.

As shown in Figure 8 C, by Streptavidin (SEQ ID NO：20) C-terminal and the N of biotin-binding protein MBP (Cd) End connects structure SA-MBP (Cd) fusion protein, its molecular weight is about 22kD.Peptide is connected as flexibility, not comprising any activity Domain, sequence GVDG.SA-MBP (Cd) is inserted into pET28a (+) carrier (Novagen, article No. 69864-3) polyclonal position The NcoI restriction enzyme sites of point.

The expression, extraction and refolding method of SA-MBP (Cd) fusion protein are identical with embodiment 3 (2).

The combination of 2.SA-MBP (Cd) fusion proteins and magnetic bead

SA-MBP (Cd) fusion proteins are identical with embodiment 3 (3) with the binding test method of magnetic bead.

As in fig. 8d, after biotinylated magnetic bead mixes incubation with SA-MBP (Cd), it is no longer able to detect in supernatant To albumen, this shows that SA-MBP (Cd) can be combined with biotinylated magnetic bead, its Avidin sample part has bioactivity.And Magnetic bead boil after supernatant in can detect albumen, and mix incubation with SA-MBP (Cd) without the control magnetic bead of biotin Afterwards, the albumen similar with addition can be still detected in supernatant, shows that SA-MBP (Cd) cannot be by not biotinylated Magnetic bead combines.In addition, SA-MBP (Cd) albumen cannot be combined explanation with control magnetic bead, it is existed in the form of inclusion body, and It is to need biotin-binding protein-biotin specific reaction to combine.

Absorption of 3.SA-MBP (Cd) fusion proteins to cadmium ion

The SA-MBP (Cd) of purifying is added into high salt TBS-T buffer solutions (final concentration of 0.1mg/ml), adds nitre The mixing of sour cadmium standard solution, cumulative volume are 500 μ l, final concentration of 0.1 μM, 1 μM and 10 μM of cadmium ion, 37 DEG C of oscillation incubation 1h. 10ml is diluted to, pipe (Millipore, article No. UFC901096), which is concentrated by ultrafiltration, using the albumen that aperture is 10kD filters SA-MBP (Cd) albumen, takes filter liquor, tests concentration of cadmium ions.As shown in figure 9, it is 0.1 μM, 1 μM or 10 μM in initial concentration of cadmium ions When, SA-MBP (Cd) albumen can be greatly reduced the remaining concentration of cadmium ions in solution with the cadmium ion in adsorbent solution.

Prepared by embodiment 5 can secret out of extracellular SA-MBP (Hg) albumen

In view of SA-MBP (Hg) folds to form inclusion body in microbial body content error-prone, in order to improve protein yield, examine Consider and secreting signal peptide is added by the N-terminal in fusion protein, fusion protein is secreted out of extracellularly.

Whole plasmid constructions are used as clone strain by the use of e.colistraindh5α (TransGen Biotech).Using withered The bacillus subtilis RIK1285 bacterial strains included in careless secreted from bacillus protein expression system [TaKaRa, article No. 3380] are made To express bacterial strain.Based on pBE-S vector construction pBE-S-LacZ plasmids (Figure 10 B, the SEQ ID NO included in the system：27). Specifically, by pAprE promoters and the His-tag of LacZ α sequence insertion pBE-S carrier of the flank with BsaI sites it Between, for using Golden Gate cloning process structure pBE-S series plasmids.Building the pBE-S series plasmids completed can be Bacillus subtilis can use His-tag antibody to carry out albumen table by western blot in middle expression SA-MBP (Hg) The detection reached.

The SA-MBP expressed in bacillus subtilis (Hg) can be secreted into extracellular signal peptide to screen, selected Take the signal peptide sequence and solution of AmyE, NprE, AprE and SacB gene of bacillus subtilis (Bacillus subtilis) The signal peptide sequence of AmyA, NprE, AprE and SacB gene of bacillus amyloliquefaciens (Bacillus amyloliquefaciens) Arrange (Figure 10 C).8 kinds of different signal peptides are respectively placed in the N-terminal of SA-MBP (Hg) using Golden Gate cloning process, are inserted Enter into pBE-S-LacZ carriers, build pBE-S series plasmids.

By 37 DEG C, the bacillus subtilis seed liquor that 200rpm concussions are incubated overnight dilutes 100 times extremely using LB culture mediums 100ml, using old terms continue culture 48 it is small when, 10000rpm centrifugations 10min takes supernatant, abandons cell precipitation.Use hole The albumen that footpath is 10kD is concentrated by ultrafiltration pipe (Millipore, article No. UFC901096) and filters SA-MBP (Hg) albumen, by supernatant 100 times of concentration takes 10 μ l to add 2 μ l sample-loading buffers (6 ×) and boils 5min, carry out western blot, use His- to 1ml SA-MBP (Hg) albumen present in tag antibody test supernatants.

As shown in Figure 10 D, No. 2 samples of the signal peptide with bacillus subtilis AmyE genes and with solution starch gemma No. 7 samples of the signal peptide of bacillus SacB genes can correct size (26kD) position detection to band, and 100 times of concentrations Band intensity in liquid is significantly increased.No. 2 and No. 7 signal peptides can guide the SA-MBP (Hg) of cell inner expression to be secreted into carefully It is extracellular.

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Claims

1. a kind of microbial cell for being used to handle heavy metal pollution, wherein, the microbial cell expresses heavy metal combination egg White and biotin-binding protein；Wherein, the heavy metal is the one or more in following heavy metal：Cadmium, mercury, lead And arsenic.

2. microbial cell as claimed in claim 1, the microbial cell has following one or more features：

The microorganism is selected from the group being made of following Institute of Micro-biology：Bacterium, actinomyces, mould, yeast and algae；It is preferred that Ground, the bacterium are bar-shaped selected from Escherichia coli, bacillus subtilis, pseudomonas aeruginosa, pseudomonas putida and glutamic acid Bacillus；

One or more of the Heavy Metal Binding Proteins in the group being made of following albumen：Cadmium-binding protein, mercury Associated proteins, lead associated proteins and arsenic associated proteins；Preferably, the Heavy Metal Binding Proteins are in following albumen It is one or more：MerR, Crs5, CadR, CmtR, PbrR, PbrR691 and ArsR；Alternatively, the Heavy Metal Binding Proteins choosing From in the heavy metal binding structural domain of following one or more albumen：MerR, Crs5, CadR, CmtR, PbrR, PbrR691 and ArsR；

The biotin-binding protein is selected from following albumen：EMA, mSA, mSA2, single stranded form Streptavidin dimer, The ovum Avidin dimer of single stranded form, the rhizavidin dimers of single stranded form, the Streptavidin four of single stranded form are poly- The ovum Avidin tetramer of body, single stranded form；Preferably, the biotin-binding protein is mSA2；Alternatively, the biotin knot Hop protein is selected from：The Streptavidin biotin binding structural domain dimer of single stranded form, the ovum Avidin life of single stranded form Rhizavidin biotin binding structural domains dimer, the chain of single stranded form of thing element binding structural domain dimer, single stranded form The ovum Avidin biotin binding structural domain tetramer of the mould Avidin biotin binding structural domain tetramer, single stranded form；It is preferred that Ground, the biotin-binding protein are the biotin binding structural domain of mSA2.

Preferably, the Heavy Metal Binding Proteins and/or the biotin-binding protein further include cross-film and film positioning knot Structure domain；Preferably, the Heavy Metal Binding Proteins and/or the biotin-binding protein are determined with cross-film and film by connecting peptide Nuclear localization sequence is connected；And

The Heavy Metal Binding Proteins and/or the biotin-binding protein further include labelled peptide, and the labelled peptide is located at The N-terminal or C-terminal of the Heavy Metal Binding Proteins and/or the biotin-binding protein；Preferably, the labelled peptide be selected from One or more in the group be made of following labelled peptide：Hexahistine label, c-Myc labels, Halo labels, Flag marks Label.

3. a kind of fusion protein for being used to handle heavy metal pollution, wherein, the fusion protein includes heavy metal binding structural domain And biotin binding structural domain；Wherein, the heavy metal is the one or more in following heavy metal：Cadmium, mercury, lead And arsenic.

4. fusion protein as claimed in claim 3, the fusion protein has following one or more features：

One or more of the heavy metal binding structural domain in the group being made of following Heavy Metal Binding Proteins：Cadmium Associated proteins, mercury-binding proteins, lead associated proteins and arsenic associated proteins；Preferably, the heavy metal binding structural domain is selected from One or more in following albumen：MerR, Crs5, CadR, CmtR, PbrR, PbrR691 and ArsR；It is alternatively, described heavy Heavy metal binding structural domain of the metal binding domain selected from following one or more albumen：MerR、Crs5、CadR、CmtR、 PbrR, PbrR691 and ArsR；

The biotin binding structural domain is selected from following albumen or its biotin binding structural domain：EMA, mSA, mSA2, strepto- Avidin, ovum Avidin and rhizavidin；Preferably, the biotin binding structural domain is Streptavidin or strepto- parent With the biotin binding structural domain of element；Alternatively, the biotin binding structural domain is selected from：The Streptavidin two of single stranded form Aggressiveness, the ovum Avidin dimer of single stranded form, the rhizavidin dimers of single stranded form, the Streptavidin of single stranded form The ovum Avidin tetramer of the tetramer, single stranded form；Streptavidin biotin binding structural domain dimer, the list of single stranded form Ovum Avidin biotin binding structural domain dimer, the rhizavidin biotins binding structural domain two of single stranded form of chain form Aggressiveness, the Streptavidin biotin binding structural domain tetramer of single stranded form, the ovum Avidin biotin of single stranded form combine Domain tetramer structure domain；

The fusion protein is further included is connected to the biotin binding structural domain by the heavy metal binding structural domain Connect peptide；

The fusion protein further includes secreting signal peptide；Preferably, the secreting signal peptide is located at the N of the fusion protein End；And

The fusion protein further includes labelled peptide, and the labelled peptide is located at the N-terminal or C-terminal of the fusion protein；Preferably, The labelled peptide is the one or more in the group being made of following labelled peptide：Hexahistine label, c-Myc marks Label, Halo labels, Flag labels.

5. a kind of kit for being used to handle heavy metal pollution, the kit include microorganism as claimed in claim 1 or 2 Cell or the fusion protein as described in claim 3 or 4, and biotinylated solid phase material；Wherein, the heavy metal is choosing From the one or more in following heavy metal：Cadmium, mercury, lead and arsenic.

6. kit as claimed in claim 5, wherein, the kit has following one or more features：

The solid phase material for surface with amino or carboxyl or with can by amino or the group of carboxyl modified (such as Hydroxyl), so as to be covalently attached with biotin to form the material of biotinylated solid phase material；Preferably, the solid phase Material has the material of albumen for glass, agarose, polystyrene or surface modification；

The biotinylated solid phase material is flaky material, it is preferable that the flaky material, which has, is more than 1mm²Surface area；

The biotinylated solid phase material is bulk material, it is preferable that the bulk material, which has, is more than 1mm³Volume；

The biotinylated solid phase material is filamentary material, it is preferable that the filamentary material has the length more than 1cm；

The biotinylated solid phase material is particle form, it is preferable that the particle has 5 μm of sizes to 1mm；And

The biotinylated solid phase material is magnetic bead form, it is preferable that the magnetic bead is glass magnetic bead, agarose magnetic bead or poly- Styrene magnetic bead, more preferably agarose magnetic bead；Preferably, the magnetic bead is with 1 μm to 100 μm, preferably 10 μm to 80 μm Size；Preferably, when carrying out biotinylation to the magnetic bead, the mass ratio of biotin and the magnetic bead is (0.5-1):1(m/ M), preferably 3:4(m/m)；Preferably, the ionic strength of biotinylation system buffer solution is 0.5-3M, preferably 1M；Preferably, it is raw Thing element system includes surfactant, and the surfactant is preferably Tween-20 or Triton X-100.

7. a kind of method for handling heavy metal pollution in sample, the described method includes：

(a) microbial cell as claimed in claim 1 or 2 or the fusion protein as described in claim 3 or 4 are provided；

(b) biotinylated solid phase material is provided；

(d) the biotinylated solid phase material of step (b) is recycled；

8. the method for claim 7, wherein, the method has following one or more features：

The solid phase material in step (b) is for surface with amino or carboxyl or with can be by amino or carboxyl modified Group (such as hydroxyl), so as to be covalently attached with biotin to form the material of biotinylated solid phase material；It is preferred that Ground, the solid phase material have the material of albumen for glass, agarose, polystyrene or surface modification；

Preferably, described in step (d) is recovered as directly recycling；The biotinylated solid phase material in step (b) is Flaky material, it is preferable that the flaky material, which has, is more than 1mm²Surface area；Alternatively, the biotinylation in step (b) Solid phase material be bulk material, it is preferable that the bulk material have is more than 1mm³Volume；Alternatively, the institute in step (b) It is filamentary material to state biotinylated solid phase material, it is preferable that the filamentary material has the length more than 1cm；

Preferably, described in step (d) is recovered as being recovered by centrifugation, and the biotinylated solid phase in step (b) Material is particle form, it is preferable that the particle has 5 μm of sizes to 1mm；Or

Preferably, described in step (d) is recovered as Magneto separate, and the biotinylated solid phase material in step (b) is Magnetic bead form, it is preferable that the magnetic bead is glass magnetic bead, agarose magnetic bead or polystyrene magnetic beads, more preferably agarose magnetic Pearl；Preferably, the magnetic bead has 1 μm to 100 μm, preferably 10 μm to 80 μm of size；Preferably, the magnetic bead is given birth to During thing elementization, the mass ratio of biotin and the magnetic bead is (0.5-1):1 (m/m), preferably 3:4(m/m)；Preferably, biotin The ionic strength of change system buffer solution is 0.5-3M, preferably 1M；Preferably, biotinylation system includes surfactant, described Surfactant is preferably Tween-20 or Triton X-100.

9. method as claimed in claim 7 or 8, wherein, the sample is the sample from environment, particularly obtained from or come From the sample of following environment：Water (including waste water), pond, river, reservoir, swimming pool, soil, food processing and/or packing factory, Agriculture ground, aquatic farm (including water planting food farm), medicine manufacturing works, fauna facility, or above-mentioned ambient source Any combination；The sample is solid, liquid or solid-liquid mixed form.