CN107828708B - Microorganism for enriching lead and related protein - Google Patents

Abstract

The invention discloses a microorganism for enriching lead and related protein. The microorganism provided by the invention contains any one of the following proteins A1) -A4): A1) the amino acid sequence comprises the proteins at the 1 st-183 th site and the 188 th-268 th site of the sequence 4 in the sequence table; A2) a polypeptide which is derived from the 1 st-183 nd and/or 188 nd-268 nd of the sequence 4 in the A1) by the substitution and/or deletion and/or addition of one or more amino acid residues and has the same function; A3) protein shown by amino acids 1-268 in a sequence 4 in a sequence table; A4) the polypeptide which is derived from the amino acid sequence of the 1 st to 268 th site of the sequence 4 in the sequence table through the substitution and/or deletion and/or addition of one or more amino acid residues and has the same function. Experiments prove that the microorganism has strong lead adsorption capacity.

Description

Microorganism for enriching lead and related protein

Technical Field

The invention relates to the field of biotechnology, and discloses a microorganism for enriching lead and related proteins.

Background

Lead is a highly toxic heavy metal naturally present in the earth crust, has a content of 13mg/kg in rocky circles, and is naturally disseminated by volcanic activity, chemical weathering, sea fog and other modes. The progress, especially in industrialization, accompanying human activities such as mining, smelting and burning fossil fuels is essentially the "redistribution" of the lead element. It is noteworthy that low concentrations of lead in the environment, as a non-naturally degradable metal poison, can be enriched by animals and plants and then transmitted to humans via the food chain. Lead does not have any physiological function to the human body but can cause serious harm to the health of the human body. Eliminating environmental lead residues is a time-consuming and costly major project, and detection, adsorption and recovery of lead-containing pollutants in the environment are always the focus of attention of scholars.

As the physical and chemical properties of the electron shells, the structures, the valence states and the like of most heavy metal ions are very close, the difficulty of obtaining the high-selectivity metal ion probe through a chemical synthesis method is high. Microorganisms living in severely polluted extreme environments evolve a set of simple and efficient inducible manipulation subsystems to adjust the content of various toxic metal ions in thalli. Around the application of biological means to treat heavy metal pollution of the environment and avoid secondary pollution to the environment possibly caused by physical and chemical methods, researchers combine DNA recombination technology and a microbial cell surface display system to try to display some target molecules on the cell surfaces of model microbial escherichia coli and yeast, and a series of microbial metal adsorbers are developed. However, due to the limitation of a microorganism display system, the difficulty of displaying macromolecular proteins is high, and the display efficiency is not high, so that research is mainly focused on the surface display of thallus cells of metal binding oligopeptides, metallothionein and the like, and application basic research is only limited to the treatment of pollution of a few heavy metals such as lead, cadmium, copper, mercury and the like in a water body. In recent years, metal binding protein has been proved to be an ideal thallus display target molecule, and various heavy metal microorganism whole cell adsorbers have been successfully developed by utilizing the high selectivity of the metal binding protein on the binding of metal ions. The biomacromolecule for efficiently and specifically identifying target metal ions is the basis for developing a novel microbial metal adsorber.

Disclosure of Invention

The invention aims to provide a microorganism for enriching lead and related protein used by the microorganism.

The present invention firstly provides a microorganism comprising any one of the following proteins A1) -A5) (this protein is named LOT-PbrR 81):

A1) the amino acid sequence comprises the proteins at the 1 st-183 th site and the 188 th-268 th site of the sequence 4 in the sequence table;

A2) a polypeptide which is derived from the 1 st-183 nd and/or 188 nd-268 nd of the sequence 4 in the A1) by the substitution and/or deletion and/or addition of one or more amino acid residues and has the same function;

A3) protein shown by amino acids 1-268 in a sequence 4 in a sequence table;

A4) the polypeptide which is derived from the amino acid sequence of the 1 st to 268 th site of the sequence 4 in the sequence table through the substitution and/or deletion and/or addition of one or more amino acid residues and has the same function;

A5) a fusion protein obtained by connecting labels at the N terminal or/and the C terminal of A1) or A2) or A3) or A4).

To facilitate purification of the protein in LOT-PbrR81, tags as shown in Table 1 can be attached to the amino terminus or the carboxyl terminus of the protein consisting of the amino acid sequence of LOT-PbrR81 of the present invention.

TABLE 1 sequence of tags

Label (R)	Residue of	Sequence of
			Poly-Arg	5-6 (typically 5)	RRRRR
Poly-His	2-10 (generally 6)	HHHHHH
			FLAG	8	DYKDDDDK
Strep-tag II	8	WSHPQFEK
			c-myc	10	EQKLISEEDL

The LOT-PbrR81 protein of the above A2), wherein the substitution and/or deletion and/or addition of one or more amino acid residues is a substitution and/or deletion and/or addition of not more than 10 amino acid residues.

The LOT-PbrR81 protein in A2) can be artificially synthesized, or can be obtained by synthesizing the coding gene and then performing biological expression.

The gene encoding the LOT-PbrR81 protein in A2) above can be obtained by deleting one or several amino acid residues from the DNA sequence encoding LOT-PbrR81 of the present invention, and/or by carrying out missense mutation of one or several base pairs, and/or by attaching a coding sequence of the tag shown in Table 1 above to its 5 'end and/or 3' end.

The microorganism may express LOT-PbrR 81. LOT-PbrR81 may be located on the surface of the microorganism.

The microorganism may be a microorganism obtained by introducing a gene encoding LOT-PbrR81 into a starting microorganism, which expresses LOT-PbrR 81.

In the above microorganism, the gene encoding LOT-PbrR81 may be any of the following genes b1) to b 4):

b1) the sequence comprises nucleic acid molecules at the 1 st 549 th site and the 562 nd 804 th site of the sequence 3 in the sequence table;

b2) a nucleic acid molecule shown in 1 st to 804 th sites of a sequence 3 in a sequence table;

b3) a nucleic acid molecule having 75% or more identity to the nucleotide sequence defined in b1) or b2) and encoding LOT-PbrR 81;

b4) a nucleic acid molecule which hybridizes under stringent conditions with the nucleotide sequence defined under b1) or b2) or b3) and encodes LOT-PbrR 81.

Wherein the nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA, etc.

Wherein, the DNA molecule shown in the sequence 3 encodes LOT-PbrR81 shown in the sequence 4.

The nucleotide sequence encoding the LOT-PbrR81 protein of the present invention can be readily mutated by one of ordinary skill in the art using known methods, such as directed evolution and point mutation. Those nucleotides which are artificially modified to have 75% or more identity to the nucleotide sequence of the LOT-PbrR81 protein isolated in the present invention are derived from the nucleotide sequence of the present invention and are identical to the sequence of the present invention as long as they encode the LOT-PbrR81 protein and have the function of the LOT-PbrR81 protein.

The term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. "identity" includes nucleotide sequences that are 75% or greater, or 85% or greater, or 90% or greater, or 95% or greater identical to the nucleotide sequence of a protein consisting of the amino acid sequence encoding LOT-PbrR81 of the present invention. Identity can be assessed visually or by computer software. Using computer software, the identity between two or more sequences can be expressed in percent (%), which can be used to assess the identity between related sequences.

In the above microorganism, the stringent conditions are hybridization and membrane washing at 68 ℃ for 2 times, 5min each, in a solution of 2 XSSC, 0.1% SDS, and hybridization and membrane washing at 68 ℃ for 2 times, 15min each, in a solution of 0.5 XSSC, 0.1% SDS; alternatively, hybridization was carried out at 65 ℃ in a solution of 0.1 XSSPE (or 0.1 XSSC), 0.1% SDS, and the membrane was washed.

The above-mentioned identity of 75% or more may be 80%, 85%, 90% or 95% or more.

In the above microorganism, the gene encoding LOT-PbrR81 can be obtained by introducing an expression vector containing the gene encoding LOT-PbrR81 into the starting microorganism.

The expression vector can be a recombinant vector which can express LOT-PbrR81 and is obtained by introducing the coding gene of the LOT-PbrR81 into an original vector. The starting vector may be a pET vector, such as pET-21a (+).

The expression vector can be specifically pLOT-PbrR81, the pLOT-PbrR81 is a recombinant plasmid obtained by replacing a small fragment between Nde I and Xho I recognition sequences of a vector pET-21a (+), by a DNA molecule shown as 1-804 th nucleotides from the 5' end in the sequence 3 of a sequence table, and the pLOT-PbrR81 can express LOT-PbrR 81.

The starting microorganism may be a bacterium. The bacterium may in particular be Escherichia coli, such as Escherichia coli BL21(DE 3).

The microorganism can be BL21/pLOT-PbrR81, and the BL21/pLOT-PbrR81 is a recombinant bacterium obtained by introducing the pLOT-PbrR81 into Escherichia coli BL21(DE 3).

The invention also provides a method for enriching lead, which comprises the following steps: and (3) utilizing the microorganism to complete lead enrichment.

The invention also provides LOT-PbrR81 or the coding gene of LOT-PbrR 81.

The invention also provides a protein named LOT, wherein the LOT is C1) or C2) or C3):

C1) protein shown by amino acids 1-183 of a sequence 4 in a sequence table;

C2) the polypeptide which is derived from the amino acid sequence of the 1 st to 183 th site of the sequence 4 in the sequence table through the substitution and/or deletion and/or addition of one or more amino acid residues and has the same function;

C3) c1) or C2) at the N-terminus or/and the C-terminus.

The invention also provides a coding gene of LOT.

The gene encoding the LOT may be d1) or d2) or d3) as follows:

d1) the coding sequence is the nucleic acid molecule of 1-549 th site of the sequence 3 in the sequence table;

d2) a nucleic acid molecule having 75% or more 75% identity to the nucleotide sequence defined by d1) and encoding a LOT;

d3) hybridizes under stringent conditions to a nucleotide sequence defined by d1) or d2) and encodes a cDNA molecule or a genomic DNA molecule of a LOT.

Wherein the nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA, etc.

The nucleotide sequence encoding the LOT protein of the present invention can be easily mutated by a person of ordinary skill in the art using known methods, such as directed evolution and point mutation. Those nucleotides which are artificially modified to have 75% or more identity to the nucleotide sequence of the LOT protein isolated in the present invention are derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention as long as they encode the LOT protein and have the function of the LOT protein.

The term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. "identity" includes nucleotide sequences that are 75% or greater, or 85% or greater, or 90% or greater, or 95% or greater, identical to the nucleotide sequence of a protein consisting of the amino acid sequence encoding a LOT of the present invention. Identity can be assessed visually or by computer software. Using computer software, the identity between two or more sequences can be expressed in percent (%), which can be used to assess the identity between related sequences.

The stringent conditions are hybridization and washing of the membrane 2 times, 5min each, at 68 ℃ in a solution of 2 XSSC, 0.1% SDS, and 2 times, 15min each, at 68 ℃ in a solution of 0.5 XSSC, 0.1% SDS; alternatively, hybridization was carried out at 65 ℃ in a solution of 0.1 XSSPE (or 0.1 XSSC), 0.1% SDS, and the membrane was washed.

The above-mentioned identity of 75% or more may be 80%, 85%, 90% or 95% or more.

The present invention also provides the microorganism, LOT-PbrR81, the gene encoding LOT-PbrR81, LOT, or any one of the following (1) to (6) for the gene encoding LOT:

(1) enriching lead;

(2) preparing a kit for enriching lead;

(3) detecting lead;

(4) preparing a kit for detecting lead;

(5) bonding lead;

(6) a kit for binding lead is prepared.

The present invention also provides a product having any one of the uses (I) to (III), said product containing said microorganism, LOT-PbrR81, a gene encoding said LOT-PbrR81, LOT, or a gene encoding said LOT:

(I) enriching lead;

(II) detecting lead;

(III) binding lead.

In the present invention, the product may be a kit.

The lead may be a lead ion.

Experiments prove that both LOT and LOT-PbrR81 can be positioned on the outer membrane of the thallus, and PbrR81 is successfully displayed on the outer membrane of the thallus. The lead content of the LOT-PbrR 81-containing microorganism of the present invention after treatment with lead ions was 75. mu. mol/g dry weight, which was significantly higher than that of the LOT-only microorganism, and that of the LOT-and LOT-PbrR 81-free microorganism. It was shown that the LOT-PbrR 81-containing microorganism of the present invention had a potent lead adsorption capacity.

Drawings

FIG. 1 shows the results of detection of each cell. The left panel shows the results of 15% SDS-PAGE, and the right panel shows the results of Western blot.

FIG. 2 shows immunofluorescence microscopy imaging analysis (left fluorescent field, right bright field, 600-fold magnification).

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The experimental procedures in the following examples are conventional unless otherwise specified. Materials, reagents, instruments and the like used in the following examples are commercially available unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.

Vector pET-21a (+): novagen, Inc., catalog number 69740. Coli BL21(DE 3): novagen Inc. catalog number 69450.

Example 1 construction of recombinant plasmid

1. Synthesizing a double-stranded DNA molecule shown in a sequence 1 in a sequence table.

2. The double-stranded DNA molecule obtained in step 1 is double-digested with restriction enzymes Nde I and Sal I, and the digested product is recovered.

3. The vector pET-21a (+) was digested with restriction enzymes Nde I and Sal I, and the vector backbone was recovered.

4. And (3) connecting the enzyme digestion product in the step (2) with the vector skeleton in the step (3), and naming the recombinant plasmid with correct sequence as pLOT. pLOT is a recombinant plasmid obtained by replacing the DNA fragment between Nde I and Sal I recognition sequences of pET-21a (+) with a DNA molecule represented by the 1-549 th nucleotide from the 5' end of the sequence 3 in the sequence table. The recombinant plasmid pLOT can express a fusion protein formed by fusing His labels at the C terminal of a protein (named LOT) shown in the 1 st to 183 th sites of a sequence 4 in a sequence table, and the fusion protein is marked as LOT-His.

5. Synthesizing a double-stranded DNA molecule shown in a sequence 2 in a sequence table.

6. Double-stranded DNA molecules obtained in the step 5 are double-digested by restriction enzymes Hind III and Xho I, and the digested products are recovered.

7. The vector backbone was recovered by double digestion of the recombinant plasmid pLOT with restriction enzymes Hind III and Xho I.

8. The cleavage product of step 6 was ligated to the vector backbone of step 7, and the correctly sequenced recombinant plasmid was designated pLOT-PbrR 81.

pLOT-PbrR81 is a recombinant plasmid obtained by substituting a small fragment between Nde I and Xho I recognition sequences of the vector pET-21a (+) for a DNA molecule represented by nucleotides 1 to 804 from the 5' end in sequence 3 of the sequence Listing. The recombinant plasmid pLOT-PbrR81 can express protein shown in a sequence 4 in a sequence table.

Wherein, the LOT shown in the 1 st to 183 th positions of the 1 st to 549 th coding sequence 4 of the sequence 3, the PbrR81 peptide segment shown in the 188 th and 268 th positions of the 562 nd and 804 th coding sequence 4 of the sequence 3, and the His tag shown in the 271 th and 276 th positions of the 811 th and 828 th coding sequence 4 of the sequence 3. The protein shown in sequence 4 was named LOT-PbrR 81-His.

Example 2 construction and culture of recombinant bacteria

1. The vector pET-21a (+) was introduced into E.coli BL21(DE3) to obtain a recombinant strain BL21/pET21 a.

2. The recombinant plasmid pLOT of example 1 was introduced into E.coli BL21(DE3) to obtain a recombinant strain BL 21/pLOT.

3. The recombinant plasmid pLOT-PbrR81 of example 1 was introduced into E.coli BL21(DE3) to give a recombinant strain BL21/pLOT-PbrR 81.

4. Induction culture of recombinant bacterium BL21/pLOT-PbrR81

(1) The recombinant strain BL21/pLOT-PbrR81 was inoculated into a liquid LB medium containing 50. mu.g/mL ampicillin, and was cultured at 37 ℃ and 250rpm with shaking to OD_600nmThe value is 0.6.

(2) After completion of step (1), IPTG (concentration of IPTG in the culture system: 0.5mM) was added to the culture system, and the mixture was cultured at 37 ℃ for 4 hours with shaking at 250 rpm.

(3) After the step (2) was completed, the whole culture system was centrifuged at 3500rpm, and BL21/pLOT-PbrR 81-induced cells were collected.

5. Induction culture of recombinant bacterium BL21/pLOT

Replacing the recombinant strain BL21/pLOT-PbrR81 with the recombinant strain BL21/pLOT, and collecting BL21/pLOT induced bacteria in the same step 4.

6. Non-induced culture of recombinant bacterium BL21/pLOT

(1) The recombinant strain BL21/pLOT was inoculated into a liquid LB medium containing 50. mu.g/mL ampicillin, and cultured at 37 ℃ with shaking at 250rpm until OD_600nmThe value is 0.6.

(2) After completion of step (1), the cells were cultured at 37 ℃ for 4 hours with shaking at 250 rpm.

(3) After the completion of step (2), the whole culture system was centrifuged at 3500rpm, and BL21/pLOT non-induced cells were collected.

7. Detection of bacteria

(1) 15% SDS-PAGE and Western-blot analysis

The thalli obtained in the step 4-6 are respectively detected according to the following steps: resuspend with lysis buffer (20mmol/L Tris-HCl, pH 8.0,2mmol/L EDTA,500mmol/L NaCl); under ice bath condition, carrying out ultrasonic cell disruption, wherein the working condition is as follows: ultrasonic treatment for 5s, interval for 5s and total time for 10 min; centrifuging at 4 deg.C and 12000rpm for 30min, and collecting precipitate; the pellet was divided into two equal portions, one for 15% SDS-PAGE analysis and the other for Western-blot analysis. In Western-blot, anti-His tag mouse monoclonal antibody is used as a primary antibody, HRP-labeled rabbit anti-mouse antibody is used as a secondary antibody, and DAB substrate is developed.

The results are shown in FIG. 1, where 1 is BL21/pLOT non-induced mycoprotein, 2 is BL21/pLOT induced mycoprotein, 3 is BL21/pLOT-PbrR81 induced mycoprotein, and M is protein Marker. After BL21/pLOT induction, the expression fusion protein LOT-His with the theoretical molecular weight of about 21.8kDa is shown; BL21/pLOT-PbrR81 was induced to express the fusion protein LOT-PbrR81-His, with a theoretical molecular weight of about 30.7 kDa. The corresponding position of the left image in FIG. 1 shows the target protein band (shown by an arrow) with the correct size, and the result is verified by the obtained Western-blot.

(2) Immunofluorescence microscopy imaging analysis:

the thalli obtained in the step 4-6 are respectively detected according to the following steps: suspending the thallus precipitate in 1mL PBS (pH7.4) containing 4% formaldehyde, slowly oscillating in a decolorizing shaker, and fixing for 1.5 hr; centrifuging at 3500rpm for 5min, and removing the supernatant; suspending the thallus precipitate in 1mL PBS (pH7.4) containing 1% fetal calf serum, slowly oscillating in a decolorizing shaker, and sealing for 30 min; centrifuging at 3500rpm for 5min, and removing the supernatant; the bacterial pellet was resuspended in 0.5mL of PBS (pH7.4), and anti-His tag mouse monoclonal antibody (1:200) was added thereto, followed by incubation at 4 ℃ overnight; the cells were washed 3 times with PBS (pH7.4); the cell pellet was resuspended in 0.5mL PBS (pH7.4), FITG-labeled donkey anti-mouse secondary antibody (1:300) was added, and the mixture was slowly shaken in a decolorization shaker for 1.5 hours in the dark, washed with PBS 3 times, and subjected to immunofluorescence observation.

As shown in FIG. 2, A was BL21/pLOT non-induced microbial cells, B was BL21/pLOT induced microbial cells, and C was BL21/pLOT-PbrR81 induced microbial cells. Through immunofluorescence detection of His tag at the C terminal of the fusion protein, it can be seen that thalli after induction of recombinant bacteria BL21/pLOT and BL21/pLOT-PbrR81 all show strong surface fluorescence, which indicates that LOT-His and LOT-PbrR81-His can be positioned on the outer membrane of the thalli, and PbrR81 is successfully displayed on the outer membrane of the thalli.

Example 3 examination of lead ion adsorption ability

The bacteria to be detected are respectively escherichia coli BL21(DE3), recombinant bacteria BL21/pET21a, BL21/pLOT and BL21/pLOT-PbrR81 of the embodiment 2, and the lead ion adsorption capacity of each bacteria is detected according to the following steps:

1. inoculating the test bacteria into liquid LB culture medium containing 50 mug/mL ampicillin and 150 mug/L lead nitrate, and performing shaking culture at 37 ℃ and 250rpm until OD_600nmThe value is 0.6.

2. After completion of step 1, IPTG (concentration of IPTG in the culture system: 0.5mM) was added to the culture system, and the mixture was cultured at 37 ℃ for 12 hours with shaking at 250 rpm.

3. After step 2, the whole culture system was centrifuged at 3500rpm for 10min, and the cells were collected.

4. And (3) cleaning the thalli obtained in the step (3) for 3 times by using sterile water, and then drying the thalli in an oven at the temperature of 80 ℃ to constant weight (weighing the thalli to obtain the dry weight of the thalli).

5. And (3) taking 1mg of the thalli dried to constant weight obtained in the step (4), adding 5ml of pure nitric acid solution, performing microwave digestion (500W, 140 ℃,2 hours), and then using double distilled water to fix the volume to 50ml to obtain the solution to be detected. And (3) detecting the lead concentration in the solution to be detected by adopting an atomic absorption method (the specific method refers to a graphite furnace atomic absorption spectrometry method for lead in WS/T18-1996 urine). The lead content of the cells per g dry weight was calculated.

The lead content of the recombinant bacterium BL21/pLOT-PbrR81 is 75 mu mol/g dry weight, the lead content of the recombinant bacterium BL21/pLOT is 16.2 mu mol/g dry weight, the lead content of the recombinant bacterium BL21/pET21a is 15.01 mu mol/g dry weight, and the lead content of escherichia coli BL21(DE3) is 14.98 mu mol/g dry weight. The result shows that the lead content of the recombinant bacterium BL21/pLOT-PbrR81 is obviously higher than that of the recombinant bacterium BL21/pLOT, the recombinant bacterium BL21/pET21a and escherichia coli BL21(DE 3). The result shows that the recombinant bacterium BL21/pLOT-PbrR81 has strong lead adsorption capacity.

<110> Shenzhen market occupational disease prevention and treatment hospital

<120> a microorganism and related protein for enriching lead

<160>4

<170>PatentIn version 3.5

<210>1

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<212>DNA

<213> Artificial sequence

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ggaattccat atgaaagcta ctaaactggt actgggcgcg gtaatcctgg gttctactct 60

gctggcaggt tgctccagca acgctaaaat cgatcaggga atttttactc ctgacaacat 120

aaatgcggac attagtcttg gaactctgag cggaaaaaca aaagagcgtg tttatctagc 180

cgaagaagga ggccgaaaag tcagtcaact cgactggaaa ttcaataacg ctgcaattat 240

taaaggtgca attaattggg atttgatgcc ccagatatct atcggggctg ctggctggac 300

aactctcggc agccgaggtg gcaatatggt cgatcaggac tggatggatt ccagtaaccc 360

cggaacctgg acggatgaaa gtagacaccc tgatacacaa ctcaattatg ccaacgaatt 420

tgatctgaat atcaaaggct ggctcctcaa cgaacccaat taccgcctgg gactcatggc 480

cggatatcag gaaagccgtt atagctttac agccagaggt ggttcctata tctacagttc 540

tgaggaggga ttcagagatg tcgacgcgt 569

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cccaagcttc agttcattcg tcactgccgg tctctggata tgccgttgag cgacgtacgg 60

accttattga gttaccggaa gcggcccgac caggattgcg gtgaagtcaa tatgctcttg 120

gatgagcaca tccgtcaggt cgaatctcgg atcggagcct tgctcgaact gaagcaccat 180

ttggtggaac tgcgcgaagc ctgttctggt gccaggcccg cccaatcgtg cgggattctg 240

cagggactgt cgctcgaggc c 261

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ggccgaaaag tcagtcaact cgactggaaa ttcaataacg ctgcaattat taaaggtgca 240

attaattggg atttgatgcc ccagatatct atcggggctg ctggctggac aactctcggc 300

agccgaggtg gcaatatggt cgatcaggac tggatggatt ccagtaaccc cggaacctgg 360

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Met Lys Ala Thr Lys Leu Val Leu Gly Ala Val Ile Leu Gly Ser Thr

1 5 10 15

Leu Leu Ala Gly Cys Ser Ser Asn Ala Lys Ile Asp Gln Gly Ile Phe

20 25 30

Thr Pro Asp Asn Ile Asn Ala Asp Ile Ser Leu Gly Thr Leu Ser Gly

35 40 45

Lys Thr Lys Glu Arg Val Tyr Leu Ala Glu Glu Gly Gly Arg Lys Val

50 55 60

Ser Gln Leu Asp Trp Lys Phe Asn Asn Ala Ala Ile Ile Lys Gly Ala

65 70 75 80

Ile Asn Trp Asp Leu Met Pro Gln Ile Ser Ile Gly Ala Ala Gly Trp

85 90 95

Thr Thr Leu Gly Ser Arg Gly Gly Asn Met Val Asp Gln Asp Trp Met

100 105 110

Asp Ser Ser Asn Pro Gly Thr Trp Thr Asp Glu Ser Arg His Pro Asp

115 120 125

Thr Gln Leu Asn Tyr Ala Asn Glu Phe Asp Leu Asn Ile Lys Gly Trp

130 135 140

Leu Leu Asn Glu Pro Asn Tyr Arg Leu Gly Leu Met Ala Gly Tyr Gln

145 150 155 160

Glu Ser Arg Tyr Ser Phe Thr Ala Arg Gly Gly Ser Tyr Ile Tyr Ser

165 170 175

Ser Glu Glu Gly Phe Arg Asp Val Asp Lys Leu Gln Phe Ile Arg His

180 185 190

Cys Arg Ser Leu Asp Met Pro Leu Ser Asp Val Arg Thr Leu Leu Ser

195 200 205

Tyr Arg Lys Arg Pro Asp Gln Asp Cys Gly Glu Val Asn Met Leu Leu

210 215 220

Asp Glu His Ile Arg Gln Val Glu Ser Arg Ile Gly Ala Leu Leu Glu

225 230 235 240

Leu Lys His His Leu Val Glu Leu Arg Glu Ala Cys Ser Gly Ala Arg

245 250 255

Pro Ala Gln Ser Cys Gly Ile Leu Gln Gly Leu Ser Leu Glu His His

260 265 270

His His His His

275

Claims (13)

1. A microorganism which is escherichia coli, said microorganism comprising a1) or a2) as follows:

A1) protein shown by amino acids 1-268 in a sequence 4 in a sequence table;

A2) protein shown in a sequence 4 in a sequence table.

2. The microorganism according to claim 1, characterized in that: the microorganism is a microorganism obtained by introducing a gene encoding the protein of claim 1 into a starting microorganism, and the microorganism expresses the protein of claim 1.

3. The microorganism according to claim 2, characterized in that: the coding gene is b1) or b2) as follows:

b1) a nucleic acid molecule shown as a sequence 3 in a sequence table;

b2) a nucleic acid molecule shown in 1 st to 804 th positions of a sequence 3 in a sequence table.

4. A method of enriching lead comprising: enrichment of lead is accomplished by using the microorganism of any one of claims 1-3.

5. The protein is shown as amino acids 1-183 of a sequence 4 in a sequence table or amino acids 1-268 of the sequence 4 or the sequence 4.

6. A gene encoding the protein according to claim 5.

7. The encoding gene of claim 6, wherein: the coding gene is a nucleic acid molecule of which the coding sequence is the 1 st to 549 th sites of a sequence 3 or the 1 st to 804 th sites of the sequence 3 or the sequence 3 in a sequence table.

8. Use of the microorganism according to any one of claims 1 to 3 in any one of the following (1) to (6):

(1) enriching lead;

(2) preparing a kit for enriching lead;

(3) detecting lead;

(4) preparing a kit for detecting lead;

(5) bonding lead;

(6) a kit for binding lead is prepared.

9. The use of the protein of claim 5 in any one of the following (1) to (6):

(1) enriching lead;

(2) preparing a kit for enriching lead;

(3) detecting lead;

(4) preparing a kit for detecting lead;

(5) bonding lead;

(6) a kit for binding lead is prepared.

10. The use of any one of the following (1) to (6) of the encoding gene according to claim 6 or 7:

(1) enriching lead;

(2) preparing a kit for enriching lead;

(3) detecting lead;

(4) preparing a kit for detecting lead;

(5) bonding lead;

(6) a kit for binding lead is prepared.

11. A product having a use according to any one of the following (I) to (III), containing the microorganism according to any one of claims 1 to 3:

(I) enriching lead;

(II) detecting lead;

(III) binding lead.

12. A product for use in any one of the following (I) to (III), containing the protein of claim 5:

(I) enriching lead;

(II) detecting lead;

(III) binding lead.

13. A product for use in any one of the following (I) to (III), comprising the gene encoding the polypeptide of claim 6 or 7:

(I) enriching lead;

(II) detecting lead;

(III) binding lead.

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