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ProteoTuner System

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Á¦Á¶»ç Á¦Ç°ÄÚµå Á¦Ç°¸í ¿ë·® °¡°Ý
(ºÎ°¡¼¼º°µµ)
ºñ°í »ç¿ëÀڸŴº¾ó
Clontech
632172
ProteoTuner System
°ü·ÃÇмú ±¸¸ÅÇϱ⠶óÀ̼±½º 
1 Kit
1,335,000¿ø  Á¦Á¶»ç ÆäÀÌÁö·Î ¹Ù·Î°¡±â x33525, x32971
Clontech
631072
ProteoTuner C System
°ü·ÃÇмú ±¸¸ÅÇϱ⠶óÀ̼±½º 
1 Kit
1,224,000¿ø  Á¦Á¶»ç ÆäÀÌÁö·Î ¹Ù·Î°¡±â x33526, x32971
Clontech
632168
ProteoTuner IRES2 System
°ü·ÃÇмú ±¸¸ÅÇϱ⠶óÀ̼±½º 
1 Kit
1,335,000¿ø  Á¦Á¶»ç ÆäÀÌÁö·Î ¹Ù·Î°¡±â x33524, x32971
Clontech
632171
Retro-X ProteoTuner System
°ü·ÃÇмú ±¸¸ÅÇϱ⠶óÀ̼±½º 
1 Kit
1,415,000¿ø  Á¦Á¶»ç ÆäÀÌÁö·Î ¹Ù·Î°¡±â x33541, x33009
Clontech
632167
Retro-X ProteoTuner IRES System
°ü·ÃÇмú ±¸¸ÅÇϱ⠶óÀ̼±½º 
1 Kit
1,415,000¿ø  Á¦Á¶»ç ÆäÀÌÁö·Î ¹Ù·Î°¡±â x33540, x33543, x33009
Clontech
632173
Lenti-X¢â ProteoTuner System
°ü·ÃÇмú ±¸¸ÅÇϱ⠶óÀ̼±½º 
1 Kit
1,399,000¿ø  Á¦Á¶»ç ÆäÀÌÁö·Î ¹Ù·Î°¡±â x33460, x32779
Clontech
631074
Lenti-X¢â ProteoTuner C System
°ü·ÃÇмú ±¸¸ÅÇϱ⠶óÀ̼±½º 
1 Kit
1,307,000¿ø  Á¦Á¶»ç ÆäÀÌÁö·Î ¹Ù·Î°¡±â x33463, x32779
Clontech
632175
Lenti-X¢â ProteoTuner Green System
°ü·ÃÇмú ±¸¸ÅÇϱ⠶óÀ̼±½º 
1 Kit
1,399,000¿ø  Á¦Á¶»ç ÆäÀÌÁö·Î ¹Ù·Î°¡±â x33459, x32779
Clontech
632189
Shield1
°ü·ÃÇмú ±¸¸ÅÇϱ⠶óÀ̼±½º 
500 ¥ìl
405,000¿ø  Á¦Á¶»ç ÆäÀÌÁö·Î ¹Ù·Î°¡±â x32971
Clontech
632188
Shield1
°ü·ÃÇмú ±¸¸ÅÇϱ⠶óÀ̼±½º 
5 mg
1,620,000¿ø  Á¦Á¶»ç ÆäÀÌÁö·Î ¹Ù·Î°¡±â
Clontech
631073
DD Monoclonal Antibody
°ü·ÃÇмú ±¸¸ÅÇϱâ
50 ¥ìl
406,000¿ø  Á¦Á¶»ç ÆäÀÌÁö·Î ¹Ù·Î°¡±â

  • ¸ñÀû ´Ü¹éÁúÀÇ ¹ßÇö ¼öÁØÀ» Á¤¹ÐÇÏ°í ½Å¼ÓÇÏ°Ô Á÷Á¢ Á¶Àý
  • ´ÜÀÏ vector ½Ã½ºÅÛ
  • ¸¹Àº ¼¼Æ÷¿Í ´Ü¹éÁú¿¡¼­ °ËÁõµÈ ½Ã½ºÅÛ
  • ¾î¶² promoter¿Íµµ ÇÔ²² »ç¿ë °¡´É
°£ÆíÇÏ°í È¿°úÀûÀÎ ±â¼ú
ProteoTuner systemÀº ¸ñÀû ´Ü¹éÁúÀ» in vivo¿¡¼­µµ ½Å¼ÓÇÏ°Ô Á÷Á¢ Á¶Àý °¡´ÉÇÑ ¸Å¿ì µ¶Ã¢ÀûÀÎ ±â¼úÀÌ´Ù. ´Ü¹éÁú ±â´É ¿¬±¸¸¦ À§ÇÑ °­·ÂÇÑ ÀÌ ±â¼úÀº 2 °¡Áö ÇÙ½É ¿ä¼Ò·Î ±¸¼º µÇ¾î ÀÖ´Ù.
  • 12 kDa destabilizing domain (DD)ÀÌ ¸ñÀû ´Ü¹éÁú°ú À¶ÇÕ ¹ßÇöµÇ¸é ´Ü¹éÁúÀ» ºÒ¾ÈÁ¤È­½ÃÄÑ proteosomal degradation °úÁ¤À¸·Î À¯µµÇÑ´Ù. DD coding sequence´Â vectorÀÇ multiple cloning site (MCS)¿¡ ÀÎÁ¢ÇÏ¿© Á¸ÀçÇÑ´Ù.
  • Shield1Àº ¼¼Æ÷¸· Åõ°ú¼ºÀÌ ÀÖ´Â 750 DaÀÇ ÀÛÀº molecular ligand·Î DD¿Í À¶ÇÕµÈ ´Ü¹éÁúÀÌ ºÐÇصÇÁö ¾Êµµ·Ï º¸È£ÇÑ´Ù.
Shield1Àº DD À¶ÇÕ ´Ü¹éÁúÀ» ¾ÈÁ¤È­½ÃÄÑ "protein on" »óÅ·ΠÀ¯µµÇÏ¿© ¼¼Æ÷ ³»¿¡ ´Ü¹éÁúÀÌ ÃàÀûµÇµµ·Ï ÇÑ´Ù. ÀÌ ¾ÈÁ¤È­´Â ´ÜÁö 15 ºÐ~30 ºÐ Á¤µµÀÇ ÂªÀº ½Ã°£ ¾È¿¡ ÀÌ·ç¾îÁø´Ù (1). ±×·¯³ª ¹èÁö±³È¯À» ÅëÇØ Shield1À» ¹èÁö¿¡¼­ Á¦°ÅÇÏ¸é ¼¼Æ÷ ³»¿¡ ÃàÀûµÇ¾î ÀÖ´ø DD À¶ÇÕ ´Ü¹éÁúÀº ºü¸£°Ô ºÐÇصǾî "Protein off" »óÅ°¡ µÈ´Ù.
µû¶ó¼­ Shield1ÀÇ ³óµµ Á¶ÀýÀ» ÅëÇØ ¼¼Æ÷ ³» ¸ñÀû ´Ü¹éÁúÀÇ ¾ÈÁ¤È­¸¦ Á¶ÀýÇÏ¿© ´Ü¹éÁúÀÇ ¹ßÇö ¾çÀ» Á¶ÀýÇÒ ¼ö ÀÖÀ¸¸ç, ´Ü¹éÁú ¹ßÇöÀÇ on/off °úÁ¤Àº °¡¿ªÀûÀÌ¸ç ¹Ýº¹ÀûÀ¸·Î Á¶ÀýÀÌ °¡´ÉÇÏ´Ù.


Figure 1. Ligand-dependent, targeted and reversible protein stabilization. A small destabilization domain (DD; blue) is fused to a target protein of interest. The small membrane-permeant ligand Shield1 (red) binds to the DD and protects it from proteasomal degradation. Removal of Shield1 causes rapid degradation of the entire fusion protein. The default pathway for the systems is degradation of the fusion protein, unless Shield1 is present.
´Ù¾çÇÑ ºÐ¾ß¿¡ Àû¿ë °¡´ÉÇÑ ½Ã½ºÅÛ
  • ¼¼Æ÷³» µµÀÔ ¹æ¹ý : ProteoTuner systemÀº lentivirus ¿ëÀ̳ª retroivirus ¿ë ±×¸®°í ÀϹÝÀûÀÎ plasmid ÇüÅ·Π±¸¼ºµÇ¾î ÀÖÀ¸¸ç, transfection control·Î Çü±¤´Ü¹éÁúÀ» ¹ßÇöÇÒ ¼ö ÀÖ´Â ÇüÅÂ¿Í ¹ßÇöÇÒ ¼ö ¾ø´Â ÇüÅ·Π±¸¼ºµÇ¾î ÀÖ´Ù.
  • DD ¼­¿­ÀÇ À¶ÇÕºÎÀ§¸¦ N-¸»´Ü ¶Ç´Â C-¸»´Ü ¼±Åà : ProteoTuner systemÀº DD domainÀÌ N-¸»´Ü¿¡ À¶Çյǰųª C-¸»´Ü¿¡ À¶ÇյǴ 2 °¡Áö ÇüÅ·ΠÁ¦°øµÇ°í ÀÖÀ¸¹Ç·Î ½ÇÇè¿¡ µû¶ó ÀûÀýÇÑ DD »ç¿ëÀÌ Áß¿äÇÏ´Ù.
    DD-C (ProteonTuner C system)´Â C-terminal tag·Î ÀûÇÕÇÏ°í ÀϹÝÀûÀÎ DD´Â N-terminal tag¸¦ ÀÌ¿ëÇÏ´Â °ÍÀÌ ÀûÇÕÇÏ´Ù.
´Ü¹éÁú ¹ßÇö ¼öÁØÀ» Á¶ÀýÇϸ鼭 Ç×ü³ª, Chemiluminescent tagÀ¸·Î °üÂû
  • DD Monoclonal Antibody´Â N-¸»´Ü°ú C-¸»´ÜÀÇ DD¸¦ ƯÀÌÀûÀ¸·Î °ËÃâÇÒ ¼ö ÀÖ´Ù. Western blotÀ̳ª immunocytochemistry·Î ¼¼Æ÷ ¿ëÇØ ÈÄ À¶ÇÕ ±¸Á¶¸¦ ½Äº°ÇÏ°í È®ÀÎÇÒ ¼ö ÀÖ´Ù. Antibody´Â DD-AcGFP1À» ÀϽÃÀûÀ¸·Î transfection ÇÑ 10,000 °³ ÀÌÇÏÀÇ ¼¼Æ÷¿¡¼­µµ DD-tag ´Ü¹éÁúÀ» °ËÃâ ÇÒ ¼ö ÀÖÀ» Á¤µµ·Î ¸Å¿ì ¹Î°¨µµ°¡ ³ô´Ù.
  • ProteoTuner Quantitiation System¿¡ Æ÷ÇÔµÈ vector´Â DD-tag (¹ßÇöÁ¶Àý)¿Í ProLabel-tag (Á¤·®)¸¦ °¡Áö°í ÀÖ´Ù. ÀÌ vectorÀÇ multicloning site (MCS)¿¡ ¸ñÀû ´Ü¹éÁúÀ» ¹ßÇöÇÏ´Â À¯ÀüÀÚ¸¦ cloningÇϸé N-¸»´Ü¿¡ DD coding sequence¿Í C-¸»´Ü¿¡ 6 kDaÀÇ ProLabel-tag°¡ °áÇÕµÈ ´Ü¹éÁúÀÌ ¹ßÇöµÈ´Ù. DD·Î Á¶ÀýµÇ´Â ´Ü¹éÁúÀÇ ¹ßÇö Á¤µµ´Â ProLabel °ËÃ⠽þàÀ¸·Î ½±°Ô °ËÃâÇÒ ¼ö ÀÖ´Ù.


Figure 2. Easy detection of DD fusions with the DD Monoclonal Antibody. Cell lysates from HeLa cells transiently expressing either DD-AcGFP1 or AcGFP1-DD, and HEK 293 cells stably expressing DD-AcGFP1, were analyzed by Western blot using the DD Monoclonal Antibody at a 1:500 dilution. Lane 1: HeLa cells transfected with pDD-AcGFP1 (e.g., DD-N).
Lane 2: Negative control (untransfected HeLa cells). Lane 3: HeLa cells transfected with pAcGFP1-DD (e.g., DD-C). Lane 4: Negative control (untransfected HEK 293 cells). Lane 5: HEK 293 cells stably expressing DD-AcGFP1.
À¯µµ¹ßÇöÀÇ ÀÌÁß Á¶Àý
pTRE-Cycle Vector´Â ´Ù¼öÀÇ ´Ü¹éÁú ¹ßÇöÀ» ÀÌÁßÀ¸·Î Á¶Àý °¡´ÉÇÏ´Ù (Figure 3). ¿ì¼± DD·Î À¶ÇÕµÈ ¸ñÀû ´Ü¹éÁúÀº Tet-inducible system¿¡ ÀÇÇÑ Àü»ç Á¶Àý°ú ProteoTuner system¿¡ ÀÇÇÑ ´Ü¹éÁú ºÐÇØ Á¶ÀýÀ» ÅëÇÏ¿© ÀÌÁßÀ¸·Î ¹ßÇöÀÌ Á¶ÀýµÇ¸ç, ´Ù¸¥ ¶Ç Çϳª´Â ¸ñÀû ´Ü¹éÁúÀ̳ª Çü±¤ ´Ü¹éÁú (mCherry ¶Ç´Â ZsGreen1)Àº Tet-inducible expression ¸¸À¸·Î ¹ßÇöÀ» Á¶ÀýÇÑ´Ù.


Figure 3. pTRE-Cycle Vectors for two-tiered expression control. pTRECycle1 allows you to coexpress two proteins of interest-one with a DD tag and one without. pTRE-Cycle2 and pTRE-Cycle3 allow you to inducibly coexpress a red or green fluorescent protein along with your DD-tagged protein of interest.
Components & Storage Conditions
°¢ Á¦Ç° ±¸¼º¹°°ú º¸°üÁ¶°ÇÀº Certificate of Analysis ¸¦ ÂüÁ¶ÇϽʽÿÀ.
References
1. Banaszynski, L. A. et al. (2006) Cell 126(5):995-1004.

Keyword :

´Ü¹éÁú ¹ßÇö·® Á¶Àý ½Ã½ºÅÛ (ProteoTuner)