CN101475952B - Preparation of phycocyanin alpha subunit fluorescent protein combined with phycoerythrobilin - Google Patents
Preparation of phycocyanin alpha subunit fluorescent protein combined with phycoerythrobilin Download PDFInfo
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Abstract
The invention discloses a method for preparing phycoerythrobilin (PEB) combined phycocyanin alpha subunit type fluorescence protein, which prepares the PEB combined phycocyanin alpha subunit type fluorescence protein by using phycobiliprotein alpha subunit lyase to catalyze the covalent binding of phycoerythrobilin and phycocyanin alpha subunit type apoprotein. The inventive method uses a biology process to produce the PEB combined phycocyanin alpha subunit type fluorescence protein, which is an environmentally friendly production method. The phycocyanin alpha subunit type fluorescence protein can be applied in the field of biology and medicine functional material, specifically in the fluorescent probes in the biology and medicine detection field.
Description
Technical field
The invention belongs to chromoprotein material field in the biotechnology, be specifically related to the preparation method of the phycocyanin alpha subunit fluorescent protein of a kind of combined with phycoerythrobilin (PEB).
Technical background
Phycobiliprotein (phycobiliprotein) is the function ingredients that blue-green algae and red algae photosynthesis are caught the recovery compound.According to its Absorption and fluorescence spectrum feature, phycobiliprotein can be divided into phycoerythrin (phycoerythrin, abbreviation CPE), phycoerythrocyanin (pec) (phycoerythrocyanin, abbreviation PEC), Phycocyanins, C-(phycocyanin, be called for short CPC) and allophycocyanin (allophycocyanin is called for short APC).CPE, PEC, CPC and APC contain alpha and beta subunit, phycobilin in each subunit (phycobilin) is by the sulfydryl covalent attachment of thioether bond and phycobiliprotein apoprotein (apo-phycobiliprotein) cysteine residues, its kind and determine the spectral quality of phycobiliprotein with the interaction of apoprotein.Phycobilin and apoprotein covalent attachment form specific conformation, so that CPE mainly absorbs the approximately visible light of 560nm, launch the approximately fluorescence of 580nm; PEC absorbs the approximately visible light of 570nm, launches the approximately fluorescence of 630nm; CPC absorbs the approximately visible light of 620nm, launches the approximately fluorescence of 640nm; APC absorbs the approximately visible light of 650～660nm, launches the approximately fluorescence of 660～670nm.The prothetic group pigment of CPE combination is phycoerythrobilin (phycoerythrobilin is called for short PEB); The prothetic group pigment of the alpha subunit combination of PEC is the purple Choline of algae (phycoviolobilin is called for short PVB); The prothetic group pigment of the beta subunit combination of PEC is phycocyanobilin (phycocyanobilin is called for short PCB); The prothetic group pigment of CPC and APC combination all is phycocyanobilin PCB.
The alpha subunit of CPC can be produced by Recombinant organism (Tooley A J, Cai Y A, GlazerA N.Biosynthesis of a fluorescent cyanobacterial C-phycocyanin holo-alpha subunitin a heterologous host[J] .Proc Natl Acad Sci USA, 2001,98 (19): 10560～10565).We find, Phycocyanins, C-alpha subunit lyase not only can be combined with Phycocyanins, C-alpha subunit sequence of phycoerythrin by catalysis PCB, can also be combined with Phycocyanins, C-alpha subunit sequence of phycoerythrin by catalysis phycoerythrobilin PEB.By genetic engineering technique, Phycocyanins, C-alpha subunit sequence of phycoerythrin gene is cloned in expression vector, can obtain Phycocyanins, C-alpha subunit class apoprotein at e. coli expression; Phycocyanins, C-alpha subunit lyase genes, PEB biological enzyme synthetic gene are cloned in expression vector.Utilize above expression vector, by genetic engineering bacterium, can directly generate the phycocyanin alpha subunit fluorescent protein in conjunction with PEB.
The phycobiliprotein type fluorescence protein can be applied to food, makeup, medicine and bioengineering field.Having the spectral response curve different from natural Phycocyanins, C-in conjunction with the phycocyanin fluorescence protein of PEB can be Biological Detection and medical treatment and detects more more options are provided.The present invention is that Phycocyanins, C-class chromoprotein matter functional materials is applied to medicine and bioengineering field, and particularly detection field is laid a good foundation.
Summary of the invention
The object of the present invention is to provide a kind of method for preparing in conjunction with the phycocyanin alpha subunit fluorescent protein of PEB.It is to use Phycocyanins, C-alpha subunit to split and close catalysis phycoerythrobilin (PEB) and Phycocyanins, C-alpha subunit class apoprotein covalent attachment, and preparation is in conjunction with the phycocyanin alpha subunit fluorescent protein of PEB.The expression plasmid that will contain Phycocyanins, C-alpha subunit lyase genes, Phycocyanins, C-alpha subunit class apoprotein gene, phycoerythrobilin biosynthetic enzyme genes changes Host Strains successively over to, obtain corresponding engineering bacteria, the genetic engineering bacterium production that obtains by this method is in conjunction with the phycocyanin alpha subunit fluorescent protein of PEB.
Technical scheme of the present invention is such: a kind of preparation method of phycocyanin alpha subunit fluorescent protein of combined with phycoerythrobilin is characterized in that comprising the steps:
(1) uses gene engineering method, alpha subunit lyase genes or its homologous gene are cloned in the expression vector, obtain alpha subunit lyase expression plasmid;
(2) use gene engineering method, Phycocyanins, C-alpha subunit class apoprotein gene or its homologous gene are cloned in second expression vector, obtain the apoprotein expression plasmid;
(3) use gene engineering method, phycoerythrobilin biosynthetic enzyme genes hol and pebB or its homologous gene are cloned in the 3rd expression vector, obtain hol and pebB expression plasmid;
(4) with gene engineering method phycoerythrobilin biosynthetic enzyme genes pebA or its homologous gene are cloned in the 4th expression vector, obtain the pebA expression plasmid;
(5) change successively alpha subunit lyase expression plasmid, apoprotein expression plasmid, hol and pebB expression plasmid and pebA expression plasmid over to supporting Host Strains, after these expression plasmids all change Host Strains over to, obtain corresponding engineering bacteria, use this engineering bacteria can be produced combined with phycoerythrobilin by fermentation engineering phycocyanin alpha subunit fluorescent protein; After the fermentation, purify protein technology routinely, purifying obtains the phycocyanin alpha subunit fluorescent protein of corresponding combined with phycoerythrobilin.
Among the preparation method of the phycocyanin alpha subunit fluorescent protein of above-mentioned combined with phycoerythrobilin, described Host Strains is intestinal bacteria; Described alpha subunit lyase genes refer to Anabaena sp.PCC7120 in the gene of cpcE and cpcF dna homolog among cpcE and cpcF gene or the M.laminosus sp.PCC7603; Described Phycocyanins, C-alpha subunit class apoprotein gene refer to Anabaena sp.PCC7120 or M.laminosus sp.PCC7603 in the gene of cpcA homology.
The present invention compared with prior art has the following advantages:
1, do not use algae as raw material but utilize intestinal bacteria to produce phycocyanin alpha subunit fluorescent protein, the intestinal bacteria breeding is fast, can greatly shorten the cycle;
2, compare with algae, the Bacillus coli cells wall is easily broken, can save the energy in the purge process;
3, produce by intestinal bacteria, target protein content is high, and the His-tag mark is arranged, and does not almost have kin albumen in the system, and it is convenient to extract;
4, generation has the spectral response curve different from natural Phycocyanins, C-alpha subunit class chromoprotein in conjunction with the novel Phycocyanins, C-alpha subunit class chromoprotein of PEB, for development fluorescence phycobiliprotein class chromoprotein matter functional materials provides more more options;
Description of drawings
Fig. 1 is the Absorption and fluorescence spectrum in conjunction with the PEB phycocyanin alpha subunit fluorescent protein that generates under the CpcE/CpcF catalysis among the present invention, and wherein solid line is absorption spectrum, the dotted line fluorescence spectrum.
Embodiment
The present invention is further detailed explanation with example for the below.
Embodiment 1
(1) can find from GeneBank, the complete sequence of algae kind Anabaena sp.PCC7120 is finished after measured, and M.laminosus sp.PCC7603 partial sequence after measured.Anabaena sp.PCC7120 is numbered in GeneBank: BA000019, and the gene order of using is numbered in GeneBank among the M.laminosus sp.PCC7603: M34254, can find required gene from finding sequence; Part order-checking of Calothrix sp.PCC7601, the gene order of using is numbered AY363679 in GeneBank, can find required gene (pebA and pebB) from finding sequence.Pass through gene engineering method, Phycocyanins, C-alpha subunit lyase genes cpcE among the Anabaena sp.PCC7120 and cpcF are cloned among the pETDuet-1 of Novagen company purchase, the gained plasmid is pETDuet-cpcE-cpcF, in intestinal bacteria, can express Phycocyanins, C-alpha subunit lyase CpcE/CpcF, obtain Phycocyanins, C-alpha subunit lyase expression plasmid.
According to the gene order of finding among the GeneBank, design primer, utilized corresponding algae kind to extract total DNA as template, obtained required fragment by pcr amplification, by the restriction enzyme site that designs in the upstream and downstream primer, target fragment is changed on the restriction enzyme site same in the corresponding plasmid.
(2) the Phycocyanins, C-alpha subunit sequence of phycoerythrin gene among the Anabaena sp.PCC7120 is cloned among the pET30 of Novagen company purchase, the gained plasmid is pET30-cpcA, in intestinal bacteria, can express Phycocyanins, C-alpha subunit sequence of phycoerythrin, obtain the expression plasmid of Phycocyanins, C-alpha subunit sequence of phycoerythrin.
(3) phycoerythrobilin biosynthetic enzyme genes (hol and pebB) is cloned among the pCDFDuet-1 of Novagen company, the gained plasmid can be expressed HOl and PebB pCDFDuet-hol-pebB simultaneously in intestinal bacteria.
(4) phycoerythrobilin biosynthetic enzyme genes (pebA) is cloned among the pACYCDuet-1 of Novagen company, the gained plasmid can be expressed PebA pACYCDuet-pebA in intestinal bacteria.
Be that apoprotein gene cpcA is in pET30 between EcoRV and two restriction enzyme sites of XhoI; Lyase genes cpcE is in first multiple clone site in pETDuet, restriction enzyme site is between NcoI and the PstI, and lyase genes cpcF is in second multiple clone site in pETDuet, and restriction enzyme site is between EcoRV and the XhoI; The PEB synthase gene is 3 (hol, pebA and pebB), hol and pebB are in same carrier pCDFDuet-1, hol is between first multiple clone site NcoI and PstI, pebB is between second multiple clone site NdeI and XhoI, and pebA is in pACYCDuet-1 between second multiple clone site BglII and the XhoI.
The step of gene insertion vector is: according to gene order design primer, and minute upstream and downstream, a pair of; The upstream downstream primer is respectively with restriction enzyme site; Utilize corresponding algae kind to extract total DNA as template; Obtain required gene fragment through pcr amplification, the gained gene fragment is carried out electrophoresis, reclaim; The gained fragment is carried out enzyme and is cut with the restriction enzyme of design, simultaneously carrier is carried out enzyme with same restriction enzyme and cuts, and reclaims respectively gene fragment and carrier; Gene fragment and carrier roughly mix at 1: 1, connect certain hour (general 4～6 hours) under the effect of T4 ligase enzyme; Connect the product conversion and enter intestinal bacteria, utilize to contain antibiotic plate screening, the picking clone, checking correctly gets final product.
(5) change pETDuet-cpcE-cpcF, pET30-cpcA, pCDFDuet-hol-pebB and pACYCDuet-pebA over to intestinal bacteria, after these plasmids all change intestinal bacteria over to, obtain colibacillus engineering; This engineering bacteria is inoculated in the LB substratum of pH7.0,37 ℃ of shaking culture are to OD
600Be 0.5 to 0.8, add isopropylthio-β-D-galactoside (isoprophylthio-β-D-galactoside, be called for short IPTG) to final concentration be 1mmol/L, 20 ℃ to 37 ℃ vibrations were expressed approximately 12 hours, produced phycocyanin alpha subunit fluorescent protein PEB-CPC A; Centrifugal collection somatic cells, behind adding damping fluid, the smudge cells, centrifugal collection supernatant liquor passes through Ni
2+Affinity chromatography, can purify obtains corresponding phycocyanin fluorescence protein PEB-CPC A.It absorbs with fluorescence spectrum sees shown in the accompanying drawing 1, and wherein solid line is the absorption spectrum after the 500nm rayed, and dotted line is fluorescence spectrum.
The transform mode that the combination of used plasmid changes in the intestinal bacteria is as follows:
The single bacterium colony of picking coli strain BL21 (DE3) from the flat board of fresh coating is inoculated in 5mL LB substratum, and 37 ℃ of shaking culture are spent the night; Get 100 μ L saturated culture, the aseptic 5mL LB substratum that is forwarded to, 37 ℃ of shaking culture are to OD
600=0.3～0.4 o'clock, bacterium liquid is transferred in the aseptic centrifuge tube of 5mL, placed on ice 10 minutes; Centrifugal 1 minute (10000g, 4 ℃) supernatant discarded, the collecting precipitation thalline; 0.1mol/L CaCl with the 1mL precooling
2The resuspended thalline of sterile solution, centrifugal 30s (10000g, 4 ℃) removes supernatant, and bacterial sediment is with the 0.1mol/L CaCl of 100 μ L precoolings
2Resuspended, be positioned on ice, add a certain amount of plasmid that builds, ice bath was placed 30 minutes behind the mixing, 42 ℃ of heat-shocked 90s, ice bath add 300 μ L LB substratum after 5 minutes, 37 ℃ of low-speed oscillations were cultivated 45 minutes, aseptic being coated on contained on the corresponding antibiotic LB culture medium flat plate, is inverted in 37 ℃ of incubators to forming visible mono-clonal bacterial plaque.
Extract 3 left and right sides bacterium colonies, be inoculated into respectively 5ml and contain in the corresponding antibiotic LB substratum, shaking culture is to saturated; Therefrom getting respectively 100 μ L is forwarded to 5ml and contains in the corresponding antibiotic LB substratum; 37 ℃ of shaking culture are to OD
600During=0.5-0.7, ice bath approximately 30 minutes adds the IPTG abduction delivering; Lucifuge, 20 ℃, 150 rev/mins were expressed approximately 12 hours.Centrifugal collecting cell, it is resuspended that cell adds damping fluid; Survey its product fluorescence volume by spectrograph, choose the high bacterial strain of fluorescence quantum yield and carry out great expression.
Embodiment 2
(1) can find from GeneBank, the complete sequence of algae kind Anabaena sp.PCC7120 is finished after measured, and M.laminosus sp.PCC7603 partial sequence after measured.Anabaena sp.PCC7120 is numbered in GeneBank: BA000019, and the gene order of using is numbered in GeneBank among the M.laminosus sp.PCC7603: M34254, can find required gene from finding sequence; Part order-checking of Calothrix sp.PCC7601, the gene order of using is numbered AY363679 in GeneBank, can find required gene (pebA and pebB) from finding sequence.Pass through gene engineering method, Phycocyanins, C-alpha subunit lyase genes cpcE among the M.laminosus sp.PCC7603 and cpcF are cloned among the pETDuet-1 of Novagen company purchase, the gained plasmid is pETDuet-cpcE-cpcF, in intestinal bacteria, can express Phycocyanins, C-alpha subunit lyase CpcE/CpcF, obtain Phycocyanins, C-alpha subunit lyase expression plasmid.
According to the gene order of finding among the GeneBank, design primer, utilized corresponding algae kind to extract total DNA as template, obtained required fragment by pcr amplification, by the restriction enzyme site that designs in the upstream and downstream primer, target fragment is changed on the restriction enzyme site same in the corresponding plasmid.
(2) the Phycocyanins, C-alpha subunit sequence of phycoerythrin gene among the Anabaena sp.PCC7120 is cloned among the pET30 of Novagen company purchase, the gained plasmid is pET30-cpcA, in intestinal bacteria, can express Phycocyanins, C-alpha subunit sequence of phycoerythrin, obtain the expression plasmid of Phycocyanins, C-alpha subunit sequence of phycoerythrin.
(3) phycoerythrobilin biosynthetic enzyme genes (hol and pebB) is cloned among the pCDFDuet-1 of Novagen company, the gained plasmid can be expressed HOl and PebB pCDFDuet-hol-pebB simultaneously in intestinal bacteria.
(4) phycoerythrobilin biosynthetic enzyme genes (pebA) is cloned among the pACYCDuet-1 of Novagen company, the gained plasmid can be expressed PebA pACYCDuet-pebA in intestinal bacteria.
Be that apoprotein gene cpcA is in pET30 between EcoRV and two restriction enzyme sites of XhoI; Lyase genes cpcE is in first multiple clone site in pETDuet, restriction enzyme site is between NcoI and the PstI, and lyase genes cpcF is in second multiple clone site in pETDuet, and restriction enzyme site is between EcoRV and the XhoI; The PEB synthase gene is 3 (hol, pebA and pebB), hol and pebB are in same carrier pCDFDuet-1, hol is between first multiple clone site NcoI and PstI, pebB is between second multiple clone site NdeI and XhoI, and pebA is in pACYCDuet-1 between second multiple clone site BglII and the XhoI.
The step of gene insertion vector is: according to gene order design primer, and minute upstream and downstream, a pair of; The upstream downstream primer is respectively with restriction enzyme site; Utilize corresponding algae kind to extract total DNA as template; Obtain required gene fragment through pcr amplification, the gained gene fragment is carried out electrophoresis, reclaim; The gained fragment is carried out enzyme and is cut with the restriction enzyme of design, simultaneously carrier is carried out enzyme with same restriction enzyme and cuts, and reclaims respectively gene fragment and carrier; Gene fragment and carrier roughly mix at 1: 1, connect certain hour (general 4～6 hours) under the effect of T4 ligase enzyme; Connect the product conversion and enter intestinal bacteria, utilize to contain antibiotic plate screening, the picking clone, checking correctly gets final product.
(5) change pETDuet-cpcE-cpcF, pET30-cpcA, pCDFDuet-hol-pebB and pACYCDuet-pebA over to intestinal bacteria, after these plasmids all change intestinal bacteria over to, obtain colibacillus engineering; This engineering bacteria is inoculated in the LB substratum of pH7.0,37 ℃ of shaking culture are to OD
600Be 0.5 to 0.8, add isopropylthio-β-D-galactoside (isoprophylthio-β-D-galactoside, be called for short IPTG) to final concentration be 1mmol/L, 20 ℃ to 37 ℃ vibrations were expressed approximately 12 hours, produced phycocyanin alpha subunit fluorescent protein PEB-CPC A; Centrifugal collection somatic cells, behind adding damping fluid, the smudge cells, centrifugal collection supernatant liquor passes through Ni
2+Affinity chromatography, can purify obtains corresponding phycocyanin fluorescence protein PEB-CPC A.
The transform mode that the combination of used plasmid is changed in the intestinal bacteria is identical with embodiment 1.
Embodiment 3
(1) can find from GeneBank, the complete sequence of algae kind Anabaena sp.PCC7120 is finished after measured, and M.laminosus sp.PCC7603 partial sequence after measured.Anabaena sp.PCC7120 is numbered in GeneBank: BA000019, and the gene order of using is numbered in GeneBank among the M.laminosus sp.PCC7603: M34254, can find required gene from finding sequence; Part order-checking of Calothrix sp.PCC7601, the gene order of using is numbered AY363679 in GeneBank, can find required gene (pebA and pebB) from finding sequence.Pass through gene engineering method, Phycocyanins, C-alpha subunit lyase genes cpcE among the Anabaena sp.PCC7120 and cpcF are cloned among the pETDuet-1 of Novagen company purchase, the gained plasmid is pETDuet-cpcE-cpcF, in intestinal bacteria, can express Phycocyanins, C-alpha subunit lyase CpcE/CpcF, obtain Phycocyanins, C-alpha subunit lyase expression plasmid.
According to the gene order of finding among the GeneBank, design primer, utilized corresponding algae kind to extract total DNA as template, obtained required fragment by pcr amplification, by the restriction enzyme site that designs in the upstream and downstream primer, target fragment is changed on the restriction enzyme site same in the corresponding plasmid.
(2) the Phycocyanins, C-alpha subunit sequence of phycoerythrin gene among the M.laminosus sp.PCC7603 is cloned among the pET30 of Novagen company purchase, the gained plasmid is pET30-cpcA, in intestinal bacteria, can express Phycocyanins, C-alpha subunit sequence of phycoerythrin, obtain the expression plasmid of Phycocyanins, C-alpha subunit sequence of phycoerythrin.
(3) phycoerythrobilin biosynthetic enzyme genes (hol and pebB) is cloned among the pCDFDuet-1 of Novagen company, the gained plasmid can be expressed HOl and PebB pCDFDuet-hol-pebB simultaneously in intestinal bacteria.
(4) phycoerythrobilin biosynthetic enzyme genes (pebA) is cloned among the pACYCDuet-1 of Novagen company, the gained plasmid can be expressed PebA pACYCDuet-pebA in intestinal bacteria.
Be that apoprotein gene cpcA is in pET30 between EcoRV and two restriction enzyme sites of XhoI; Lyase genes cpcE is in first multiple clone site in pETDuet, restriction enzyme site is between NcoI and the PstI, and lyase genes cpcF is in second multiple clone site in pETDuet, and restriction enzyme site is between EcoRV and the XhoI; The PEB synthase gene is 3 (hol, pebA and pebB), hol and pebB are in same carrier pCDFDuet-1, hol is between first multiple clone site NcoI and PstI, pebB is between second multiple clone site NdeI and XhoI, and pebA is in pACYCDuet-l between second multiple clone site BglII and the XhoI.
The step of gene insertion vector is: according to gene order design primer, and minute upstream and downstream, a pair of; The upstream downstream primer is respectively with restriction enzyme site; Utilize corresponding algae kind to extract total DNA as template; Obtain required gene fragment through pcr amplification, the gained gene fragment is carried out electrophoresis, reclaim; The gained fragment is carried out enzyme and is cut with the restriction enzyme of design, simultaneously carrier is carried out enzyme with same restriction enzyme and cuts, and reclaims respectively gene fragment and carrier; Gene fragment and carrier roughly mix at 1: 1, connect certain hour (general 4～6 hours) under the effect of T4 ligase enzyme; Connect the product conversion and enter intestinal bacteria, utilize to contain antibiotic plate screening, the picking clone, checking correctly gets final product.
(5) change pETDuet-cpcE-cpcF, pET30-cpcA, pCDFDuet-hol-pebB and pACYCDuet-pebA over to intestinal bacteria, after these plasmids all change intestinal bacteria over to, obtain colibacillus engineering; This engineering bacteria is inoculated in the LB substratum of pH7.0,37 ℃ of shaking culture are to OD
600Be 0.5 to 0.8, add isopropylthio-β-D-galactoside (isoprophylthio-β-D-galactoside, be called for short IPTG) to final concentration be 1mmol/L, 20 ℃ to 37 ℃ vibrations were expressed approximately 12 hours, produced phycocyanin alpha subunit fluorescent protein PEB-CPC A; Centrifugal collection somatic cells, behind adding damping fluid, the smudge cells, centrifugal collection supernatant liquor passes through Ni
2+Affinity chromatography, can purify obtains corresponding phycocyanin fluorescence protein PEB-CPC A.
The transform mode that the combination of used plasmid is changed in the intestinal bacteria is identical with embodiment 1.
Embodiment 4
(1) can find from GeneBank, the complete sequence of algae kind Anabaena sp.PCC7120 is finished after measured, and M.laminosus sp.PCC7603 partial sequence after measured.Anabaena sp.PCC7120 is numbered in GeneBank: BA000019, and the gene order of using is numbered in GeneBank among the M.laminosus sp.PCC7603: M34254, can find required gene from finding sequence; Part order-checking of Calothrix sp.PCC7601, the gene order of using is numbered AY363679 in GeneBank, can find required gene (pebA and pebB) from finding sequence.Pass through gene engineering method, Phycocyanins, C-alpha subunit lyase genes cpcE among the M.laminosus sp.PCC7603 and cpcF are cloned among the pETDuet-1 of Novagen company purchase, the gained plasmid is pETDuet-cpcE-cpcF, in intestinal bacteria, can express Phycocyanins, C-alpha subunit lyase CpcE/CpcF, obtain Phycocyanins, C-alpha subunit lyase expression plasmid.
According to the gene order of finding among the GeneBank, design primer, utilized corresponding algae kind to extract total DNA as template, obtained required fragment by pcr amplification, by the restriction enzyme site that designs in the upstream and downstream primer, target fragment is changed on the restriction enzyme site same in the corresponding plasmid.
(2) the Phycocyanins, C-alpha subunit sequence of phycoerythrin gene among the M.laminosus sp.PCC7603 is cloned among the pET30 of Novagen company purchase, the gained plasmid is pET30-cpcA, in intestinal bacteria, can express Phycocyanins, C-alpha subunit sequence of phycoerythrin, obtain the expression plasmid of Phycocyanins, C-alpha subunit sequence of phycoerythrin.
(3) phycoerythrobilin biosynthetic enzyme genes (hol and pebB) is cloned among the pCDFDuet-1 of Novagen company, the gained plasmid can be expressed HOl and PebB pCDFDuet-hol-pebB simultaneously in intestinal bacteria.
(4) phycoerythrobilin biosynthetic enzyme genes (pebA) is cloned among the pACYCDuet-1 of Novagen company, the gained plasmid can be expressed PebA pACYCDuet-pebA in intestinal bacteria.
Be that apoprotein gene cpcA is in pET30 between EcoRV and two restriction enzyme sites of XhoI; Lyase genes cpcE is in first multiple clone site in pETDuet, restriction enzyme site is between NcoI and the PstI, and lyase genes cpcF is in second multiple clone site in pETDuet, and restriction enzyme site is between EcoRV and the XhoI; The PEB synthase gene is 3 (hol, pebA and pebB), hol and pebB are in same carrier pCDFDuet-1, hol is between first multiple clone site NcoI and PstI, pebB is between second multiple clone site NdeI and XhoI, and pebA is in pACYCDuet-1 between second multiple clone site BglII and the XhoI.
The step of gene insertion vector is: according to gene order design primer, and minute upstream and downstream, a pair of; The upstream downstream primer is respectively with restriction enzyme site; Utilize corresponding algae kind to extract total DNA as template; Obtain required gene fragment through pcr amplification, the gained gene fragment is carried out electrophoresis, reclaim; The gained fragment is carried out enzyme and is cut with the restriction enzyme of design, simultaneously carrier is carried out enzyme with same restriction enzyme and cuts, and reclaims respectively gene fragment and carrier; Gene fragment and carrier roughly mix at 1: 1, connect certain hour (general 4～6 hours) under the effect of T4 ligase enzyme; Connect the product conversion and enter intestinal bacteria, utilize to contain antibiotic plate screening, the picking clone, checking correctly gets final product.
(5) change pETDuet-cpcE-cpcF, pET30-cpcA, pCDFDuet-hol-pebB and pACYCDuet-pebA over to intestinal bacteria, after these plasmids all change intestinal bacteria over to, obtain colibacillus engineering; This engineering bacteria is inoculated in the LB substratum of pH7.0,37 ℃ of shaking culture are to OD
600Be 0.5 to 0.8, add isopropylthio-β-D-galactoside (isoprophylthio-β-D-galactoside, be called for short IPTG) to final concentration be 1mmol/L, 20 ℃ to 37 ℃ vibrations were expressed approximately 12 hours, produced phycocyanin alpha subunit fluorescent protein PEB-CPC A; Centrifugal collection somatic cells, behind adding damping fluid, the smudge cells, centrifugal collection supernatant liquor passes through Ni
2+Affinity chromatography, can purify obtains corresponding phycocyanin fluorescence protein PEB-CPC A.
The transform mode that the combination of used plasmid is changed in the intestinal bacteria is identical with embodiment 1.
Embodiment 5
(1) can find from GeneBank, the complete sequence of algae kind Anabaena sp.PCC7120 is finished after measured, and M.laminosus sp.PCC7603 partial sequence after measured.Anabaena sp.PCC7120 is numbered in GeneBank: BA000019, and the gene order of using is numbered in GeneBank among the M.laminosus sp.PCC7603: M34254, can find required gene from finding sequence; Part order-checking of Calothrix sp.PCC7601, the gene order of using is numbered AY363679 in GeneBank, can find required gene (pebA and pebB) from finding sequence.Pass through gene engineering method, Phycocyanins, C-alpha subunit lyase genes cpcE among the Anabaena sp.PCC7120 and cpcF are cloned among the pETDuet-1 of Novagen company purchase, the gained plasmid is pETDuet-cpcE-cpcF, in intestinal bacteria, can express Phycocyanins, C-alpha subunit lyase CpcE/CpcF, obtain Phycocyanins, C-alpha subunit lyase expression plasmid.
According to the gene order of finding among the GeneBank, design primer, utilized corresponding algae kind to extract total DNA as template, obtained required fragment by pcr amplification, by the restriction enzyme site that designs in the upstream and downstream primer, target fragment is changed on the restriction enzyme site same in the corresponding plasmid.
(2) the Phycocyanins, C-alpha subunit sequence of phycoerythrin gene among the Anabaena sp.PCC7120 is cloned among the pCOLADuet-1 of Novagen company purchase, the gained plasmid is pCOLADuet-cpcA, in intestinal bacteria, can express Phycocyanins, C-alpha subunit sequence of phycoerythrin, obtain the expression plasmid of Phycocyanins, C-alpha subunit sequence of phycoerythrin.
(3) phycoerythrobilin biosynthetic enzyme genes (hol and pebB) is cloned among the pCDFDuet-1 of Novagen company, the gained plasmid can be expressed HOl and PebB pCDFDuet-hol-pebB simultaneously in intestinal bacteria.
(4) phycoerythrobilin biosynthetic enzyme genes (pebA) is cloned among the pACYCDuet-1 of Novagen company, the gained plasmid can be expressed PebA pACYCDuet-pebA in intestinal bacteria.
The fragment that is apoprotein gene cpcA is in pCOLADuet-1 between the EcoRI of first multiple clone site and two restriction enzyme sites of PstI; Lyase genes cpcE is in first multiple clone site in pETDuet, restriction enzyme site is between NcoI and the PstI, and lyase genes cpcF is in second multiple clone site in pETDuet, and restriction enzyme site is between EcoRV and the XhoI; The PEB synthase gene is 3 (hol, pebA and pebB), hol and pebB are in same carrier pCDFDuet-1, hol is between first multiple clone site NcoI and PstI, pebB is between second multiple clone site NdeI and XhoI, and pebA is in pACYCDuet-1 between second multiple clone site BglII and the XhoI.
The step of gene insertion vector is: according to gene order design primer, and minute upstream and downstream, a pair of; The upstream downstream primer is respectively with restriction enzyme site; Utilize corresponding algae kind to extract total DNA as template; Obtain required gene fragment through pcr amplification, the gained gene fragment is carried out electrophoresis, reclaim; The gained fragment is carried out enzyme and is cut with the restriction enzyme of design, simultaneously carrier is carried out enzyme with same restriction enzyme and cuts, and reclaims respectively gene fragment and carrier; Gene fragment and carrier roughly mix at 1: 1, connect certain hour (general 4～6 hours) under the effect of T4 ligase enzyme; Connect the product conversion and enter intestinal bacteria, utilize to contain antibiotic plate screening, the picking clone, checking correctly gets final product.
(5) change pETDuet-cpcE-cpcF, pCOLADuet-cpcA, pCDFDuet-hol-pebB and pACYCDuet-pebA over to intestinal bacteria, after these plasmids all change intestinal bacteria over to, obtain colibacillus engineering; This engineering bacteria is inoculated in the LB substratum of pH7.0,37 ℃ of shaking culture are to OD
600Be 0.5 to 0.8, add isopropylthio-β-D-galactoside (isoprophyl thio-β-D-galactoside, be called for short IPTG) to final concentration be 1mmol/L, 20 ℃ to 37 ℃ vibrations were expressed approximately 12 hours, produced phycocyanin alpha subunit fluorescent protein PEB-CPC A; Centrifugal collection somatic cells, behind adding damping fluid, the smudge cells, centrifugal collection supernatant liquor passes through Ni
2+Affinity chromatography, can purify obtains corresponding phycocyanin fluorescence protein PEB-CPC A.
The transform mode that the combination of used plasmid is changed in the intestinal bacteria is identical with embodiment 1.
Embodiment 6
(1) can find from GeneBank, the complete sequence of algae kind Anabaena sp.PCC7120 is finished after measured, and M.laminosus sp.PCC7603 partial sequence after measured.Anabaena sp.PCC7120 is numbered in GeneBank: BA000019, and the gene order of using is numbered in GeneBank among the M.laminosus sp.PCC7603: M34254, can find required gene from finding sequence; Part order-checking of Calothrix sp.PCC7601, the gene order of using is numbered AY363679 in GeneBank, can find required gene (pebA and pebB) from finding sequence.Pass through gene engineering method, Phycocyanins, C-alpha subunit lyase genes cpcE among the M.laminosus sp.PCC7603 and cpcF are cloned among the pETDuet-1 of Novagen company purchase, the gained plasmid is pETDuet-cpcE-cpcF, in intestinal bacteria, can express Phycocyanins, C-alpha subunit lyase CpcE/CpcF, obtain Phycocyanins, C-alpha subunit lyase expression plasmid.
According to the gene order of finding among the GeneBank, design primer, utilized corresponding algae kind to extract total DNA as template, obtained required fragment by pcr amplification, by the restriction enzyme site that designs in the upstream and downstream primer, target fragment is changed on the restriction enzyme site same in the corresponding plasmid.
(2) the Phycocyanins, C-alpha subunit sequence of phycoerythrin gene among the Anabaena sp.PCC7120 is cloned among the pCOLADuet-1 of Novagen company purchase, the gained plasmid is pCOLADuet-cpcA, in intestinal bacteria, can express Phycocyanins, C-alpha subunit sequence of phycoerythrin, obtain the expression plasmid of Phycocyanins, C-alpha subunit sequence of phycoerythrin.
(3) phycoerythrobilin biosynthetic enzyme genes (hol and pebB) is cloned among the pCDFDuet-1 of Novagen company, the gained plasmid can be expressed HOl and PebB pCDFDuet-hol-pebB simultaneously in intestinal bacteria.
(4) phycoerythrobilin biosynthetic enzyme genes (pebA) is cloned among the pACYCDuet-1 of Novagen company, the gained plasmid can be expressed PebA pACYCDuet-pebA in intestinal bacteria.
The fragment that is apoprotein gene cpcA is in pCOLADuet-1 between the EcoRI of first multiple clone site and two restriction enzyme sites of PstI; Lyase genes cpcE is in first multiple clone site in pETDuet, restriction enzyme site is between NcoI and the PstI, and lyase genes cpcF is in second multiple clone site in pETDuet, and restriction enzyme site is between EcoRV and the XhoI; The PEB synthase gene is 3 (hol, pebA and pebB), hol and pebB are in same carrier pCDFDuet-1, hol is between first multiple clone site NcoI and PstI, pebB is between second multiple clone site NdeI and XhoI, and pebA is in pACYCDuet-1 between second multiple clone site BglII and the XhoI.
The step of gene insertion vector is: according to gene order design primer, and minute upstream and downstream, a pair of; The upstream downstream primer is respectively with restriction enzyme site; Utilize corresponding algae kind to extract total DNA as template; Obtain required gene fragment through pcr amplification, the gained gene fragment is carried out electrophoresis, reclaim; The gained fragment is carried out enzyme and is cut with the restriction enzyme of design, simultaneously carrier is carried out enzyme with same restriction enzyme and cuts, and reclaims respectively gene fragment and carrier; Gene fragment and carrier roughly mix at 1: 1, connect certain hour (general 4～6 hours) under the effect of T4 ligase enzyme; Connect the product conversion and enter intestinal bacteria, utilize to contain antibiotic plate screening, the picking clone, checking correctly gets final product.
(5) change pETDuet-cpcE-cpcF, pCOLADuet-cpcA, pCDFDuet-hol-pebB and pACYCDuet-pebA over to intestinal bacteria, after these plasmids all change intestinal bacteria over to, obtain colibacillus engineering; This engineering bacteria is inoculated in the LB substratum of pH7.0,37 ℃ of shaking culture are to OD
600Be 0.5 to 0.8, add isopropylthio-β-D-galactoside (isoprophyl thio-β-D-galactoside, be called for short IPTG) to final concentration be 1mmol/L, 20 ℃ to 37 ℃ vibrations were expressed approximately 12 hours, produced phycocyanin alpha subunit fluorescent protein PEB-CPC A; Centrifugal collection somatic cells, behind adding damping fluid, the smudge cells, centrifugal collection supernatant liquor passes through Ni
2+Affinity chromatography, can purify obtains corresponding phycocyanin fluorescence protein PEB-CPC A.
The transform mode that the combination of used plasmid is changed in the intestinal bacteria is identical with embodiment 1.
Embodiment 7
(1) can find from GeneBank, the complete sequence of algae kind Anabaena sp.PCC7120 is finished after measured, and M.laminosus sp.PCC7603 partial sequence after measured.Anabaena sp.PCC7120 is numbered in GeneBank: BA000019, and the gene order of using is numbered in GeneBank among the M.laminosus sp.PCC7603: M34254, can find required gene from finding sequence; Part order-checking of Calothrix sp.PCC7601, the gene order of using is numbered AY363679 in GeneBank, can find required gene (pebA and pebB) from finding sequence.Pass through gene engineering method, Phycocyanins, C-alpha subunit lyase genes cpcE among the Anabaena sp.PCC7120 and cpcF are cloned among the pETDuet-1 of Novagen company purchase, the gained plasmid is pETDuet-cpcE-cpcF, in intestinal bacteria, can express Phycocyanins, C-alpha subunit lyase CpcE/CpcF, obtain Phycocyanins, C-alpha subunit lyase expression plasmid.
According to the gene order of finding among the GeneBank, design primer, utilized corresponding algae kind to extract total DNA as template, obtained required fragment by pcr amplification, by the restriction enzyme site that designs in the upstream and downstream primer, target fragment is changed on the restriction enzyme site same in the corresponding plasmid.
(2) the Phycocyanins, C-alpha subunit sequence of phycoerythrin gene among the M.laminosus sp.PCC7603 is cloned among the pCOLADuet-1 of Novagen company purchase, the gained plasmid is pCOLADuet-cpcA, in intestinal bacteria, can express Phycocyanins, C-alpha subunit sequence of phycoerythrin, obtain the expression plasmid of Phycocyanins, C-alpha subunit sequence of phycoerythrin.
(3) phycoerythrobilin biosynthetic enzyme genes (hol and pebB) is cloned among the pCDFDuet-1 of Novagen company, the gained plasmid can be expressed HOl and PebB pCDFDuet-hol-pebB simultaneously in intestinal bacteria.
(4) phycoerythrobilin biosynthetic enzyme genes (pebA) is cloned among the pACYCDuet-1 of Novagen company, the gained plasmid can be expressed PebA pACYCDuet-pebA in intestinal bacteria.
The fragment that is apoprotein gene cpcA is in pCOLADuet-1 between the EcoRI of first multiple clone site and two restriction enzyme sites of PstI; Lyase genes cpcE is in first multiple clone site in pETDuet, restriction enzyme site is between NcoI and the PstI, and lyase genes cpcF is in second multiple clone site in pETDuet, and restriction enzyme site is between EcoRV and the XhoI; The PEB synthase gene is 3 (hol, pebA and pebB), hol and pebB are in same carrier pCDFDuet-1, hol is between first multiple clone site NcoI and PstI, pebB is between second multiple clone site NdeI and XhoI, and pebA is in pACYCDuet-1 between second multiple clone site BglII and the XhoI.
The step of gene insertion vector is: according to gene order design primer, and minute upstream and downstream, a pair of; The upstream downstream primer is respectively with restriction enzyme site; Utilize corresponding algae kind to extract total DNA as template; Obtain required gene fragment through pcr amplification, the gained gene fragment is carried out electrophoresis, reclaim; The gained fragment is carried out enzyme and is cut with the restriction enzyme of design, simultaneously carrier is carried out enzyme with same restriction enzyme and cuts, and reclaims respectively gene fragment and carrier; Gene fragment and carrier roughly mix at 1: 1, connect certain hour (general 4～6 hours) under the effect of T4 ligase enzyme; Connect the product conversion and enter intestinal bacteria, utilize to contain antibiotic plate screening, the picking clone, checking correctly gets final product.
(5) change pETDuet-cpcE-cpcF, pCOLADuet-cpcA, pCDFDuet-hol-pebB and pACYCDuet-pebA over to intestinal bacteria, after these plasmids all change intestinal bacteria over to, obtain colibacillus engineering; This engineering bacteria is inoculated in the LB substratum of pH7.0,37 ℃ of shaking culture are to OD
600Be 0.5 to 0.8, add isopropylthio-β-D-galactoside (isoprophyl thio-β-D-galactoside, be called for short IPTG) to final concentration be 1mmol/L, 20 ℃ to 37 ℃ vibrations were expressed approximately 12 hours, produced phycocyanin alpha subunit fluorescent protein PEB-CPC A; Centrifugal collection somatic cells, behind adding damping fluid, the smudge cells, centrifugal collection supernatant liquor passes through Ni
2+Affinity chromatography, can purify obtains corresponding phycocyanin fluorescence protein PEB-CPC A.
The transform mode that the combination of used plasmid is changed in the intestinal bacteria is identical with embodiment 1.
Embodiment 8
(1) can find from GeneBank, the complete sequence of algae kind Anabaena sp.PCC7120 is finished after measured, and M.laminosus sp.PCC7603 partial sequence after measured.Anabaena sp.PCC7120 is numbered in GeneBank: BA000019, and the gene order of using is numbered in GeneBank among the M.laminosus sp.PCC7603: M34254, can find required gene from finding sequence; Part order-checking of Calothrix sp.PCC7601, the gene order of using is numbered AY363679 in GeneBank, can find required gene (pebA and pebB) from finding sequence.Pass through gene engineering method, Phycocyanins, C-alpha subunit lyase genes cpcE among the M.laminosus sp.PCC7603 and cpcF are cloned among the pETDuet-1 of Novagen company purchase, the gained plasmid is pETDuet-cpcE-cpcF, in intestinal bacteria, can express Phycocyanins, C-alpha subunit lyase CpcE/CpcF, obtain Phycocyanins, C-alpha subunit lyase expression plasmid.
According to the gene order of finding among the GeneBank, the design primer utilizes corresponding algae kind to extract total DNA as template, obtains required fragment by pcr amplification, by the restriction enzyme site that designs in the upstream and downstream primer, target fragment is changed on the restriction enzyme site same in the corresponding plasmid.
(2) the Phycocyanins, C-alpha subunit sequence of phycoerythrin gene among the M.laminosus sp.PCC7603 is cloned among the pCOLADuet-1 of Novagen company purchase, the gained plasmid is pCOLADuet-cpcA, in intestinal bacteria, can express Phycocyanins, C-alpha subunit sequence of phycoerythrin, obtain the expression plasmid of Phycocyanins, C-alpha subunit sequence of phycoerythrin.
(3) phycoerythrobilin biosynthetic enzyme genes (hol and pebB) is cloned among the pCDFDuet-1 of Novagen company, the gained plasmid can be expressed HOl and PebB pCDFDuet-hol-pebB simultaneously in intestinal bacteria.
(4) phycoerythrobilin biosynthetic enzyme genes (pebA) is cloned among the pACYCDuet-1 of Novagen company, the gained plasmid can be expressed PebA pACYCDuet-pebA in intestinal bacteria.
The fragment that is apoprotein gene cpcA is in pCOLADuet-1 between the EcoRI of first multiple clone site and two restriction enzyme sites of PstI; Lyase genes cpcE is in first multiple clone site in pETDuet, restriction enzyme site is between NcoI and the PstI, and lyase genes cpcF is in second multiple clone site in pETDuet, and restriction enzyme site is between EcoRV and the XhoI; The PEB synthase gene is 3 (hol, pebA and pebB), hol and pebB are in same carrier pCDFDuet-1, hol is between first multiple clone site NcoI and PstI, pebB is between second multiple clone site NdeI and XhoI, and pebA is in pACYCDuet-1 between second multiple clone site BglII and the XhoI.
The step of gene insertion vector is: according to gene order design primer, and minute upstream and downstream, a pair of; The upstream downstream primer is respectively with restriction enzyme site; Utilize corresponding algae kind to extract total DNA as template; Obtain required gene fragment through pcr amplification, the gained gene fragment is carried out electrophoresis, reclaim; The gained fragment is carried out enzyme and is cut with the restriction enzyme of design, simultaneously carrier is carried out enzyme with same restriction enzyme and cuts, and reclaims respectively gene fragment and carrier; Gene fragment and carrier roughly mix at 1: 1, connect certain hour (general 4～6 hours) under the effect of T4 ligase enzyme; Connect the product conversion and enter intestinal bacteria, utilize to contain antibiotic plate screening, the picking clone, checking correctly gets final product.
(5) change pETDuet-cpcE-cpcF, pCOLADuet-cpcA, pCDFDuet-hol-pebB and pACYCDuet-pebA over to intestinal bacteria, after these plasmids all change intestinal bacteria over to, obtain colibacillus engineering; This engineering bacteria is inoculated in the LB substratum of pH 7.0,37 ℃ of shaking culture are to OD
600Be 0.5 to 0.8, add isopropylthio-β-D-galactoside (isoprophyl thio-β-D-galactoside, be called for short IPTG) to final concentration be 1mmol/L, 20 ℃ to 37 ℃ vibrations were expressed approximately 12 hours, produced phycocyanin alpha subunit fluorescent protein PEB-CPC A; Centrifugal collection somatic cells, behind adding damping fluid, the smudge cells, centrifugal collection supernatant liquor passes through Ni
2+Affinity chromatography, can purify obtains corresponding phycocyanin fluorescence protein PEB-CPC A.
The transform mode that the combination of used plasmid is changed in the intestinal bacteria is identical with embodiment 1.
Above-mentioned preparation method is applicable to adopt the alpha subunit lyase in the various blue-green algaes, Phycocyanins, C-alpha subunit class apoprotein and PEB biosynthetic enzyme genes or its homologous gene prepare the phycocyanin alpha subunit fluorescent protein in conjunction with PEB, but relate to the disclosed problem of microbial strains, the present invention has only selected openly the blue-green algae Anabaena sp.PCC7120 of complete sequence and openly the blue-green algae M.laminosus sp.PCC7603 of partial sequence among the GenBank, Calothrixsp.PCC7601 is that example is illustrated the inventive method, and those skilled in the art can adopt other raw material to implement the present invention according to above-mentioned disclosed content.
Claims (2)
1. the preparation method of the phycocyanin alpha subunit fluorescent protein of a combined with phycoerythrobilin is characterized in that comprising the steps:
(1) uses gene engineering method, with alpha subunit lyase genes: among the Anabaena sp. PCC7120 among cpcE and cpcF gene or the M.Laminosus sp.PCC7603 cpcE and cpcF gene clone in expression vector, obtain alpha subunit lyase expression plasmid;
(2) use gene engineering method, with Phycocyanins, C-alpha subunit class apoprotein gene: the cpcA gene clone obtains the apoprotein expression plasmid among Anabaena sp. PCC7120 or the M.Laminosus sp.PCC7603 in the second expression vector;
(3) use gene engineering method, phycoerythrobilin biosynthetic enzyme genes hol and pebB are cloned in the 3rd expression vector, obtain hol and pebB expression plasmid;
(4) with gene engineering method phycoerythrobilin biosynthetic enzyme genes pebA is cloned in the 4th expression vector, obtains the pebA expression plasmid;
(5) change successively alpha subunit lyase expression plasmid, apoprotein expression plasmid, hol and pebB expression plasmid and pebA expression plasmid over to supporting Host Strains, after these expression plasmids all change Host Strains over to, obtain corresponding engineering bacteria, use this engineering bacteria can produce combined with phycoerythrobilin by fermentation engineering phycocyanin alpha subunit fluorescent protein; After the fermentation, purify protein technology routinely, purifying obtains the phycocyanin alpha subunit fluorescent protein of corresponding combined with phycoerythrobilin;
Described phycoerythrobilin biosynthetic enzyme genes: wherein the pebB gene in the step (3) and the pebA gene in the step (4) derive from Calothrix sp.PCC7601.
2. method according to claim 1 is characterized in that, described Host Strains is intestinal bacteria.
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| CN1434057A (en) * | 2003-02-26 | 2003-08-06 | 华中科技大学 | Process for preparing reversible photochromic biliprotein |
| CN1443849A (en) * | 2003-04-11 | 2003-09-24 | 华中科技大学 | Preparation method of reversible photochromic biliprotein |
| US6740507B2 (en) * | 2001-07-31 | 2004-05-25 | The Regents Of The University Of California | Engineering of living cells for the expression of holo-phycobiliprotein-based constructs |
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| US6740507B2 (en) * | 2001-07-31 | 2004-05-25 | The Regents Of The University Of California | Engineering of living cells for the expression of holo-phycobiliprotein-based constructs |
| CN1434057A (en) * | 2003-02-26 | 2003-08-06 | 华中科技大学 | Process for preparing reversible photochromic biliprotein |
| CN1443849A (en) * | 2003-04-11 | 2003-09-24 | 华中科技大学 | Preparation method of reversible photochromic biliprotein |
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| Keiko Mukougawa等.Metabolic engineering to produce phytochromes with phytochromobilin,phycocyanobilin, or phycoerythrobilin chromophore in Escherichia coli.FEBS Letters.Elsevier B.V.,2006,5801333-1338. * |
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