SE547227C2 - Identification of (poly)peptide-(poly)peptide interactions - Google Patents

Identification of (poly)peptide-(poly)peptide interactions

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Publication number
SE547227C2
SE547227C2 SE2351223A SE2351223A SE547227C2 SE 547227 C2 SE547227 C2 SE 547227C2 SE 2351223 A SE2351223 A SE 2351223A SE 2351223 A SE2351223 A SE 2351223A SE 547227 C2 SE547227 C2 SE 547227C2
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Prior art keywords
poly
peptide
nucleotide sequence
sequence encoding
promoter
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SE2351223A
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Swedish (sv)
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SE2351223A1 (en
Inventor
Markus Schmid
Silvio Collani
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Markus Schmid
Silvio Collani
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Publication date
Application filed by Markus Schmid, Silvio Collani filed Critical Markus Schmid
Priority to SE2351223A priority Critical patent/SE547227C2/en
Priority to PCT/SE2024/050895 priority patent/WO2025090001A1/en
Publication of SE2351223A1 publication Critical patent/SE2351223A1/en
Publication of SE547227C2 publication Critical patent/SE547227C2/en

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    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
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    • 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
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/02—Libraries contained in or displayed by microorganisms, e.g. bacteria or animal cells; Libraries contained in or displayed by vectors, e.g. plasmids; Libraries containing only microorganisms or vectors
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/04—Libraries containing only organic compounds
    • C40B40/06—Libraries containing nucleotides or polynucleotides, or derivatives thereof
    • C40B40/08—Libraries containing RNA or DNA which encodes proteins, e.g. gene libraries
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • G01N33/5023—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects on expression patterns
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    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6845—Methods of identifying protein-protein interactions in protein mixtures
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    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
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Abstract

A method for identifying (poly)peptide-(poly)peptide interactions comprises transforming a cell with a first expression vector encoding a first fusion protein between a first (poly)peptide and a first inactive part of Cre recombinase and a second expression vector encoding a second fusion protein between a second (poly)peptide and a second inactive part of Cre recombinase and comprising a selectable marker not operatively linked to any promoter. Interaction between the first and second (poly)peptides of two fusion proteins induces activation of the Cre recombinase and in trans recombination of the expression vectors into a first recombined vector and in cis recombination of the first recombined vector to bring the selectable marker operatively linked to a promoter, wherein the expression of the selectable marker indicates interaction between the first and second (poly)peptides in the cell. The invention also relates to a kit and expression vector that can be used in the method.

Claims (21)

Claims
1. A method for identifying (poly)peptide-(poly)peptide interaction (PPI), the method comprises: transforming (S1) a cell with a first expression vector and a second expression vector, wherein the first expression vector comprises: a first promoter constitutiveiy or inducibiy active in the cell; and a first open reading frame operatively linked to the first promoter and comprising: a nucleotide sequence encoding a first inactive part of Cre recombinase; a first recombinase recognition site; and a nucleotide sequence encoding a first (poly)peptide; the second expression vector comprises: a second promoter constitutively or inducibly active in the cell; a second open reading frame operatively linked to the second promoter and comprising: a nucleotide sequence encoding a second inactive part of Cre recombinase; a second recombinase recognition site; and a nucleotide sequence encoding a second (poly)peptide; a selectable marker not operatively linked to any promoter; and a third recombinase recognition site; expressing (S2) the first open reading frame and the second open reading frame in the cell to obtain a first fusion protein comprising the first inactive part of Cre recombinase and the first (poly)peptide and a second fusion protein comprising the second inactive part of Cre recombinase and the second (poly)peptide, wherein an interaction betvveen the first (poly)peptide of the first fusion protein and the second (poly)peptide of the second fusion protein induces reconstitution of an enzymatically active Cre recombinase from the first inactive part of Cre recombinase of the first fusion protein and the second inactive part of Cre recombinase of the second fusion protein; the enzymatically active Cre recombinase induces in trans recombination of the first expression vector and the second expression vector at the first recombinase recognition site and the second recombinase recognition site into a first recombined vector; the enzymatically active Cre recombinase induces in cis recombination of the first recombined vector at the third recombinase recognition site and a recombinase recognition site formed in the in trans recombination into a second recombined vector comprising the selectable marker operatively linked to the first promoter; and identifying (S3) PPI betvveen the first (poly)peptide and the second (poly)peptide if the cell expresses the selectable marker.
2. The method according to claim 1, wherein one of the first inactive part of Cre recombinase and the second inactive part of Cre recombinase is an inactive N-terminal part of Cre recombinase and the other of the first inactive part of Cre recombinase and the second inactive part of Cre recombinase is an inactive C-terminal part of Cre recombinase.
3. The method according to claim 2, wherein the inactive N-terminal part of Cre recombinase comprises, preferabIy consists of, the amino acid sequence according to SEQ ID NO:
4. The method according to claim 2 or 3, wherein the inactive C-terminal part of Cre recombinase comprises, preferabIy consists of, the amino acid sequence according to SEQ ID NO:
5. The method according to any one of cIaims 1 to 4, wherein the second recombinase recognition site is an inverted recombinase recognition site.
6. The method according to any one of cIaims 1 to 5, wherein one of the first recombinase recognition site and the second recognition site is a Iox 66 site and the other of the first recombinase recognition site and the second recognition site is a Iox 71 site.
7. The method according to claim 6, wherein the Iox 66 site comprises, preferabIy consists of, the nucleotide sequence according to SEQ ID NO:
8. The method according to claim 6 or 7, wherein the Iox 71 site comprises, preferabIy consists of, the nucleotide sequence according to SEQ ID NO:
9. The method according to any one of cIaims 1 to 8, wherein the third recombinase recognition site is a loxP site, preferabIy comprising, more preferabIy consisting of, the nucleotide sequence according to SEQ ID NO:
10. The method according to any one of cIaims 1 to 9, wherein the seIectabIe marker Iacks a start codon for translation.
11. The method according to claim 10, wherein the second recombined vector comprises the seIectabIe marker Iinked in frame to the nucleotide sequence encoding the first inactive part of Crerecombinase comprising a start codon for translation, wherein the nucleotide sequence encoding the first inactive part of Cre recombinase and the selectable marker are operatively linked to the first promoter.
12. The method according to any one of claims 1 to 11, wherein the first open reading frame comprises: a nucleotide sequence encoding an inactive N-terminal part of Cre recombinase; a Iox 66 site; and the nucleotide sequence encoding the first (po|y)peptide.
13. The method according to any one of claims 1 to 12, wherein the second open reading frame comprises: a nucleotide sequence encoding an inactive C-terminal part of Cre recombinase; an inverted Iox 71 site; and the nucleotide sequence encoding the second (po|y)peptide.
14. The method according to any one of claims 1 to 13, wherein the selectable marker is a third selectable marker; the first expression vector comprises: the first promoter; the first open reading frame operatively linked to the first promoter; and a first selectable marker operatively linked to the first promoter or a third promoter of the first expression vector, preferably operatively linked to the third promoter; and the second expression vector comprises: the second promoter; the second open reading frame operatively linked to the second promoter; the third selectable marker not operatively linked to any promoter; the third recombinase recognition site; and a second selectable marker operatively linked to the second promoter or a fourth promoter of the second expression vector, preferably operatively linked to the fourth promoter.
15. The method according to any one of claims 1 to 14, further comprising sequencing (S4) at least a portion of the second recombined vector or at least one fragment thereof, wherein the at least a portion of the second recombined vector or the at least one fragment thereof comprises at least a portion of thenucleotide sequence encoding the first (po|y)peptide and at least a portion of the nucleotide sequence encoding the second (po|y)peptide.
16. The method according to any one of claims 1 to 15, wherein the first open reading frame comprises: the nucleotide sequence encoding the first inactive part of Cre recombinase; one of a Iox 66 site and a Iox 71 site; a first type IIS restriction site; the nucleotide sequence encoding the first (po|y)peptide; and the second open reading frame comprises: the nucleotide sequence encoding the second inactive part of Cre recombinase; the other of the Iox 66 site and the Iox 71 site; a second type IIS restriction site; and the nucleotide sequence encoding the second (po|y)peptide; the method further comprising: treating (S10) the second recombined vector with a type IIS restriction enzyme to generate a fragment of the second recombined vector comprising a portion of the nucleotide sequence encoding the first (po|y)peptide, a Iox 66/71 site and a portion of the nucleotide sequence encoding the second (po|y)peptide; sequencing (S11) the fragment to determine the nucleotide sequence of the portion of the nucleotide sequence encoding first (po|y)peptide and of the portion of the nucleotide sequence encoding the second (po|y)peptide; and identifying (S12) the first (po|y)peptide and the second (po|y)peptide based on the determined nucleotide sequence.
17. The method according to any one of claims 1 to 16, further comprising: providing (S20) multiple first expression vectors, wherein each first expression vector of the multiple first expression vectors comprises: the first promoter; and the first open reading frame operatively linked to the first promoter and comprising: the nucleotide sequence encoding the first inactive part of Cre recombinase; the first recombinase recognition site; and a nucleotide sequence encoding a respective first test (po|y)peptide of multiple first test (poly)peptides; andproviding (S21) multiple second expression vectors, wherein each second expression vector of the multiple second expression vectors comprises: the second promoter; the second open reading frame operatively linked to the second promoter and comprising: the nucleotide sequence encoding the second inactive part of Cre recombinase; the second recombinase recognition site; and a nucleotide sequence encoding a respective second test (poly)peptide of multiple second test (poly)peptides; the selectable marker not operatively linked to any promoter; and the third recombinase recognition site, wherein transforming (S1) the cell comprises transforming (S1) cells with a respective first expression vector of the multiple first expression vectors and a respective second expression vector of the multiple second expression vectors; and identifying (S3) PPI comprises: identifying (S22) any cell expressing the selectable marker; isolating (S23) the second recombined vector from the identified cell; and identifying (S24) PPI betvveen a first test (poly)peptide of the multiple first test (poly)peptides and a second test (poly)peptide of the multiple second test (poly)peptides by sequencing at least a portion of the isolated second recombined vector or at least one fragment thereof, wherein the at least a portion of the isolated second recombined vector or the at least one fragment thereof comprises at least a portion of the nucleotide sequence encoding a first test (poly)peptide of the multiple first test (poly)peptides and at least a portion of the nucleotide sequence encoding a second test (poly)peptide of the multiple second test (poly)peptides.
18. The method according to any one of claims 1 to 16, further comprising: providing (S20) multiple first expression vectors, wherein each first expression vector of the multiple first expression vectors comprises: the first promoter; and the first open reading frame operatively linked to the first promoter and comprising: the nucleotide sequence encoding the first inactive part of Cre recombinase; the first recombinase recognition site; and a nucleotide sequence encoding a respective first test (poly)peptide of multiple first test (poly)peptides, wherein transforming (S1) the cell comprises transforming (S1) cells with a respective first expression vector of the multiple first expression vectors and the second expression vector; and identifying (S3) PPI comprises: identifying (S22) any cell expressing the selectable marker; isolating (S23) the second recombined vector from the identified cell; and identifying (S24) PPI betvveen a first test (poly)peptide of the multiple first test (poly)peptides and the second (poly)peptide by sequencing at least a portion of the isolated second recombined vector or at least one fragment thereof, wherein the at least a portion of the isolated second recombined vector or the at least one fragment thereof comprises at least a portion of the nucleotide sequence encoding a first test (poly)peptide of the multiple first test (poly)peptides and at least a portion of the nucleotide sequence encoding the second (poly)peptide.
19. The method according to anyone of claims 1 to 16, further comprising: providing (S21) multiple second expression vectors, wherein each second expression vector of the multiple second expression vectors comprises: the second promoter; the second open reading frame operatively linked to the second promoter and comprising: the nucleotide sequence encoding the second inactive part of Cre recombinase; the second recombinase recognition site; and a nucleotide sequence encoding a respective second (poly)peptide of multiple second test (poly)peptides; the selectable marker not operatively linked to any promoter; and the third recombinase recognition site, wherein transforming (S1) the cell comprises transforming (S1) cells with the first expression vector and a respective second expression vector of the multiple second expression vectors; and identifying (S3) PPI comprises: identifying (S22) any cell expressing the selectable marker; isolating (S23) the second recombined vector from the identified cell; and identifying (S24) PPI betvveen the first (poly)peptide and a second test (poly)peptide of the multiple second test (poly)peptides by sequencing at least a portion of the isolated second recombined vector or at least one fragment thereof, wherein the at least a portion of the isolated second recombined vector or the at least one fragment thereof comprises at least a portion of the nucleotide sequence encoding the first (poly)peptide and at least a portion of the nucleotide sequence encoding a second test (poly)peptide of the multiple second test (poly)peptides.
20. A kit for identifying (poly)peptide-(poly)peptide interaction, the kit comprising: a first expression vector comprising: a first promoter; and a first open reading frame operatively linked to the first promoter and comprising: a nucleotide sequence encoding a first inactive part of Cre recombinase; a first recombinase recognition site; and a first cloning site or a nucleotide sequence encoding a first (poly)peptide; and a second expression vector comprising: a second promoter; a second open reading frame operatively linked to the second promoter and comprising: a nucleotide sequence encoding a second inactive part of Cre recombinase; a second recombinase recognition site; and a second cloning site or a nucleotide sequence encoding a second (poly)peptide; a selectable marker not operatively linked to any promoter; and a third recombinase recognition site.
21. An expression vector comprising: a promoter; an open reading frame operatively linked to the promoter and comprising: a nucleotide sequence encoding an inactive part of Cre recombinase; a first recombinase recognition site; and a cloning site or nucleotide sequence encoding a (poly)peptide; a selectable marker not operatively linked to any promoter; and a second recombinase recognition site.
SE2351223A 2023-10-27 2023-10-27 Identification of (poly)peptide-(poly)peptide interactions SE547227C2 (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
SE2351223A SE547227C2 (en) 2023-10-27 2023-10-27 Identification of (poly)peptide-(poly)peptide interactions
PCT/SE2024/050895 WO2025090001A1 (en) 2023-10-27 2024-10-22 Identification of (poly)peptide-(poly)peptide interactions

Applications Claiming Priority (1)

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SE2351223A SE547227C2 (en) 2023-10-27 2023-10-27 Identification of (poly)peptide-(poly)peptide interactions

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SE547227C2 true SE547227C2 (en) 2025-06-10

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Citations (7)

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WO2009009908A1 (en) * 2007-07-19 2009-01-22 Mcmaster University A recombmase based screening method for detecting molecular interactions comprising a single plasmid vector with two unique recombmase sites
US8349619B2 (en) * 2002-03-13 2013-01-08 Sygnis Bioscience Gmbh & Co. Kg Method for detecting and analyzing protein interactions in-vivo
US20150010932A1 (en) * 2013-07-02 2015-01-08 Salk Institute For Biological Studies Methods for assaying protein-protein interactions
EP2825650A1 (en) * 2012-03-12 2015-01-21 DSM IP Assets B.V. Recombination system
WO2015163744A1 (en) * 2014-04-25 2015-10-29 재단법인 의약바이오컨버젼스연구단 Vector for simultaneously analyzing plurality of protein-protein interactions
US20200157514A1 (en) * 2017-04-10 2020-05-21 The University Of Tokyo Polypeptide Set to Be Used in Light Dependent Gene Recombination

Patent Citations (7)

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Publication number Priority date Publication date Assignee Title
US8349619B2 (en) * 2002-03-13 2013-01-08 Sygnis Bioscience Gmbh & Co. Kg Method for detecting and analyzing protein interactions in-vivo
US7323313B2 (en) * 2003-05-09 2008-01-29 Health Research, Inc. Methods for protein interaction determination
WO2009009908A1 (en) * 2007-07-19 2009-01-22 Mcmaster University A recombmase based screening method for detecting molecular interactions comprising a single plasmid vector with two unique recombmase sites
EP2825650A1 (en) * 2012-03-12 2015-01-21 DSM IP Assets B.V. Recombination system
US20150010932A1 (en) * 2013-07-02 2015-01-08 Salk Institute For Biological Studies Methods for assaying protein-protein interactions
WO2015163744A1 (en) * 2014-04-25 2015-10-29 재단법인 의약바이오컨버젼스연구단 Vector for simultaneously analyzing plurality of protein-protein interactions
US20200157514A1 (en) * 2017-04-10 2020-05-21 The University Of Tokyo Polypeptide Set to Be Used in Light Dependent Gene Recombination

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Title
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Wen M et al., 'Split-Cre complementation restores combination activity on transgene excision in hair roots of transgenic tobacco', PLoS One, 2014, 9(10):e110290 *

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