# US 9,434,765 B2 · High affinity SUMO traps > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 9,434,765 B2 - Document ID: US9434765B2 - Status: Granted - Kind: B2 - Application number: 14/119,343 - Filed: 2012-03-08 - Published: 2014-06-12 - Granted: 2016-09-06 - Earliest claimed: 2011-05-24 - Assignees: AsociacióN Centro De InvestigacióN Cooperativa En Biociencias-Cic Biogune - Applicants: Manuel Salvador Rodríguez Medina - Inventors: Manuel Salvador Rodríguez Medina - Web page: https://patentgrep.com/patent/US9434765B2 ## CPC - C07K 7/08: having 12 to 20 amino acids (gastrins C07K14/595; somatostatins C07K14/655; melanotropins C07K14/68) - C07K 14/47: from mammals - C12N 9/93: {Ligases (6)} - C12Y 603/02019: Ubiquitin-protein ligase (6.3.2.19), i.e. ubiquitin-conjugating enzyme - G01N 33/566: using specific carrier or receptor proteins as ligand binding reagents {where possible specific carrier or receptor proteins are classified with their target compounds} - C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00) - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - C12Y: ENZYMES - G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q) ## Abstract The potential use of these “SUMO receptors” to isolate SUMOylated targets has been considered using the SIM sequences of the SUMO-dependent ubiquitin-protein ligase RNF4. RFN4 contains 4 SIM sequences known to interact with SUMOylated proteins. The capacity of the SIM2 and SIM3 of RNF4 to purify SUMOylated proteins was increased when in the present invention it was disposed in tandem up to 4 SIM sequences. In a preferred embodiment to increase flexibility and functionality of this SUMO-trap, we have changed the natural linkers resulting in a broader capture of SUMOylated proteins. This Tandem SUMO Interacting Motifs (TSIMs) or SUMO-Binding Entities (SUBEs) system disclosed in the present invention is useful to capture polySUMOylated proteins from cell extracts. Therefore, in another embodiment of the present invention, TSIMs or SUBEs can be used for the identification SUMO substrates and the study of SUMO-regulated processes. ## Claims ### Claim 1 A polypeptide comprising at least eight SUMO interacting motifs (SIM) arranged in tandem, wherein said SUMO interacting motifs are linked to each other by a natural amino acid sequence linker. ### Claim 2 (depends on 1) The polypeptide according to claim 1, wherein the at least eight SUMO interacting motifs (SIMs) of claim 1 are present in a protein selected from the group consisting of: PML, Daxx, UBA2, PIAS, RNF4 ligase. ### Claim 3 (depends on 1) The polypeptide according to claim 1, wherein the at least eight SUMO interacting motifs (SIMs) of claim 1 are selected from the group consisting of: SIM1, SIM2, SIM3, SIM4, and combinations thereof. ### Claim 4 (depends on 1) The polypeptide according to claim 1, wherein the at least eight SUMO interacting motifs (SIMs) of claim 1 are SIM2 and/or SIM3, from the RNF4 ligase protein. ### Claim 5 (depends on 1) The polypeptide according to claim 1, wherein the polypeptide comprises four SIM2 and four SIM3 motifs. ### Claim 6 (depends on 1) The polypeptide according to claim 1, consisting of the amino acid sequence encoded by the nucleotide sequence SEQ ID NO: 7. ### Claim 7 (depends on 1) The polypeptide according to claim 1, wherein the natural sequence linker is present in RFN4. ### Claim 8 (depends on 1) A process for obtaining a polypeptide according to claim 1, which comprises culturing a cell comprising a nucleotide encoding at least eight SUMO interacting motifs (SIM) arranged in tandem and linked to each other by a natural amino acid sequence linker, under the control of a promoter wherein the polypeptide is expressed, and, recovering said polypeptide from the culture medium. ### Claim 9 (depends on 1) An in vitro and/or ex vivo method for the isolation of a SUMOylated protein from a sample which comprises i) incubating the polypeptide of claim 1 with said sample in conditions allowing said polypeptide to interact with at least one SUMOylated protein present in said sample; and ii) recovering the SUMOylated protein which is bound to said polypeptide. ### Claim 10 (depends on 1) A kit comprising the polypeptide of claim 1 and instructions for use of said polypeptide. ### Claim 11 (depends on 10) The kit according to claim 10 further comprising a solid support. ### Claim 12 (depends on 1) An in vitro and/or ex vivo method for identifying SUMO substrates from a sample which comprises i) incubating the polypeptide of claim 1 with said sample in conditions allowing said polypeptide to interact with at least one substrate of SUMO proteins present in said sample; ii) recovering any SUMO substrates which is bound to said polypeptide; and iii) identifying SUMO substrates recovered in the previous step. ### Claim 13 (depends on 12) The method according to claim 12 wherein the SUMO substrates are selected from: tumor suppressor p53 and NF-κB inhibitor IκBα. ### Claim 14 A nucleotide sequence encoding a polypeptide comprising at least eight SUMO interacting motifs (SIM) arranged in tandem, wherein said SUMO interacting motifs are linked to each other by a natural amino acid sequence linker. ### Claim 15 (depends on 14) The nucleotide sequence according to claim 14, wherein said nucleotide sequence is SEQ ID NO: 6. ### Claim 16 (depends on 14) A gene construct comprising the nucleotide sequence of claim 14, operatively linked to transcription control elements. ### Claim 17 (depends on 14) A vector comprising the nucleotide sequence of claim 14. ### Claim 18 (depends on 14) A cell comprising the nucleotide sequence of claim 14.