EP1180145A1 - Biosynthese de proteines etrangeres au moyen de microalgues transformees - Google Patents

Biosynthese de proteines etrangeres au moyen de microalgues transformees

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Publication number
EP1180145A1
EP1180145A1 EP00911471A EP00911471A EP1180145A1 EP 1180145 A1 EP1180145 A1 EP 1180145A1 EP 00911471 A EP00911471 A EP 00911471A EP 00911471 A EP00911471 A EP 00911471A EP 1180145 A1 EP1180145 A1 EP 1180145A1
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EP
European Patent Office
Prior art keywords
microalgae
protein
gene
transformed
chlorella
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EP00911471A
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German (de)
English (en)
Inventor
Tae-Jin Choi
Young-Tae Kim
Dae-Hyun Kim
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Algenetech
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Algenetech
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
    • A01G33/00—Cultivation of seaweed or algae
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management

Definitions

  • the present invention relates to a method for biosynthesis of foreign protein using transformed microalgae. More particularly, it relates to a method for biosynthesis of foreign proteins by transforming microalgae protoplasts with DNA vector containing intended foreign protein gene, and then culturing it in a large scale.
  • Escherichia coli is the most widely used heterologous expression system, but the bacterium has some limitations including; i) poor or no expression of certain proteins, ii) some recombinant proteins lack biological activity, iii) some recombinant proteins are toxic to Escherichia coli, and iv) some recombinant proteins form insoluble inclusion bodies. Similar problems can occur with yeast expression systems. Cultured mammalian and insect cells have been used to solve these problems, but these systems can be expensive because of the cost of media, equipment and requirement for extensive purification procedures.
  • microalgae expression system was more economical than cell culture or animal or plant expression system because the microalgae had simpler metabolic pathway than those of animals or plants had and could be cultured in a large scale using an aquarium with light and carbon dioxide.
  • microalgae has post-translational modification process unlike Escherichia coli indicates that the biological activity of foreign protein expressed in microalgae should be more similar to that of naturally occurring protein. Under this circumstance, we intended to develop microalgae overexpression system for producing foreign proteins.
  • Jarvis and Brown described the transient expression of luciferase in protoplasts of Chlorella ellipsoidea (Jarvis, E. E., and Brown, L. M. 1991. Transient expression of firefly luciferase in protoplasts of the green alga Chlorella ellipsoidea, Current Genetics 19, 317-321) and Dawson et al. found that nitrate reductase- deficient mutants of Chlorella sorokiniana could be rescued by transforming them with nitrate reductase gene isolated from Chlorella vulgaris(Oawson, H.
  • the object of present invention is to provide a method for the stable expression of a foreign protein in a microalgae overexpression system.
  • the method of the present invention is characterized in that which comprises the steps of; (i) obtaining protoplast of microalgae; (ii) preparing a vector containing genes coding desired proteins, said genes originated from organisms other than microalgae; (iii) introducing the vector into the protoplast to give transformed protoplast and (iv) culturing the transformed microalgae to produce the desired protein. Also, the method of the present invention could further comprise another step of selecting transformed cells with antibiotics between step (iii) and step (iv) other than the above steps.
  • Figure 1 shows photographs of Chlorella ellipsoidea cells stained with calcofluor white before (a) and after (b) enzyme treatment for cell wall removal observed by fluorescent microscope.
  • Figure 2 shows a photograph of expression of GFP in transformed Chlorella ellipsoidea observed by fluorescent microscope.
  • Figure 3 shows a schematic diagram of the transformation vector pCTV.
  • Figure 4 shows the growth of transformed and non-transformed Chlorella ellipsoidea cultured in medium containing or not containing phleomycin.
  • Figure 5 shows the results of PCR amplification and Southern blot analysis of flounder growth hormone(hereinafter fGH) gene and Sh ble gene inserted into genomic DNA of transformed Chlorella ellipsoidea.
  • panel A shows the result of PCR amplification and Southern blot analysis for fGH
  • panel B shows the result of PCR amplification and Southern blot analysis of Sh ble
  • lane 1 shows molecular weight size marker
  • lane 2 shows transformed Chlorella ellipsoidea
  • lane 3 shows non-transformed Chlorella ellipsoidea
  • lane 4 shows fGH and Sh ble gene fragments digested from pBluescript SK+.
  • Figure 6 shows the result of Western blot analysis of fGH expressed in tranformed Chlorella ellipsoidea.
  • lane 1 shows molecular weight size marker and lane 2 shows the Glutathione-S-transferase(hereinafter, GST)-fGH fusion protein used for production of antibody and
  • lane 3 shows the total protein isolated from non-transformed Chlorella ellipsoidea and
  • lane 4 shows the total protein isolated from transformed Chlorella ellipsoidea.
  • Figure 7 shows the result of Western blot analysis showing the amount of fGH expressed in transformed Chlorella ellipsoidea.
  • lane M shows molecular weight size marker and lane 1 & 2 show the lO ⁇ g of GST-fGH fusion protein and lane 3 & 4 show the fGH isolated from 10ml transformed Chlorella ellipsoidea and lane 5 & 6 show the lO ⁇ g of GST protein.
  • Figure 8 shows the results of Western blot analysis showing fGH accumulated on Brachionus plicatilis and Artemia naupilus, both of which were fed with Chlorella ellipsoidea transformed with fGH.
  • lane 1 ⁇ 4 show Brachionus plicatilis 30, 60, 90 and 120 minutes, respectively after feeding with transformed Chlorella ellipsoidea
  • lane 6-8 show Artemia naupilus 30, 60 and 90 minutes, respectively after feeding with transformed Chlorella ellipsoidea.
  • Figure 9 shows the growth promotion of flounder by Chlorella ellipsoidea transformed with fGH.
  • open bars indicate the growth promotion by transformed Chlorella ellipsoidea
  • filled bars indicate the growth promotion by non-transformed Chlorella ellipsoidea
  • vertical line indicates standard deviation, and lower cases indicate significant differences (p ⁇ 0.05).
  • Figure 10 shows the growth promotion of flounder fries after 30 days feeding of Brachionus plicatilis and Artemia naupilus that had been fed for 1 hour with Chlorella ellipsoidea transformed with fGH.
  • foreign protein is used to mean any protein originated from organisms different from host microalgae, and it includes its active fragments, variants, and analogues as long as they retain its original biological activity.
  • foreign gene is used to indicate any nucleic acid sequence, regardless of its source(natural or synthetic), coding the foreign protein as defined above, and may include, DNA, RNA, cDNA or their variants resulting form base deletion, substitution or insertion, as long as they still code for the foreign protein having its biological activity.
  • Chlorella ellipsoidea is an attractive organism for the production of complex proteins because of its eukaryotic characteristics and low cost for large-scale culture.
  • the inventors report the first functional expression of a foreign protein, the flounder growth hormone(fGH) in Chlorella ellipsoidea, and the growth promotion of fish by feeding them this transformed chlorella.
  • Protoplasts of Chlorella ellipsoidea were transformed with a vector containing the fGH gene under the control of the cauliflower mosaic virus 35S promoter and the phleomycin resistance Sh ble gene under the control of the Chlamydomonas RBCS2 gene promoter.
  • the green fluorescence from chlorella transformed with the GFP gene and the phleomycin resistance of chlorella transformed with Sh ble gene indicate the functional expression of these proteins.
  • the biological activity of the recombinant fGH was confirmed by feeding flounder fry.
  • the microalgae transformed with flounder growth hormone gene could express the hormone in a biologically active form. Therefore, it's possible to produce valuable proteins for medicine and industry from transformed microalgae.
  • microalgae could be produced with simple equipment and low cost, and a method of isolation and purification of expressed proteins therefrom is also simple, so that the cost of producing protein could be significantly reduced.
  • this invention describes the successful use of the Sh ble gene as a selectable marker for Chlorella ellipsoidea transformation, the first demonstration of stable gene integration and expression of a biologically active foreign protein in the transformed Chlorella ellipsoidea.
  • the results indicate that Chlorella ellipsoidea can be used to produce proteins of scientific or pharmacological use.
  • the microalgae used in the present invention are not particularly limited but the technique can be applied to other algae including Chlorella from sea and fresh water such as Chlorella ellipsoidea, Chlorella sorokiniana and Chlorella vulgaris, Chlamydomonas, Volvox, Cheatoceros, Phaeodactylum, Skeletonema, Navicula, Caloneise, Nitzschia, Thalassiosira, Amphora, Nannochloris, Nannochloropsis, Tetraselmis, Dunaliella, Spirulina, Microcystis, Oscillatoria, Tricodesminus, Isochryosis, Pavlova, Dinophyceae and the like.
  • the foreign protein gene used in the present invention is the flounder growth hormone gene. However, other genes originated from bacteria, fungi, virus, animals, plants or fishes could be used for overexpression by using the present invention.
  • vector production, cloning, transformation of host by vector, selection and culture of transformant, and the recovering process of the desired protein after culture are known to those of skilled in the art.
  • Chlorella epillsoidea was obtained from the Korea Marine Microalgae Culture Center of Pukyong National University (Strain No. KMCC C-20). Cells were inoculated in fresh f/2 medium(Guillard, R. R. L., and Ryther, J. H. 1962. Studies on marine planktonic diatoms. I. Cyclotella nana Hustedt and Detonula confervacea(Cleve) Gran. Can. J. Microbiol. 3, 229-239) containing 50 ⁇ g/ml each of chloramphenicol and streptomycin at an initial concentration of l lO 6 cells/ml and cultured at 25°C, 18:6 hour photoperiod under a 3000 lux fluorescent lamp.
  • Cells were harvested for protoplast formation 8-9 days after inoculation when the cell count reached 1-2 ⁇ l0 8 cells/ml.
  • Cells (50ml) were centrifuged for 5minutes at l,500 ⁇ g, washed once with 25mM phosphate buffer (pH 6.0), and suspended in 5ml of phosphate buffer containing 0.6M sorbitol, 0.6M mannitol, 4% (w/v) cellulase(Calbiochem, USA), 2%(w/v) macerase(Calbiochem), and 50 units pectinase(Sigma Chemicals, USA). The cell suspension was incubated at 25°C for 16 hours in the dark with gentle shaking.
  • a small 5 kb binary vector was constructed from the plant transformation vector Binl9(Bevan, M. 1984. Binary Agrobacterium vectors for plant transformation. Nucleic Acids Res. 12, 8711-8712).
  • the new vector called pMIN contains the oriV origin for replication in both E. coli and Agrobacterium, the npt II gene for kanamycin resistance, the trfA gene for DNA replication, and the right and left border T-DNA elements for integration.
  • the subsequent cloning of a DNA fragment containing the cauliflower mosaic virus 35S promoter to direct expression of the green fluorescent protein (GFP) produced a vector pMinGFP for use in higher plant and algae transformation.
  • GFP green fluorescent protein
  • a flounder cDNA library was constructed with the Lambda ZAP-II cDNA synthesis kit (Stratagene, USA) using total mRNA isolated from the Japanese flounder pituitary gland.
  • the titer of the amplified library was 3 ⁇ l0 9 pfu/ml and a l ⁇ l aliquot was used for PCR amplification.
  • the growth hormone gene from the Japanese flounder, Paralichthys olivaceus, the major aquaculture fish in Korea, was used to transform chlorella.
  • the fGH gene was cloned by PCR amplification of a flounder pituitary cDNA library, using the fGH-N primer (5'-CGGGATCCGGTCAGTCCCTTATGCAGCCAATCACA-3') and fGH- C primer (5'-AAAAGCTCGAGCTCTTGGCGGAG-3') (Watahiki, M., Yamamoto, M., Yamakawa, M., Tanaka, M.
  • Sh ble gene originated from Streptoalloteichus hindustamus, which encodes a small protein (13.7kDa) that confers resistance to tallysomycin, bleomycin, phleomycin, and zeomycin by binding to the antibiotics and inhibiting their DNA cleaving activities.
  • the alga was cultured in f72 medium containing different concentrations of phleomycin; Reduced growth occurred in media containing 0.1 or 0.5 ⁇ g/ml phleomycin, and the alga failed to grow in media containing more than 1 ⁇ g/ml phleomycin.
  • the Sh ble gene that confers resistance to phleomycin is suitable to select transformed chlorella
  • the Sh ble coding region and upstream Chlamydomonas reinhardtu RBCS2 promoter were amplified from the plasmid pSP109(Lumbreras, N, Stevens, D. R., & Purton, S. 1998. Efficient foreign gene expression in Chlamydomonas reinhardtu mediated by an endogenous intron. Plant J. 14, 441-447) with ble- ⁇ primer (5'-AAACTCGAGGGCGCGCCAGAAGGAGC- 3') and ble-C primer (5'-AAACTCGAGAATTCGAGGTCGGTACC-3').
  • Chlorella protoplasts(l ⁇ l0 8 ) were centrifuged at 400 ⁇ g for 5 minutes, resuspended in 5 ml of f/2 medium containing 0.6M sorbitol/mannitol, centrifuged at 400 ⁇ g for 5 minutes, and resuspended in 1ml of 0.6 M sorbitol/mannitol solution containing 0.05M CaCl 2 . Then, l ⁇ l0 8 protoplasts in 0.4ml were placed in a fresh microcentrifuge tube and 5 ⁇ g of pCTN vector was added with 25 ⁇ g calf thymus D ⁇ A(Sigma Chemicals).
  • Approximately 3xl0 8 transformed cells were pelleted from 3 ml of culture, resuspended in 500 ⁇ l of CTAB buffer[250ml: hexadecyltrimethylammonium bromide(CTAB) 5g, 1M Tris(pH 8.0) 25ml, NaCl 20.45g, EDTA 1.68g, ⁇ - mercaptoethanol(2%)] and incubated at 65°C for 1 hour, and then extracted with an 0 equal volume of phenol/chloroform. The aqueous phase recovered after 5 minutes centrifugation at 3,000xg was extracted several times and chromosomal DNA was precipitated with ethanol, pelleted and resuspended in 30 ⁇ l of TE buffer.
  • CTAB buffer 250ml: hexadecyltrimethylammonium bromide(CTAB) 5g, 1M Tris(pH 8.0) 25ml, NaCl 20.45g, EDTA 1.68g,
  • PCR products of the expected size were produced only with DNA isolated from transformed chlorella. These DNA fragments were identified by Southern analyses with probes specific to the fGH or Sh ble genes(see Fig. 5). The stability of the integrated DNA was confirmed by PCR amplification of the two genes from the chromosomal DNA isolated from chlorella after seven serial transfers into medium lacking phleomycin.
  • EXAMPLE 8 Expression of fGH in tranformed Chlorella ellipsoidea fGH expression was tested by Western analysis as described hereinafter. Transformed Chlorella ellipsoidea was harvested from 3 ml of culture containing 10 8 to 10 9 cells by centrifuging for 5 minutes at 17,000 x g.
  • the cells were homogenized in liquid nitrogen, resuspended in 20 ⁇ l of sample loading buffer[lmM EDTA, 250mM Tris-Cl (pH 6.8), 4 % SDS, 2 % ⁇ - mercaptoethanol, 0.2 % bromophenyl blue, 50 % glycerol], and boiled for 10 minutes.
  • sample loading buffer [lmM EDTA, 250mM Tris-Cl (pH 6.8), 4 % SDS, 2 % ⁇ - mercaptoethanol, 0.2 % bromophenyl blue, 50 % glycerol]
  • the sample was centrifuged for 10 minutes at 12,000 x g and the supernatant was electrophoresed on a 15 % SDS-PAGE.
  • protein extracts prepared from non-transformed chlorella were separated by SDS-PAGE. Western blot analysis was conducted by standard procedures.
  • Protein extracts separated from tramsformed and non-transformed chlorella by SDS-PAGE were transferred onto nitrocellulose membranes.
  • the final dilution of polyclonal antibody against fGH was 1:3,000 and alkaline phosphatase- conjugated anti-mouse IgG was used as the secondary antibody.
  • the 20kDa fGH was present in transformed chlorella but absent in non-transformed cells (see Fig. 6).
  • fGH expressed in transformed chlorella was determined by an Enzyme Linked Immunosorbent Assay (ELISA) and Western blots with purified GST, GST-fGH fusion protein and extract from transformed chlorella using polyclonal antibody against GST-fGH fusion protein(see Fig. 7).
  • ELISA Enzyme Linked Immunosorbent Assay
  • About 400ng of fGH was obtained from l ⁇ l0 8 stationary phase cells (400 ⁇ g of total protein in 1ml culture). The yield is equivalent to 400 ⁇ g fGH per litter of cultured chlorella assuming a final cell count of lxl 0 8 cells /ml.
  • this system could be used to produce eukaryotic proteins, especially proteins of pharmaceutical importance.
  • chlorella can not be directly fed to fish and crustacean larvae because of the high cellulose content in their cell walls, chlorella have been used to mass culture zooplanktons, which contain cellulase. Also it is known that fish can take up proteins in feed by pinocytosis and there are reports of fish growth promotion by the oral administration of recombinant mammalian and fish growth hormone. Thus, four day old flounder larvae were grouped into 1000 fish each in a 300 litter tank filled with 200 litter of sea water. Rotifers (Brachionus plicatilis) and brine shrimp (Artemia nauplius) were used to accumulate the growth hormone and to remove the cellulose from chlorella cell wall.
  • Zooplanktons were starved for one day after hatching and provided with 3x108 cells/ml of transformed and non- transformed chlorella for one hour.
  • Western analysis confirmed that the fGH in the alga accumulated in zooplankton bodies by 1 hour of feeding; after 1 hour fGH was degraded and disappeared 2 hours after feeding (see Fig. 8).
  • the flounder larvae were fed once a day with the rotifers for 10 days and then with a mixture of the rotifers and the brine shrimp for 5 days, followed by 15 days feeding with the brine shrimp.
  • the final counts of the rotifer and the brine shrimp were 10 and 5 individuals/ml, respectively.

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Abstract

L'invention concerne un procédé économique de biosynthèse de protéines étrangères cibles, au moyen de microalgues transformées. L'invention porte notamment sur un procédé d'utilisation de microalgues transformées en tant que bioréacteur, permettant une biosynthèse économique de protéines étrangères par la transformation de protoplaste de microalgues, telles que Chlorella ellipsoidea, à l'aide d'un vecteur d'ADN contenant le gène de la protéine étrangère cible et par sa mise en culture à grande échelle. En particulier, le gène Sh Ble, qui est résistant à la phléomycine, est utilisé comme marqueur de sélection dans le procédé de l'invention.
EP00911471A 1999-05-28 2000-03-17 Biosynthese de proteines etrangeres au moyen de microalgues transformees Withdrawn EP1180145A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
KR1019990019439A KR20000075076A (ko) 1999-05-28 1999-05-28 형질전환된 미세조류를 이용하여 외래 단백질을 생산하는 방법
KR9919439 1999-05-28
PCT/KR2000/000233 WO2000073455A1 (fr) 1999-05-28 2000-03-17 Biosynthese de proteines etrangeres au moyen de microalgues transformees

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EP1180145A1 true EP1180145A1 (fr) 2002-02-20

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EP (1) EP1180145A1 (fr)
JP (1) JP2003501031A (fr)
KR (2) KR20000075076A (fr)
CN (1) CN1354792A (fr)
AU (1) AU3333900A (fr)
CA (1) CA2374402A1 (fr)
WO (1) WO2000073455A1 (fr)

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JP2014193153A (ja) * 2013-02-28 2014-10-09 Euglena Co Ltd ユーグレナへの遺伝子導入方法
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CN113699046A (zh) * 2021-08-10 2021-11-26 吴信忠 含有aif1细胞因子抗体的小球藻及其制备方法和应用

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