WO2006101283A1 - Developpement de vecteur d'expression a l'aide de penetratine en tant que methode pour ameliorer l'administration transdermique de proteines recombinees - Google Patents

Developpement de vecteur d'expression a l'aide de penetratine en tant que methode pour ameliorer l'administration transdermique de proteines recombinees Download PDF

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Publication number
WO2006101283A1
WO2006101283A1 PCT/KR2005/000872 KR2005000872W WO2006101283A1 WO 2006101283 A1 WO2006101283 A1 WO 2006101283A1 KR 2005000872 W KR2005000872 W KR 2005000872W WO 2006101283 A1 WO2006101283 A1 WO 2006101283A1
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Prior art keywords
penetratin
expression vector
protein
fusion
proteins
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PCT/KR2005/000872
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English (en)
Inventor
Deok Hoon Park
Jong Sung Lee
Kwang Sun Jung
Sung Taek Hong
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Biospectrum, Inc.
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Publication of WO2006101283A1 publication Critical patent/WO2006101283A1/fr

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B02—CRUSHING, PULVERISING, OR DISINTEGRATING; PREPARATORY TREATMENT OF GRAIN FOR MILLING
    • B02C—CRUSHING, PULVERISING, OR DISINTEGRATING IN GENERAL; MILLING GRAIN
    • B02C11/00—Other auxiliary devices or accessories specially adapted for grain mills
    • 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
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43563—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects
    • C07K14/43577—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects from flies
    • C07K14/43581—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects from flies from Drosophila
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B03—SEPARATION OF SOLID MATERIALS USING LIQUIDS OR USING PNEUMATIC TABLES OR JIGS; MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C—MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C1/00—Magnetic separation
    • B03C1/02—Magnetic separation acting directly on the substance being separated
    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/67—General methods for enhancing the expression
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention relates to an expression vector for expressing a fusion protein in E. coli using penetratin, which is a portion of a Drosophila transcription factor, as a fusion partner. More particularly, the present invention relates to an expression vector of a fusion protein using, as a fusion partner, a specific region of a transcription factor, penetratin, which has been shown to be expressed in the eukaryote Drosophila, and to translocate through the cellular membrane. The present invention is also concerned with the use of the expression vector for producing and enhancing the cellular permeability of a protein of interest.
  • a method of expressing a target protein in a fusion form has the following advantages.
  • this method stabilizes in a host a foreign protein, such as a peptide, which has a low molecular weight and is thus susceptible to degradation by host enzymes.
  • the method facilitates purification of the expressed product, resulting in efficient yield of the product.
  • the expressed product is secreted into a specific region of a host, that is, the cytoplasm or cell wall, or into the culture medium.
  • a target protein can be provided in the form of being deficient in methionine in the amino terminus in order to maintain the activity of the target protein.
  • Such expression systems employ the following fusion partners: proteins, such as J3 - galactosidase, chloramphenicol acetyltransferase (CAT) , glutathione S-transferase (GST) , and maltose binding protein (MBP) , and synthetic peptides, such as polyhistidine, polycysteine, and Flag peptide.
  • proteins such as J3 - galactosidase, chloramphenicol acetyltransferase (CAT) , glutathione S-transferase (GST) , and maltose binding protein (MBP)
  • synthetic peptides such as polyhistidine, polycysteine, and Flag peptide.
  • expression vectors using proteins such as ⁇ - galactosidase, CAT, GST, and MBP, as fusion partners are disadvantageous in that the fusion partners are very large in size and in that a target protein is thus actually obtained in a relatively low amount even though the fusion protein is expressed at a high level.
  • expression vectors using synthetic peptides such as polyhistidine, polycysteine, and Flag peptide, are disadvantageous in that when a target protein is a peptide or has a low molecular weight, an expressed fusion protein has a small size and is thus easily degraded in host cells.
  • the fusion protein has a remarkably low ability to permeate the cellular membrane, and thus does not substantially exhibit its activity.
  • the present inventors intend to develop a vector system capable of maximizing the activity of a target protein using penetratin in order to mass produce the target protein and enhance the cellular permeability of the target protein.
  • penetratin which is a 16- amino-acid peptide derived from a Drosophila transcription factor, can be used as a suitable fusion partner.
  • the penetratin gene In order to prepare the penetratin gene to be used as a fusion partner, the nucleotide sequence coding for the 16 amino acids of the penetratin peptide was identified, and two strands of the nucleotide sequence were then synthesized.
  • oligonucleotides also called oligomers
  • oligomers were allowed to anneal and were inserted into Ndel/Xhol sites of pET17b, thereby yielding pET-Pen N and PET-Pen C.
  • Various proteins may be inserted into the constructed vectors to produce penetratin-fused recombinant proteins having enhanced skin permeability.
  • the present invention provides an expression vector of a fusion protein using penetratin, which is a portion of a Drosophila transcription factor, as a fusion partner, and a method of mass producing a target protein and increasing the cellular permeability of the protein to maximize the activity of the protein using the expression vector.
  • penetratin which is a portion of a Drosophila transcription factor
  • Fig. 1 is a genetic map of the expression vector pETl7b.
  • FIG. 2 is a schematic diagram showing a process for constructing a recombinant expression vector, pET-Pen N, into which penetratin is inserted as a fusion partner according to the present invention.
  • Fig. 3 is a schematic diagram showing a process for constructing a recombinant expression vector, pET-Pen C.
  • Fig. 4 is a schematic diagram showing a process for constructing expression vectors of recombinant human interferon-gamma using penetratin as a fusion partner.
  • Fig. 5 is a photograph of electrophoresis gels showing expression patterns of fusion proteins of interferon-gamma and penetratin in microorganisms transformed with pPenIFNg and pIFNgPen.
  • Fig. 6 is a schematic diagram showing a process for constructing expression vectors of recombinant human EGF using penetratin as a fusion partner.
  • Fig. 7 is a photograph of electrophoresis gels showing expression patterns of EGF fusion proteins with penetratin, expressed in microorganisms transformed with pPenEGF and pEGFPen .
  • Fig. 8 illustrates the results of Western blotting showing the activity of recombinant human interferon-gamma according to the present invention.
  • Fig. 9 is a graph showing the activity of recombinant human EGF according to the present invention.
  • Fig. 10 illustrates the intracellular translocation of recombinant human interferon-gamma according to the present invention (left panel: COS-7 cells; right panel: NIH3T3 cells) .
  • Fig. 11 illustrates the permeation of recombinant human interferon-gamma according to the present invention through the skin tissue.
  • the nucleotide sequence coding for 16 amino acids of the penetratin peptide was identified, and two strands of the nucleotide sequence were then synthesized.
  • the resulting two oligonucleotides (also called oligomers) were allowed to anneal and were inserted into Ndel/Xhol sites of pET17b, thereby yielding pET-Pen N and PET-Pen C.
  • the expression vector of a fusion protein into which penetratin is inserted as a fusion partner may be effectively used when a variety of target proteins is desired to be mass produced.
  • the expression vector may be useful in the production of a broad range of target proteins having biological activity, such as human hormones, various cytokines and antigens.
  • the penetratin peptide may be inserted into vectors inducible with something other than IPTG to be used as a fusion partner .
  • EXAMPLE 1 Construction of pET-Pen N expression vector
  • the nucleotide sequence coding for 16 amino acids of penetratin was identified, and two strands of the nucleotide sequence were then synthesized.
  • the two synthesized oligonucleotides were allowed to anneal, generating a Nhel site and an Xhol site at the 5 '-end and 3 '-end, respectively, of the resulting DNA duplex.
  • a pET17b vector was digested with Ndel/Xhol and ligated with the annealed oligonucleotides.
  • a vector for expressing a fusion protein as shown in Fig. 2, was constructed. This vector produces a protein in which penetratin is fused to the N terminus of a target protein.
  • the synthesized sense- and antisense-strand oligonucleotides had the following sequences: sense: 5 ' -TAT gCg CCA gAT AAA gAT TTg gTT CCC gAA TCg gCg CAT gAA gTg gAA gAA gAg gCC-3'; antisense: 5 ' -TAT gCg CCA gAT AAA gAT TTg gTT CCC gAA TCg gCg CAT gAA gTg gAA gAA gAg gCC-3'.
  • EXAMPLE 2 Construction of pET-Pen C expression vector
  • Two strands of the nucleotide sequence coding for the penetratin peptide were synthesized and phosphorylated.
  • the two synthesized oligonucleotides were allowed to anneal.
  • a pET17b vector was digested with Ndel/Xhol, dephosphorylated, and ligated with the annealed oligonucleotides.
  • a vector for expressing a fusion protein as shown in Fig. 3, was constructed. This vector produces a protein in which penetratin is fused to the C terminus of a target protein. The successful construction of the vector was confirmed using Nrul digestion and full DNA sequencing.
  • the synthesized phosphorylated sense- and antisense-strand oligonucleotides had the following sequences: sense: 5 ' -TAT gTC gCg ACA gAT AAA gAT TTg gTT CCC gAA TCg gCg CAT gAA gTg gAA gAA gTg ACS'; and antisense: 5 ' -TCg AgT CAC TTC TTC CAC TTC ATg CgC CgA TTC ggg AAC CAA ATC TTT ATCTgT CgC gAC A-3 ' .
  • EXAMPLE 3 Construction and expression of pPen-IFN gamma and piFN gamma-Pen
  • Phosphorylated 5 ' -end and 3 ' -end primers for interferon gamma were prepared.
  • the 3 ' -end primer used for the construction of pPen-IFN gamma was designed to contain a stop codon
  • the 3 ' -end primer used for the construction of pIFN gamma-Pen was designed to have a sequence in which the stop codon of IFN Y was deleted.
  • RT- PCR was then carried out using total RNA from a human cell line, HaCat, and each set of the 5 ' -end and 3 ' -end primers, thereby obtaining two IFN gamma DNA fragments.
  • the pPen N vector was digested with Stu I for the construction of pPen-IFN gamma, and the pPen C with Nrul for the construction of pIFN gamma-Pen.
  • the linearized vectors were dephosphorylated and ligated with the IFN gamma DNA fragments obtained by RT-PCR.
  • the resulting vectors were confirmed by PCR and DNA sequencing using T7 primer and the 3 ' -end primer for IFN gamma (Fig. 4) .
  • the 5 ' -end primer for the construction of pPen-IFN gamma had the sequence of 5'-P tgt tac tgc cag gac cca tat gta aaa g-3 ' .
  • the 3 ' -end primer for the construction of pPen-IFN gamma had the sequence of 5 ' -P tta ctg gga tgc tct teg ace teg aaa c-3 ' .
  • the 5 ' -end primer for the construction of pIFN gamma-Pen had the sequence of 5'-PATg TgT TAC TgC CAg gAC CCA TAT gTA CAA g-3 ' .
  • the 3 ' - end primer for the construction of piFN gamma-Pen had the sequence of 5'-PCTg ggT TgC TCT TCg ACC TCg AAA CAg-3 ' .
  • the prepared expression vectors, pPen-IFN gamma and pIFN gamma- Pen, were introduced into E.
  • coli BL21 (DE3) using Kushner's method (see, Kushner, Genetic Engineering, pl7, Elsevier/North-Holland, Amsterdam, 1978) .
  • the transformants (which were deposited under accession number KCCM 10523) were inoculated in LB broth supplemented with ampicillin (5 g of NaCl, 5 g of yeast extract, 10 g of bactotrypton and 50 mg of ampicillin per 1 L) , and were incubated at 37 ° C overnight with shaking at 250 rpm.
  • the saturated culture was inoculated in fresh ampicillin-containing LB broth in a final concentration of 1% and incubated with shaking.
  • IPTG IP-linked glycoprotein
  • lane 1 is a protein sample from cells not treated with IPTG
  • lanes 2, 3 and 4 are protein samples from cells induced with IPTG for 2 hrs, 4 hrs and 7 hrs, respectively.
  • EXAMPLE 4 Construction and expression of pPen-EGF and pEGF-Pen
  • Phosphorylated 5 ' -end and 3 ' -end primers for EGF were prepared.
  • the 3 ' -end primer used for the construction of pPen-EGF was designed to contain a stop codon
  • the 3 ' -end primer used for the construction of pEGF-Pen was designed to have a sequence in which the stop codon of EGF was deleted.
  • RT-PCR was then carried out using total RNA from the human cell line HaCat, and each set of the 5 ' -end and 3 ' -end primers, thereby obtaining two EGF DNA fragments.
  • the pPen N vector was digested with Stu I for the construction of pPen-EGF, and the pPen C with Nrul for the construction of pEGF-Pen.
  • the linearized vectors were dephosphorylated and ligated with the EGF DNA fragments obtained by RT-PCR.
  • the resulting vectors were confirmed by PCR and DNA sequencing using T7 primer and the 3 ' -end primer for EGF (Fig. 6) .
  • the 5 ' -end primer for the construction of pPen-EGF had the sequence of 5'- Paat agt gac tct gaa tgt ccc ctg-3 ' .
  • the 3 ' -end primer for the construction of pPen-EGF had the sequence of 5 ' - Ptta gcg cag ttc cca cca ctt cag g-3 ' .
  • the 5 ' -end primer for the construction of pEGF-Pen had the sequence of 5 ' - Patg aat agt gac tct gaa tgt ccc ctg-3 ' .
  • the 3 ' -end primer for the construction of pEGF-Pen had the sequence of 5 ' - Pgcg cag ttc cca cca ctt cag g-3 ' .
  • the prepared expression vectors were transformed into E.
  • Example 3 The transformants (which were deposited under accession number KCCM 10524) were grown with shaking in LB broth supplemented with ampicillin, and treated with IPTG for a predetermined period of time to induce protein expression.
  • the expressed proteins were analyzed using SDS-PAGE. As shown in Fig. 7, fusion proteins were expressed when cells were treated with IPTG for 2 hrs and 4 hrs, but they were not expressed when IPTG induction was carried out for 7 hrs .
  • Fusion proteins expressed according to the method described in Example 3 were purified and assessed for their activity using cells.
  • HeLa cells were cultured in an incubator and treated with 20 ng/ml of the fusion protein. After being further cultured for 16 hrs, the cells were harvested. The collected cells were lysed with lysis buffer, and cell lysates were electrophoresed on an SDS- PAGE gel. The separated proteins were then transferred onto a nitrocellulose membrane. The membrane was subjected to Western blotting using an antibody against IRF-I, whose intracellular expression is induced by interferon gamma. As a result, the interferon gamma expressed in the form fused to penetratin was found to retain its activity (Fig. 8) .
  • beta actin which is intracellularly expressed, was used as a quantitative control.
  • lane 1 is a sample not treated with interferon gamma
  • lane 2 is interferon gamma as a positive control
  • lane 3 is a sample treated with the penetratin-INF gamma fusion protein
  • lane 4 is a sample treated with the INF gamma-penetratin fusion protein.
  • Fusion proteins expressed according to the method described in Example 3 were purified and assessed for their activity using cells.
  • HeLa cells were cultured in an incubator and transfected with an AP-luciferase gene using Superfect. After being incubated for 24 hrs, the cells were treated with 20 ng/ml of the EGF fusion protein with penetratin. After being further cultured for 16 hrs, the cells were harvested. The cells were then lysed with lysis buffer, and luciferase activity was measured using a luminometer . As a result, EGF expressed in the form fused with penetratin was found to retain its activity (Fig. 9) .
  • EXAMPLE 7 Evaluation of the intracellular translocation of the penetratin-fused human INF gamma
  • the fusion protein produced according to the method described in Example 3 was assessed for intracellular translocation using an immunofluorescence assay (IFA) .
  • IFA immunofluorescence assay
  • NIH3T3 cells and HeLa cells were grown in culture plates and treated with the penetratin-fused INF gamma. After being cultured for 16 hrs, the cells were washed with PBS and treated with an anti-INF gamma antibody for 30 min. After being washed, the cells were treated with an FITC- conjugated secondary antibody. Then, the cells were observed under a fluorescence microscope to determine whether the fusion protein had translocated into the cells (Fig. 10) .
  • panel A shows the result of an IFA with COS-7 cells
  • panel B shows the result of an IFA with NIH3T3 cells.
  • EXAMPLE 8 Evaluation of the skin permeation of the penetratin-fused human INF gamma
  • the fusion protein produced according to the method described in Example 3 was assessed for penetration through the skin using immunohistochemistry .
  • the penetratin-fused human INF gamma was sprayed onto mouse skin. After 1 hr or 2 hrs, skin tissues were collected, and subjected to immunohistochemistry using an anti-INF gamma antibody to measure the skin permeation of the fusion protein. As shown in Fig. 11, the penetratin-fused human INF gamma was found to permeate into the dermis remarkably well compared to INF gamma not fused with penetratin.
  • the expression vector of the present invention which employs penetratin as a fusion partner, remarkably enhances the skin permeability of proteins, target proteins can penetrate into the skin without processing upon direct application onto the skin, leading to increased efficacy of the proteins.
  • a system using such a feature may be useful in the development of cosmetics or quasi-drugs using proteins.

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Abstract

L'invention concerne un vecteur d'expression pour l'expression d'une protéine de fusion à l'aide de pénétratine, qui est dérivée de l'homéodomaine d'un facteur de transcription de drosophile, en tant que partenaire de fusion. L'invention concerne également un procédé d'amélioration de la perméabilité cellulaire d'une protéine à étudier par production de la protéine à l'aide d'un vecteur d'expression. On a découvert que lorsque des gènes codant pour des protéines cibles présentant une variété de tailles sont insérés individuellement dans le vecteur d'expression, les protéines cibles fusionnées avec la pénétratine sont exprimées à des niveaux élevés et présentent une perméabilité cellulaire élevée. Ainsi, le vecteur d'expression d'une protéine de fusion, dans lequel la pénétratine est insérée en tant que partenaire de fusion, peut être efficacement utilisé pour produire en masse une variété de protéines cibles et pour améliorer la perméabilité des protéines dans des cellules présentes dans ou isolées à partir de la peau et d'autres organes. En particulier, le vecteur d'expression peut être efficace dans la production et l'utilisation clinique d'un large éventail de protéines présentant une activité biologique, telles que des hormones humaines, diverses cytokines, ainsi que des antigènes.
PCT/KR2005/000872 2005-03-24 2005-03-25 Developpement de vecteur d'expression a l'aide de penetratine en tant que methode pour ameliorer l'administration transdermique de proteines recombinees WO2006101283A1 (fr)

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KR1020050024702A KR100729830B1 (ko) 2005-03-24 2005-03-24 페너트라틴을 이용하여 단백질의 세포투과를 증진시키는대장균용발현벡터
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Cited By (1)

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EP2253326A1 (fr) * 2008-02-28 2010-11-24 Toray Industries, Inc. Composition pharmaceutique pour une administration transnasale

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KR102013788B1 (ko) 2016-11-04 2019-08-23 한국과학기술연구원 인간상피세포 성장인자 또는 피부침투성 인간상피세포 성장인자 발현용 벡터 및 상기 벡터로 형질전환된 미세조류

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2253326A1 (fr) * 2008-02-28 2010-11-24 Toray Industries, Inc. Composition pharmaceutique pour une administration transnasale
EP2253326A4 (fr) * 2008-02-28 2013-01-09 Toray Industries Composition pharmaceutique pour une administration transnasale
AU2009218060B2 (en) * 2008-02-28 2014-08-28 Toray Industries, Inc. Pharmaceutical composition for transnasal administration
US8895503B2 (en) 2008-02-28 2014-11-25 Toray Industries, Inc. Pharmaceutical composition for transnasal administration of peptide hormones or cytokines
KR101585046B1 (ko) * 2008-02-28 2016-01-13 도레이 카부시키가이샤 경비 투여용 의약 조성물

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