Truncated potato glycoprotein Patatin and preparation method thereof
阅读说明：本技术 一种截短的马铃薯糖蛋白Patatin及其制备方法 (Truncated potato glycoprotein Patatin and preparation method thereof ) 是由 周景文 余世琴 戴梓桥 曾伟主 陈坚 于 2021-09-26 设计创作，主要内容包括：本发明公开了一种截短的马铃薯糖蛋白Patatin及其制备方法,属于生物工程领域。本发明在马铃薯来源的糖蛋白Patatin-1的基础上对N端进行了截短,分别截短了N端第2～23位、2～37位、2～60位或2～71位氨基酸,本发明将构建的携带N端截短的糖蛋白基因的重组质粒在转化至毕赤酵母、酿酒酵母及乳酸克鲁维酵母等宿主中,使发酵后的重组酵母Patatin产量达225mg/L。本发明的方法为马铃薯Patatin在食品生产中的应用奠定了基础。(The invention discloses a truncated potato glycoprotein Patatin and a preparation method thereof, belonging to the field of bioengineering. The N end is truncated on the basis of glycoprotein Patatin-1 from potato, and the 2 nd to 23 th, 2 nd to 37 th, 2 nd to 60 th or 2 th to 71 th amino acids of the N end are truncated respectively. The method lays a foundation for the application of the potato Patatin in food production.)
1. The glycoprotein Patatin-1 mutant is characterized by having an amino acid sequence shown as any one of SEQ ID NO. 2-5.
2. A gene encoding the glycoprotein Patatin-1 mutant according to claim 1.
3. An expression vector comprising the gene of claim 2.
4. The expression vector of claim 3, comprising the pPIC9K plasmid, the pXW55 plasmid or the pKLAC1 plasmid.
5. A recombinant microbial cell expressing the glycoprotein Patatin-1 mutant according to claim 1.
6. The recombinant microbial cell of claim 5, comprising but not limited to Pichia, Saccharomyces cerevisiae, or Kluyveromyces lactis.
7. A recombinant Pichia pastoris is characterized in that Pichia pastoris GS115 is taken as a host, pPIC9K plasmid is taken as an expression vector, and a gene of a coding glycoprotein mutant shown in SEQ ID No.7 is expressed.
8. A method for improving glycoprotein Patatin-1 synthesis amount in yeast cells is characterized in that N-terminal amino acid truncation is carried out on Patatin-1 from potato, and then the truncated sequence is expressed in the yeast cells; the truncation refers to truncation of 22, 36, 59, or 70 amino acids following the first methionine at the N-terminus.
9. A method for synthesizing glycoprotein Patatin-1, which is characterized in that the recombinant microorganism cell of claim 5 or 6 or the recombinant Pichia pastoris of claim 7 is fermented in a culture medium at the temperature of 28-32 ℃ and the rpm of 200-250 for at least 96 h.
10. The glycoprotein Patatin-1 mutant of claim 1, or the gene of claim 2, or the recombinant microbial cell of any one of claims 5 to 6, or the recombinant pichia pastoris of claim 7, for use in the preparation of glycoprotein Patatin or products containing glycoprotein Patatin.
Technical Field
The invention relates to a truncated potato glycoprotein Patatin and a preparation method thereof, belonging to the field of bioengineering.
Background
The world population is growing rapidly and the demand for food proteins is increasing, which poses a challenge to food safety. In recent years, new, alternative and sustainable sources of proteins have been sought from plants, algae and insects. One of the plant proteins of interest is potato protein, and the potato tuber protein Patatin is a major component of potato protein. Like other proteins, Patatin has nutritional value, and also has excellent functional properties such as gelling property, foaming property, ester acyl hydrolysis activity, antioxidant activity and the like. The researches of Creusot and the like find that the rheological properties of the Patatin, the beta-lactoglobulin, the ovalbumin and the soybean protein are highly similar, but the initial gel temperature and the ionic strength required in the gel forming process are obviously lower than those of other three common gel proteins, and the Patatin has the rheological property of small deformation in the gel forming process. Patatin has unique thermal polymerization and gelling characteristics, and can become an excellent food gelling agent.
In China, potatoes are mainly used for producing starch, and a large amount of waste liquid generated in the process is called potato juice (PFJ). According to statistics, the process of producing starch by 1000kg of potatoes can produce 5-12 m3The waste liquid of (2) contains 1 to 2 mass percent of potato protein. Currently, there are many physical and chemical methods for extracting potato protein from PFJ, such as Na2SO3Aqueous solution leaching, thermal polymerization, acid precipitation and membrane recovery. Various extraction methods have advantages and disadvantages, but cannot simultaneously achieve the effects of no toxicity, safety, high recovery rate, low cost and protein functional property maintenance. In the industrial production process, a large amount of germinated potatoes are difficult to avoid to be mixed into processing raw materials, and toxins such as solanine and the like in the potatoes are combined with protein, so that the edible safety of the potato protein isolate is influenced. In recent years, potato Patatin is expressed in escherichia coli by means of genetic engineering, but inclusion bodies are often generated in escherichia coli expression, the safety cannot be guaranteed, and the potato Patatin is difficult to apply to the food industry.
Pichia pastoris is a heterologous protein expression host strain which is widely applied at present, is easy to culture, grows rapidly, has high exogenous gene expression amount, is safe and nontoxic, has a post-translational processing modification system, can correctly post-translationally process eukaryotic genes, and can effectively secrete exogenous gene expression products out of cells, so that the Pichia pastoris is considered as a synthetic host strain of potato Patatin. In addition, other microbial expression hosts with certain food safety characteristics are selected, and the production and application of the potato Patatin are well promoted.
Disclosure of Invention
The invention aims to provide a glycoprotein Patatin-1 mutant which has an amino acid sequence shown in any one of SEQ ID NO. 2-5.
In one embodiment, the glycoprotein Patatin-1 mutant is obtained by respectively truncating amino acid sequences at positions 2-23, 2-37, 2-60 and 2-71 on the basis of the potato-derived glycoprotein Patatin-1 shown in SEQ ID NO. 1.
In one embodiment, the glycoprotein Patatin-1 mutant is obtained by truncating amino acids 2-23 on the basis of a potato-derived glycoprotein Patatin-1 shown in SEQ ID No.1 to obtain an amino acid sequence shown in SEQ ID No. 2.
In one embodiment, the glycoprotein Patatin-1 mutant is obtained by truncating amino acids 2-37 on the basis of a potato-derived glycoprotein Patatin-1 shown in SEQ ID No.1 to obtain an amino acid sequence shown in SEQ ID No. 3.
In one embodiment, the glycoprotein Patatin-1 mutant is obtained by truncating amino acids 2-60 on the basis of a potato-derived glycoprotein Patatin-1 shown in SEQ ID No.1 to obtain an amino acid sequence shown in SEQ ID No. 4.
In one embodiment, the glycoprotein Patatin-1 mutant is obtained by truncating amino acids 2-71 on the basis of a potato-derived glycoprotein Patatin-1 shown in SEQ ID No.1 to obtain an amino acid sequence shown in SEQ ID No. 5.
The invention also provides a gene for coding the glycoprotein Patatin-1 mutant.
In one embodiment, the gene comprises the nucleotide sequence set forth in SEQ ID NO. 7.
The invention also provides an expression vector containing the gene.
In one embodiment, the expression vector is obtained by ligating the gene shown in SEQ ID NO.7 between the SnaBI and AvrII sites on the pPIC9K plasmid.
In one embodiment, the expression vector further comprises a pXW55 plasmid or a pKLAC1 plasmid.
The invention also provides a recombinant strain expressing the glycoprotein Patatin-1 mutant.
In one embodiment, the host cell of the recombinant strain includes, but is not limited to, pichia, saccharomyces cerevisiae, or kluyveromyces lactis.
In one embodiment, the recombinant strain takes pichia pastoris GS115 as a host, takes pPIC9K plasmid as an expression vector, and expresses a gene which is shown as SEQ ID NO.7 and codes for the glycoprotein mutant.
In one embodiment, the recombinant strain takes saccharomyces cerevisiae BJ5464 as a host and pXW55 plasmid as an expression vector to express a gene which is shown as SEQ ID NO.7 and used for encoding glycoprotein mutants.
In one embodiment, the recombinant strain is a Kluyveromyces lactis GG799 host, and a pKLAC1 plasmid is an expression vector to express a gene encoding the glycoprotein mutant shown in SEQ ID No. 7.
The invention also provides a method for improving the synthesis amount of glycoprotein Patatin-1 in yeast cells, which comprises the steps of carrying out amino acid truncation on the N end of Patatin-1 from potatoes and expressing the truncated glycoprotein Patatin-1 in the yeast cells.
In one embodiment, the truncation refers to truncation by 22, 36, 59, or 70 amino acids following the first methionine at the N-terminus.
The invention also provides a method for synthesizing glycoprotein Patatin-1, which is to ferment the recombinant strain in a culture medium at 28-32 ℃ and 200-250 rpm for at least 96 h.
The invention also provides application of the recombinant strain in preparation of glycoprotein Patatin or products containing glycoprotein Patatin.
The invention has the beneficial effects that:
the invention provides glycoprotein Patatin-1 with an amino acid sequence shown as SEQ ID No. 2-5, which is obtained by truncating amino acids at positions 2-23, 2-37, 2-60 or 2-71 at the N end on the basis of the glycoprotein shown as SEQ ID No. 1. Expressing the truncated glycoprotein in Pichia pastoris GS115 by taking a recombinant plasmid pPIC9K-Patatin-1 as a vector; or respectively taking Saccharomyces cerevisiae BJ5464 and Kluyveromyces lactis GG799 as hosts, expressing truncated glycoprotein by recombinant plasmid with His tag, and purifying with nickel ion affinity chromatography column to obtain target protein Patatin-1. The strategy provided by the invention lays a foundation for the application of glycoprotein Patatin.
Drawings
FIG. 1: pPIC9K-Patatin-1 plasmid map.
FIG. 2: the recombinant truncated potato glycoprotein Patatin-1 pichia pastoris expression result.
Detailed Description
(I) culture Medium
LB culture medium: 10g/L of peptone, 5g/L of yeast powder and 10g/L of sodium chloride. 20g/L agar powder was added to prepare an LB solid medium.
YPD medium: peptone 20g/L, yeast powder 10g/L, and glucose 20 g/L. 20g/L agar powder was added to prepare a YPD solid medium.
MD culture medium: 798mL/L of deionized water, and 20g/L of agar powder is added; before plate inversion, 10% by volume of 10% basic nitrogen source mother liquor of yeast XYNB, 10% by volume of basic nitrogen source mother liquor of yeast XYNB, and 20g/L glucose were added to prepare an MD solid medium.
BMGY medium: peptone 20g/L, yeast powder 10g/L, glycerin 20g/L, 50mmol/L potassium phosphate buffer solution, 10% volume fraction of 10 XYNB yeast basic nitrogen source mother liquor, biotin 4X 10-4g/L。
BMMY medium: peptone 20g/L, yeast powder 10g/L, methanol 10g/L, 50mmol/L potassium phosphate buffer solution, 10% volume fraction of 10 XYNB yeast basic nitrogen source mother liquor, biotin 4X 10-4g/L。
EXAMPLE 1 construction of recombinant plasmid pPIC9K-Patatin-1 and recombinant plasmid containing truncated glycoprotein gene
(1) Construction of recombinant plasmid pPIC9K-Patatin-1
The Patatin storage protein derived from Solanum chacoense has the capability of synthesizing glycoprotein Patatin, synthesizes a gene which has a nucleotide sequence shown as SEQ ID NO.6 and codes for the glycoprotein Patatin, is connected between two sites of SnaBI and AvrII on a pPIC9K plasmid, is named as pPIC9K-Patatin-1 and is synthesized by Jinwei company of Suzhou.
(2) Construction of mutant plasmids
Taking the recombinant plasmid pPIC9K-Patatin-1 constructed in the step (1) as a template, and the sequences of the primers are shown in Table 1, and respectively constructing mutant plasmids of 22 (amino acids at positions 2-23), 36 (amino acids at positions 2-37), 59 (amino acids at positions 2-60) and 70 (amino acids at positions 2-71) truncated at the N end of the glycoprotein Patatin-1: pPIC9K-Patatin-1-t22, pPIC9K-Patatin-1-t36, pPIC9K-Patatin-1-t59 and pPIC9K-Patatin-1-t 70. Each truncated sequence corresponds to one or several alpha-helices or beta-sheets, respectively. Taking the Patatin-1-T22 as an example, T22-F/T22-R as a primer and pPIC9K-Patatin-1 as a template, and amplifying the recombinant plasmid pPIC9K-Patatin-1-T22 containing 22 amino acid stages of Patatin-1 fragments.
And (3) PCR reaction system: the PCR total was 50. mu.l, which included 25. mu.l of high fidelity DNA polymerase:
2 × Phanta Max Master Mix (available from Nanjing Novowed Biotech Co., Ltd.), 20. mu.l of ultrapure water, 2. mu.l of each of the upstream and downstream primers, and 1. mu.l of each of the templates.
And (3) PCR reaction conditions: pre-denaturation: the reaction steps comprise that the temperature is 95 ℃ and the time is 3 min: (1) denaturation: 95 ℃, 15s, (2) annealing: 55-60 ℃, 15s, (3) extension: setting 25-32 cycles for 1-5 min at 72 ℃. And (3) final extension stage: and 5-10 min at 72 ℃.
After the PCR product is verified by 1% -2% agarose gel electrophoresis, adding Dpn I enzyme into the PCR product, and incubating for 1-2 h at 37 ℃ to eliminate the circular plasmid template; and then purifying and recovering the PCR product, converting the linearized plasmid group into a circular plasmid by using a Gibson assembly method, transferring the assembled plasmid into escherichia coli JM109 competence, coating the competent escherichia coli JM109 competence in an LB solid culture medium added with corresponding antibiotics, overnight culturing at 37 ℃, selecting positive clone and extracting the plasmid, and leaving the plasmid as recombinant pichia pastoris for construction and use after correct sequencing.
TABLE 1 primers for construction of truncated Patatin-1 expression vectors
Example 2 construction of recombinant truncated glycoprotein Patatin-1 expressing Pichia pastoris
The recombinant plasmid pPIC9K-Patatin-1 and the mutant plasmids pPIC9K-Patatin-1-t22, pPIC9K-Patatin-1-t36, pPIC9K-Patatin-1-t59 and pPIC9K-Patatin-1-t70 which are correctly sequenced in the example 1 are incubated for 1h at 37 ℃ by using SalI enzyme for plasmid linearization treatment, and are respectively transformed into pichia pastoris GS115 competent cells through pichia pastoris shock transformation, coating on MD solid plate, culturing at 30 deg.C for 2-3 days, selecting positive clone, constructing recombinant Pichia pastoris GS115-pPIC9K-Patatin-1, GS115-pPIC9K-Patatin-1-t22, GS115-pPIC9K-Patatin-1-t36, GS115-pPIC9K-Patatin-1-t59 and GS115-pPIC9K-Patatin-1-t 70.
Carrying out shake flask fermentation on the recombinant pichia pastoris, wherein the specific method comprises the following steps:
1) inoculating YPD plate activated Pichia pastoris GS115 in a 25mL/250mL triangular flask, and culturing overnight at 30 ℃; 1% inoculation of the aboveThe culture solution was cultured in a 50mL/500mL Erlenmeyer flask at the OD cell concentration6001.3 to 1.5;
2) centrifuging at 4 ℃ for 10min at 5000r/min, collecting thalli, and suspending the cells with 50mL and 25mL of sterile water respectively;
3) suspending the cells by using 5mL of 1M D-sorbitol solution, centrifuging at 5000r/min at 4 ℃ for 10min, and collecting thalli;
4) suspending the cells with 500. mu.L of 1M D-sorbitol solution, and dispensing 80. mu.L/1.5 mL of EP tube for electroporation of competent cells;
5) mixing 20 μ L linearized plasmid with 80 μ L competent cells, and standing on ice for 15 min;
6) adding the mixture into a pre-cooled sterile electric conversion cup (0.2cm), performing electric shock at 1500V, 25F and 200Q once, and adding 1mL of 1M D-sorbitol solution;
7) coating the mixture 150u L on an MD solid plate, and culturing at 30 ℃ for 2-3 days;
8) white colonies from the plates were picked and the correct transformants were selected. The individual colonies were spotted on 1, 2, 3, 4mg/mL (G418 geneticin) YPD plates, and single colonies on 4mg/mLG418 geneticin plates were selected for 250mL shake flask fermentations.
Example 3 recombinant Pichia Sharp flask fermentation
The high-copy transformants selected in example 2 and grown on 4mg/mL G418 geneticin plates were picked, inoculated into 250mL Erlenmeyer flasks containing 50mL YPD, and cultured at 30 ℃ for 1-2 days.
Adding 500 mu L YPD strain activation solution into 50mL BMGY medium, and culturing at 30 ℃ for 15-16 h. And (3) centrifuging the bacterial liquid, washing the bacteria by using normal saline, removing supernatant, collecting the bacteria, adding the bacteria into 50mL of BMMY culture medium for heavy suspension, and performing shaking table culture at 30 ℃. Samples were taken every 24h after inoculation and 1% methanol was added.
The yield of the recombinant saccharomyces cerevisiae A22 for expressing the glycoprotein with 22 truncated amino acids after fermentation for 96 hours can reach more than 157 mg/L. The results of 120h fermentation of recombinant yeast with different amino acid numbers truncated are shown in FIG. 2. Compared with recombinant pichia pastoris GS115-pPIC9K-Patatin-1(15mg/L) for expressing complete glycoprotein, the recombinant pichia pastoris for expressing glycoprotein (A22) with truncated amino acids at positions 2-23 has the advantages that the yield of the Patatin is increased by 1500 percent and can reach 225 mg/L. No production of Patatin was detected in recombinant Pichia pastoris with three amino acid positions, G36, N59 and D70, truncated. Therefore, the effect of the truncated amino acids at the 2 nd to 23 th positions on the synthesis of Patatin by pichia pastoris is better.
Example 4 purification of the Potato derived glycoprotein Patatin-1
The supernatant after fermentation of example 3 was collected by centrifugation, and the recombinant protein was purified using a nickel ion affinity chromatography column according to the protocol. And (3) buffer solution A: 20mM imidazole, 300mM NaCl, 50mM NaH2PO4The concentration of imidazole in the eluent B was adjusted to 500mM in the loading buffer A. Desalting with desalting column, wherein the desalting buffer solution is Tris-HCl buffer solution with pH of 7.4, and detecting the content of purified glycoproteins, namely, Patatin-1-t22, Patatin-1-t36, Patatin-1-t59 and Patatin-1-t 70.
Example 5 expression of the Potato derived glycoprotein Patatin-1 in Saccharomyces cerevisiae
The pXW55 plasmid containing glycoprotein Patatin-1 coding gene is constructed according to the same strategy of the embodiment 1-3, and the constructed recombinant plasmids are respectively expressed in saccharomyces cerevisiae BJ 5464. The expression of Patatin-1-t22 can be detected by culturing the recombinant saccharomyces cerevisiae in YNB culture medium for a period of time.
Example 6 expression of the Potato-derived glycoprotein Patatin-1 in Kluyveromyces lactis
The pKLAC1 plasmid containing the glycoprotein Patatin-1 coding gene is constructed according to the same strategy of the embodiment 1-3, and the constructed recombinant plasmids are respectively expressed in Kluyveromyces lactis GG 799. The recombinant Kluyveromyces lactis is cultured in YPD medium for a period of time, and the expression of Patatin-1-t22 can be detected.
Optionally, a mode of co-expression of the Patatin-1 and chaperonin can be adopted to effectively improve the soluble expression level of the Patatin-1, so that the subsequent separation and purification of the protein are facilitated.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
SEQUENCE LISTING
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Val Asp Gly Ala Val Ala Thr Val Gly Asp Pro Ala Leu Leu Ser Leu
145 150 155 160
Ser Val Ala Thr Arg Leu Ala Gln Glu Asp Pro Ala Phe Ser Ser Ile
165 170 175
Lys Ser Leu Asp Tyr Lys Gln Met Leu Leu Leu Ser Leu Gly Thr Gly
180 185 190
Thr Asn Ser Glu Phe Asp Lys Thr Tyr Thr Ala Gln Glu Ala Ala Lys
195 200 205
Trp Gly Pro Leu Arg Trp Leu Leu Ala Ile Gln Gln Met Thr Asn Ala
210 215 220
Ala Ser Ser Tyr Met Thr Asp Tyr Tyr Ile Ser Thr Val Phe Gln Ala
225 230 235 240
His His Ser Gln Asn Asn Tyr Leu Arg Val Gln Glu Asn Ala Leu Thr
245 250 255
Gly Thr Thr Thr Glu Met Asp Asp Ala Ser Glu Ala Asn Met Glu Leu
260 265 270
Leu Val Gln Val Gly Glu Thr Leu Leu Lys Lys Pro Val Ser Lys Asp
275 280 285
Ser Pro Glu Thr Tyr Glu Glu Ala Leu Lys Arg Phe Ala Lys Leu Leu
290 295 300
Ser Asp Arg Lys Lys Leu Arg Ala Asn Lys Ala Ser Tyr
305 310 315
<210> 6
<211> 1161
<212> DNA
<213> Artificial sequence
<400> 6
atggccacca ccaaatcttt tctgattctg ttttttatga ttctggccac cacctcttct 60
acctgtgcca aactggaaga aatggttacc gttctgtcta ttgatggtgg cggtattaaa 120
ggtattattc cggccattat tctggaattt ctggaaggtc agctgcaaga agttgataat 180
aataaagatg cccgtctggc cgattatttt gatgttattg gtggtacctc taccggtggt 240
ctgctgaccg ccatgattac caccccgaat gaaaataatc gtccgattgc cgctgccaaa 300
gattttgttc cgttttattt tgaacatggt ccgcatattt ttaattcttc tggtcgtccg 360
atttttggtc cgatgtatga tggtaattat ctgctgcaag ttctgcaaga aaaactgggt 420
gaaacccgtg ttcatcaagc cctgaccgaa gttgccattt cttcttttga tattaaaacc 480
aataaaccgg ttatttttac caaatctaat ctggccaaat ctccggaact ggatgccaaa 540
atgtatgata tttgttattc taccgccgct gccccgatgt attttccgcc gcattatttt 600
attacccata cctctaatgg tgatatttat gaatttaatc tggttgatgg tgccgttgcc 660
accgttggtg atccggccct gctgtctctg tctgttgcca cccgtctggc ccaagaagat 720
ccggcctttt cttctattaa atctctggac tataaacaga tgctgttgct gtctttgggt 780
accggtacca attctgaatt tgataaaacc tataccgccc aagaagccgc caaatggggt 840
ccgctgcgtt ggctgctggc cattcagcag atgaccaatg ccgcctcttc ttatatgacc 900
gattactata tttctaccgt ttttcaagcc catcattctc agaataatta tctgcgtgtt 960
caagaaaatg ccctgaccgg taccactacc gaaatggatg atgcctctga agccaatatg 1020
gaactgctgg ttcaagttgg tgaaaccctg ctgaaaaaac cggtttctaa agattctccg 1080
gaaacctatg aagaagccct gaaacgtttt gccaaactgc tgtctgatcg taaaaaactg 1140
cgtgccaata aagcctctta t 1161
<210> 7
<211> 1122
<212> DNA
<213> Artificial sequence
<400> 7
atgaaactgg aagaaatggt taccgttctg tctattgatg gtggcggtat taaaggtatt 60
attccggcca ttattctgga atttctggaa ggtcagctgc aagaagttga taataataaa 120
gatgcccgtc tggccgatta ttttgatgtt attggtggta cctctaccgg tggtctgctg 180
accgccatga ttaccacccc gaatgaaaat aatcgtccga ttgccgctgc caaagatttt 240
gttccgtttt attttgaaca tggtccgcat atttttaatt cttctggtcg tccgattttt 300
ggtccgatgt atgatggtaa ttatctgctg caagttctgc aagaaaaact gggtgaaacc 360
cgtgttcatc aagccctgac cgaagttgcc atttcttctt ttgatattaa aaccaataaa 420
ccggttattt ttaccaaatc taatctggcc aaatctccgg aactggatgc caaaatgtat 480
gatatttgtt attctaccgc cgctgccccg atgtattttc cgccgcatta ttttattacc 540
catacctcta atggtgatat ttatgaattt aatctggttg atggtgccgt tgccaccgtt 600
ggtgatccgg ccctgctgtc tctgtctgtt gccacccgtc tggcccaaga agatccggcc 660
ttttcttcta ttaaatctct ggactataaa cagatgctgt tgctgtcttt gggtaccggt 720
accaattctg aatttgataa aacctatacc gcccaagaag ccgccaaatg gggtccgctg 780
cgttggctgc tggccattca gcagatgacc aatgccgcct cttcttatat gaccgattac 840
tatatttcta ccgtttttca agcccatcat tctcagaata attatctgcg tgttcaagaa 900
aatgccctga ccggtaccac taccgaaatg gatgatgcct ctgaagccaa tatggaactg 960
ctggttcaag ttggtgaaac cctgctgaaa aaaccggttt ctaaagattc tccggaaacc 1020
tatgaagaag ccctgaaacg ttttgccaaa ctgctgtctg atcgtaaaaa actgcgtgcc 1080
aataaagcct cttatcacca ccaccaccac cactaaccta gg 1122