WO2004016648A1 - Proteine fluorescente - Google Patents

Proteine fluorescente Download PDF

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Publication number
WO2004016648A1
WO2004016648A1 PCT/DE2003/002436 DE0302436W WO2004016648A1 WO 2004016648 A1 WO2004016648 A1 WO 2004016648A1 DE 0302436 W DE0302436 W DE 0302436W WO 2004016648 A1 WO2004016648 A1 WO 2004016648A1
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WO
WIPO (PCT)
Prior art keywords
amino acid
sequence
protein
amino acids
proteins
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PCT/DE2003/002436
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German (de)
English (en)
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WO2004016648A8 (fr
Inventor
Ludger Altrogge
Tatjana Males
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Amaxa Gmbh
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
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Publication date
Application filed by Amaxa Gmbh filed Critical Amaxa Gmbh
Priority to AU2003257392A priority Critical patent/AU2003257392A1/en
Priority to DE10393454T priority patent/DE10393454D2/de
Publication of WO2004016648A1 publication Critical patent/WO2004016648A1/fr
Publication of WO2004016648A8 publication Critical patent/WO2004016648A8/fr

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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
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention relates to an artificially produced autofluorescent protein and a method for its production.
  • FACS fluorescence activated cell sorting
  • WO 99/49019 A2 discloses green fluorescent proteins from anthozoa of the genera Renilla and Ptilosarcus, as well as the isolated nucleic acids which code for these proteins.
  • WO 00/46233 A1 also discloses a fluorescent protein from corals, as well as the genes coding therefor and possible uses for the proteins.
  • WO 01/32688 A1 discloses the amino acid sequences of green fluorescent proteins from Renilla reniformis (Anthozoa, Coelenterata), as well as the nucleotide sequences of the nucleic acids derived therefrom and a multitude of possible uses.
  • WO 01/34824 A2 shows a sequence comparison (“alignment”) of different fluorescent proteins from Aequorea victoria, Ptilosarcus gurneyi and Renilla mulleri. This sequence comparison serves to determine homologies of the proteins, ie to determine the relative similarities between these proteins, and does not lead for the production of new fluorescent proteins. The efforts to isolate new autofluorescent proteins from ever new sources show that there is a very great need for new fluorescent proteins with advantageous properties.
  • the object is achieved according to the invention by a fluorescent protein which has an amino acid sequence which has at least 80% homology with one of the amino acid sequences according to Seq ID Nos. 1, 15, 17, 19 and 21.
  • new autofluorescent proteins are made available which are detectable in cells due to their fluorescence and can therefore be used as markers for gene expression and protein localization, for example in cell, development and molecular biology.
  • the proteins according to the invention also sometimes have new properties, such as, for example, the ability of the fluorescence to be regenerated after it has bleached out by irradiation with light of a specific wavelength.
  • fluorescent proteins are also to be understood as fusion proteins and multimers which contain at least one fluorescent protein according to the invention. This applies in particular to fusion proteins, since the proteins according to the invention can be used, inter alia, as expression markers and thus a fusion with other proteins is appropriate.
  • homology means the degree of agreement between two protein sequences, ie the number of amino acid positions in the proteins that match in percent.
  • One or more gaps can be inserted into one or both protein sequences so that the highest possible number of identical amino acids in Are assigned to each other with respect to their respective position.
  • a conventional data processing program can, for example, also be used to determine the homology.
  • the fluorescent protein according to the invention has at least 90% homology with one of the amino acid sequences according to Seq ID Nos. 1, 15, 17, 19 and 21.
  • the properties of the protein according to the invention can be advantageously influenced by targeted or undirected mutation. It has proven to be particularly advantageous that, based on the amino acid sequence according to Seq ID No. 1, the amino acid at position 2 is valine or glutamic acid, the amino acid at position 3 is alanine or leucine, the amino acid at position 4 is lysine or cysteine, the amino acid Position 6 lysine or valine or glutamic acid, the amino acid at position 7 asparagine or alanine, the amino acid at position 10 lysine or threonine, the amino acid at position 44 threonine or alanine, the amino acid at position 98 isoleucine or phenylalanine, the amino acid at position 108 isoleucine or aianin, the amino acid at position 125 leucine or phenylalanine, the amino acid at position 128 valine or alanine, the amino acid at position 150 lysine or glutamic acid, the amino acid at position
  • the presence of these amino acids at the respective positions has an advantageous effect, for example, on the expression, stability, solubility and fluorescence intensity of the proteins according to the invention.
  • Some of the fluorescent proteins according to the invention have the advantageous and surprising property that the fluorescence decays after a short time when irradiated with light and is suitable for fluorescence and can be regenerated by subsequent irradiation with light of a different wavelength.
  • the irradiation to excite the fluorescence with light of a wavelength between 375 and 580 nm and the irradiation to regenerate the fluorescence with light of shorter wavelength, in particular a wavelength between 320 and 400 nm, can take place.
  • this fluorescent protein can advantageously be used, for example, for the detection of time-dependent cellular processes, such as protein diffusion or transport, or for optical information storage.
  • the invention further relates to a nucleic acid molecule with a nucleotide sequence which codes for a fluorescent protein, the nucleotide sequence being selected from the group consisting of a) an isolated or artificial nucleotide sequence which codes for the fluorescent protein according to the invention. b) a nucleotide sequence according to Seq ID No. 2, 16, 18, 20 or 22, or c) a nucleotide sequence which differs from the nucleotide sequences according to a) or b) by the exchange of at least one codon for a synonymous codon, ie in that has at least one silent mutation.
  • the invention further relates to a vector for expressing a fluorescent protein in a suitable cell, which contains the previously described nucleic acid molecule in an expressible form.
  • the invention also relates to cells which contain the protein according to the invention, the nucleic acid molecule and / or the vector mentioned, and a kit which contain the protein according to the invention, the nucleic acid molecule described, the vector described and / or at least one of the cells mentioned.
  • a pharmaceutical composition is also provided which contains the protein according to the invention, the nucleic acid molecule and / or the vector mentioned, and preferably conventional auxiliaries and / or carriers.
  • the invention further relates to a method for producing a fluorescent protein according to the invention.
  • the amino acid sequences of at least three known autofluorescent proteins are compared in that they are initially arranged next to one another in such a way that, with the introduction of gaps, the positions of invariant amino acids or similar regions for all proteins may match.
  • the amino acid sequences can, for example, be aligned with one another in such a way that the highest possible number of identical amino acids are assigned to one another in relation to their respective positions, this preferably being done while maintaining the respective order of the amino acids.
  • Similar areas are all sequence sections, preferably a section of at least three consecutive amino acids which have an essentially identical primary structure or which form a domain in the folded protein, the function of which is already known.
  • the similar areas can also be in different positions for different proteins. In this case, the similar areas can be compared, for example, by inserting gaps or moving these areas in the sequence.
  • an average sequence over at least a substantial part of the total length of the amino acid sequence is determined.
  • the amino acid that occurs most frequently in the underlying amino acid sequences at this position is selected, with a gap being treated like an amino acid. This results in a sequence of amino acids and gaps with positions in between where no amino acid occurs most frequently or where two or more amino acids occur with the same frequency.
  • an amino acid must now be selected according to previously defined, meaningful, reproducible and generally applicable criteria. The method according to the invention provides several options for this.
  • an amino acid at such a position is determined by first establishing a ranking of the underlying proteins before comparing the amino acid sequences, i.e. Rank numbers from 1 to n are assigned to the individual proteins. If the most common amino acid cannot be clearly identified at a position, the amino acid that belongs to the protein sequence with the lowest rank number among the most common amino acids is used.
  • an amino acid is determined at such a position in that the amino acids are divided into groups on the basis of functional and / or structural criteria and, in the case of several amino acids, an amino acid is selected with the same frequency from the group that is present on the respective Position occurs most frequently.
  • an amino acid at such a position is determined by selecting the amino acid in the case of several amino acids with the same frequency on the basis of functional and / or structural criteria, taking into account the properties of the known proteins.
  • the selection of an amino acid with several amino acids with the same frequency within an amino acid sequence can also take place on the basis of different criteria, ie the aforementioned embodiments can be combined with one another in an advantageous manner. Furthermore, functional and structural criteria or other knowledge of the known proteins also requires, or at least influences, the aforementioned determination of the ranking of the amino acid sequences before their comparison.
  • the method according to the invention surprisingly enables the production of new fluorescent proteins which can be expressed in a suitable system.
  • the process steps described above lead to a complete, artificial amino acid sequence which represents a protein which shows autofluorescence and can be expressed in a suitable system.
  • an artificial nucleic acid sequence which codes for the determined average sequence, is first created and at least one corresponding nucleic acid molecule is synthesized.
  • This nucleic acid molecule is linked to a suitable promoter and the protein is then expressed in a suitable system.
  • the method according to the invention thus leads to a new, fully functional fluorescent protein.
  • the method can be carried out easily and without great expenditure on equipment.
  • the method according to the invention not only is a known protein changed, but rather it is based on several known proteins, so that the positive properties of the different proteins can be combined in the new protein. In this way, new fluorescent proteins can be produced without having to isolate and clone previously unknown natural proteins with great effort.
  • the average sequence is modified before the creation of the artificial nucleic acid sequence at the N-terminus and / or C-terminus by exchanging at least one amino acid.
  • the additional amino acid valine can be introduced, for example, after the start amino acid methionine.
  • additional hydrophilic amino acid serine can be added to improve the solubility of the protein.
  • the method according to the invention subsequently, i.e. after creation and expression of the average sequence, at least one codon is exchanged and / or changed by mutagenesis in the artificial nucleic acid molecule and thereby at least one amino acid of the average sequence is exchanged.
  • certain properties of the artificial protein such as stability, solubility, compatibility or functionality, can be changed in an advantageous manner.
  • the fluorescence i.e. the common function of the originally known proteins can be influenced or improved in a targeted manner.
  • the invention it was possible to generate other mutants of the fluorescent average protein (average - FP), which are significantly improved with regard to the solubility in the cell and the brightness of the fluorescence.
  • the deviations of the mutant clones from the average FP with respect to the amino acid sequence are a maximum of 10%, i.e. the homology of the proteins according to the invention with one another is at least 90%.
  • the protein is isolated and / or purified after expression so that it can be used in a suitable form for further use.
  • FIG. 1 shows a comparison of the amino acid sequences of 9 fluorescent proteins (FPs), in which the sequences are arranged with one another in such a way that an optimal match of results in invariant amino acids or similar regions for all proteins. Rank numbers were assigned to the individual FPs:
  • Rank 1 Aequorea victoria GFP (Gene (1992) 111, 229)
  • Rank 2 Zoanthus sp. zFP506 (Nature Biotechnol. (1999) 17, 969)
  • Rank 3 Zoanthus sp. zFP538 (Nature Biotechnol. (1999) 17, 969)
  • Rank 4 Discosoma striata dsFP483 (Nature Biotechnol. (1999) 17, 969)
  • Rank 5 Discosoma sp. "red” dsFP583 (Nature Biotechnol. (1999) 17,969)
  • Rank 6 Anemonia majano amFP486 (Nature Biotechnol.
  • the average sequence or, by adding additional modifications, the average FP was determined in accordance with the method according to the invention.
  • the numbering of the positions refers to the average sequence determined. At the points where there is a gap in the average sequence, the positions are not numbered consecutively, but are given the additions "b" or "c". Exceptions to this are only positions 1b and 226b, where additional amino acids are inserted in the average FP compared to the average sequence determined.
  • FIG. 2 shows a schematic representation of the structure of the plasmids which are used to carry out the described method and to express the fluorescent proteins according to the invention in Escherichia coli and mammalian cells.
  • FIG. 3 shows fluorescence microscopic images of NIH3T3 cells which were transfected with expression plasmids for various mutagenesis products of the proteins according to the invention. A: 9 hours after transfection, B: 24 hours after transfection.
  • FIG. 4 shows fluorescence microscopic images of NIH3T3 cells (fluorescein filter set) after transfection with an expression plasmid for a mutagenesis product of a protein produced according to the invention (p2A9-c15m3).
  • FIG. 5 shows fluorescence microscopic images of Escherichia coli cells (DH5), each with 7.1 ⁇ g of a plasmid without fluorescent protein (pMCS5, negative control; A) and of an expression plasmid (pExpAcFP; B) which contains the gene for the average FP contains were transformed. 2 ml of a culture of the transformed cells were pelleted, the supernatant was decanted, the pellet was resuspended with the last drop and 10 ⁇ l of this suspension were placed on a slide with a cover slip.
  • the amino acid sequences were initially arranged in the form of an "alignments", in which, with the introduction of gaps, the positions of invariant amino acids were matched for all proteins, the amino acid sequences being aligned with one another in such a way that the highest possible order was maintained while maintaining the respective amino acid sequence Number of identical amino acids are assigned to one another in relation to their respective position (cf. Matz et al. (1999), Nat. Biotechnol. 17, 969-973).
  • the amino acid sequences of Renilla mulleri GFP and Ptilosarcus gurneyi GFP were then also taken into account obviously invariant amino acids added (see Figure 1 a - e).
  • FP fluorescent protein
  • 12 partially complementary DNA oligonucleotides (Seq ID Nos. 3-14) were used, from which the entire FP gene was amplified in two successive PCR reactions .
  • Subsequent restriction digestion with the restriction enzymes Xba I and Not I gave a DNA fragment which, after ligation with the Xba I / Not I fragment from pExpA, gave the expression plasmid pExpA-cFP (see FIG. 2A).
  • PCR products obtained in this way were digested with Xba I and Not I and then ligated with the appropriate fragment of pExpA and transformed into Escherichia co // lines (DH5). Of the ampicillin-resistant colonies, those that were significantly lighter when viewed under a fluorescence microscope were selected. The increase in fluorescence intensity was verified by repeated transformation into Escherichia coli cells, the plasmid DNA was prepared and the sequence of the FP gene was determined.
  • the improved pExpA-cFP plasmids were combined and again amplified in the presence of Mn 2+ ions. This time, however, the restriction digested PCR products were ligated with the Xba I and Not I cut p2A9 fragment in order to obtain plasmids which are suitable for expression both in Escherichia coli cells and in mammalian cells.
  • PCR reactions were carried out to specifically change the N- and C-terminal areas.
  • the ⁇ '-primer hybridized with the FP gene and contained a mixture of all 4 bases instead of the first 6 codons at 18 positions, as well as the 3'-primer instead of the last 6 codons, so that in the translated protein at the N- and C-terminus a random sequence of 6 arbitrary amino acids was created.
  • These PCR products were also digested with Xba I and Not I and ligated to the corresponding fragment of p2A9.
  • the plasmids were produced by modification of pMCS5 (MoBiTec, Göttingen, Germany).
  • pMCS5 has a similar structure to pBluescript SK (-) (Stratagene) and differs from it only in the 5 'region of the coding sequence for the lacZ ⁇ fragment.
  • pMCS5 therefore has the lac promoter and, downstream, the lac operator, as a result of which the expression of an inserted coding sequence depends on the absence of an active lac repressor.
  • pMCS5 was modified in such a way that the lac operator is no longer included.
  • the early mRNA polyadenylation signal from SV40 was used to prepare a reconstruction of this expression plasmid for Escherichia coli into an expression plasmid for mammalian cells (p2A9-cFP, see FIG. 2B). This was done by additionally inserting regulatory promoter sequences which cause expression of the downstream gene in mammalian cells.
  • CMV cytomegalovirus
  • NIH3T3 cells (adherent, 2.5 x 10 5 cells per well in a 12-hole dish, cultivated to 70-80% confluence) were transfected with 1 ⁇ g vector DNA with Exgene (MBI Fermentas).
  • the plasmids p2A9-c15m2 (Seq ID No. 15), p2A9-c15m3 (Seq ID No. 17), p2A9-c15m12 (Seq ID No. 19) and p2A9-c15m48 (Seq ID No. 21) contained differently mutated genes, c15m2 (Seq ID No. 16), c15m3 (Seq ID No. 18), c15m12 (Seq ID No.
  • NIH3T3 cells (adherent, 2.5 x 10 5 cells per well in a 12-hole dish, cultivated to 70-80% confluence) were transfected with 1 ⁇ g vector DNA with Exgene (MBI Fermentas).
  • Table 1 shows the differences in the amino acid sequence between the individual modified proteins cFP2, cFP3, cFP12 and cFP48, which are caused by the mutated genes c15m2 (Seq ID No. 16), c15m3 (Seq ID No. 18), c15m12 (Seq ID No. 20) and c15m48 (Seq ID No. 22) are encoded, in comparison with the Dur.chnitt-FP (D-FP).
  • D-FP Dur.chnitt-FP
  • Table 2 shows that the differences between the new fluorescent proteins according to the invention and the known proteins, which were the starting point for determining the average sequence, are relatively high. There are only sequence homologies here between 32% and 69%. It was therefore possible to provide a completely new group of amino acid sequences or proteins which show clear autofluorescence and offer advantageous possible uses. In addition, an additional function or advantageous property could be generated in the proteins according to the invention.
  • Table 1 Differences in the amino acid sequences between the average FP (D-FP) and the individual mutated fluorescent proteins (clones cFP2 / Seq. ID No. 15, cFP3 / Seq ID No. 17, cFP12 / Seq ID No. 19 and cFP48 / Seq ID No. 21) based on the positions of the amino acids in the average FP according to Seq ID No. 1.

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  • Health & Medical Sciences (AREA)
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  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Gastroenterology & Hepatology (AREA)
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  • Proteomics, Peptides & Aminoacids (AREA)
  • Tropical Medicine & Parasitology (AREA)
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  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne des protéines autofluorescentes fabriquées de façon artificielle. Lesdites protéines autofluorescentes peuvent être détectées dans des cellules en raison de leur fluorescence et peuvent par conséquent servir de marqueur pour l'expression génétique et la localisation protéique, par exemple en biologie cellulaire, moléculaire et de développement. Les protéines selon l'invention présentent partiellement de nouvelles propriétés comme par exemple la capacité de régénération de la fluorescence après décoloration, par rayonnement de lumière à une certaine longueur d'onde. L'invention concerne également un procédé de fabrication desdites protéines fluorescentes.
PCT/DE2003/002436 2002-07-19 2003-07-19 Proteine fluorescente WO2004016648A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
AU2003257392A AU2003257392A1 (en) 2002-07-19 2003-07-19 Fluorescent protein
DE10393454T DE10393454D2 (de) 2002-07-19 2003-07-19 Fluoreszierendes Protein

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE2002133082 DE10233082A1 (de) 2002-07-19 2002-07-19 Fluoreszierendes Protein
DE10233082.4 2002-07-19

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WO2004016648A1 true WO2004016648A1 (fr) 2004-02-26
WO2004016648A8 WO2004016648A8 (fr) 2004-05-06

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0897985A2 (fr) * 1997-07-24 1999-02-24 F.Hoffmann-La Roche Ag Phytases consensus
WO2001029225A1 (fr) * 1999-10-21 2001-04-26 Panorama Research, Inc. Procede general permettant d'ameliorer l'expression des proteines heterologues
WO2001034824A2 (fr) * 1999-11-10 2001-05-17 Rigel Pharmaceuticals, Inc. Procedes et compositions contenant des proteines fluorescentes vertes (gfp) de renilla

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0897985A2 (fr) * 1997-07-24 1999-02-24 F.Hoffmann-La Roche Ag Phytases consensus
WO2001029225A1 (fr) * 1999-10-21 2001-04-26 Panorama Research, Inc. Procede general permettant d'ameliorer l'expression des proteines heterologues
WO2001034824A2 (fr) * 1999-11-10 2001-05-17 Rigel Pharmaceuticals, Inc. Procedes et compositions contenant des proteines fluorescentes vertes (gfp) de renilla

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
CRAMERI A ET AL: "IMPROVED GREEN FLUORESCENT PROTEIN BY MOLECULAR EVOLUTION USING DNA SHUFFLING", NATURE BIOTECHNOLOGY, NATURE PUBLISHING, US, vol. 14, 14 March 1996 (1996-03-14), pages 315 - 319, XP000791095, ISSN: 1087-0156 *

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DE10393454D2 (de) 2005-06-16
AU2003257392A1 (en) 2004-03-03
DE10233082A1 (de) 2004-03-04
WO2004016648A8 (fr) 2004-05-06

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