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´Ù¾çÇÑ ¿Âµµ¿¡¼­ ¹ÝÀÀÇÒ ¼ö ÀÖ´Â Capping enzyme

Faustovirus Capping Enzyme (S17)

-

Á¦Á¶»ç Á¦Ç°ÄÚµå Á¦Ç°¸í ¿ë·® °¡°Ý
(ºÎ°¡¼¼º°µµ)
ºñ°í »ç¿ëÀڸŴº¾ó
Takara
2480A
Faustovirus Capping Enzyme (S17)
°ü·ÃÇмú ±¸¸ÅÇϱ⠶óÀ̼±½º 
500 U
160,000¿ø  2480a_ds_j.pdf
Takara
2480B
Faustovirus Capping Enzyme (S17)
°ü·ÃÇмú ±¸¸ÅÇϱ⠶óÀ̼±½º 
2,000 U (2480Ax4)
576,000¿ø 

Á¦Ç°¼³¸í
Faustovirus Capping Enzyme (S17) (;FCE)Àº Faustovirus S17±ÕÁÖ À¯·¡ÀÇ È¿¼Ò·Î ´ÜÀÏ subunitÀ» °¡Áø ´Ü¹éÁúÀÌ´Ù. ÀÌ È¿¼Ò´Â ±âÁ¸¿¡ »ç¿ëµÇ´ø vaccinia ¹ÙÀÌ·¯½º À¯·¡ÀÇ Vaccinia Capping Enzyme (Code 2460A) (;VCE)°ú À¯»çÇÑ ÀÛ¿ë ±âÀüÀ» °¡Áö°í ÀÖ¾î RNA (5'-triphosphate RNA)ÀÇ 5¡¯ ¸»´Ü¿¡ 7-methylguanylate cap ±¸Á¶ (Cap 0)¸¦ ºÎ°¡ÇÏ´Â È°¼ºÀ» °¡Áø´Ù. ÀÌ È¿¼Ò¸¦ »ç¿ëÇϸé IVT¿¡¼­ È¿°úÀûÀ¸·Î ¿ÏÀüÇÑ Cap 0 ±¸Á¶¸¦ °¡Áö´Â RNA¸¦ ÇÕ¼ºÇÒ ¼ö ÀÖ°í ¿©±â¿¡ mRNA Cap 2-O-Methyltransferase (Code 2470A)¸¦ °°ÀÌ »ç¿ëÇϸé Cap 0ÀÇ ±¸Á¶ RNA¿¡¼­ ù¹ø° ´ºÅ¬·¹¿ÀŸÀ̵åÀÇ 2¡¯-O ºÎºÐÀ» ¸ÞÆ¿È­ÇÏ¿© Cap 1ÀÇ ±¸Á¶ÀÇ RNA¸¦ single stepÀ¸·Î ÇÕ¼ºÇÏ´Â °Í ¿ª½Ã °¡´ÉÇÏ´Ù*.

Vaccinia Capping Enzyme (Code 2460A)º¸´Ù ´Ù¾çÇÑ ¿Âµµ¿¡¼­ ³ôÀº È°¼ºÀ» º¸ÀÌ°í (30~50¡É) (±×¸² 1), ´Ù¾çÇÑ RNA¿¡ Àû¿ë °¡´ÉÇϱ⠶§¹®¿¡ mRNA ¹é½Å ¹× Ä¡·áÁ¦ µî ºÐ¾ßÀÇ ¿¬±¸ °³¹ß¿¡ À¯¿ëÇÏ´Ù.

*Uncapped RNA¸¦ 1 stepÀ¸·Î Cap-1 RNA·Î ÇÕ¼ºÇÏ°íÀÚ ÇÏ´Â °æ¿ì, mRNA Cap 2-O-Methyltransferase (Code 2470A)¿¡ Æ÷ÇÔµÈ ¹öÆÛ ´ë½Å Faustovirus Capping Enzyme¿¡ Æ÷ÇÔµÈ 10¡¿Capping Buffer 2 ¸¦ »ç¿ëÇϼ¼¿ä.


±×¸² 1. ¹ÝÀÀ¿Âµµ¿¡ µû¸¥ FCE¿Í VCEÀÇ capping È¿À²
FCE¿Í VCE µÎ Á¾·ùÀÇ capping enzymeÀ» »ç¿ëÇØ ´Ù¾çÇÑ ¿Âµµ¿¡¼­ capping ¹ÝÀÀÀ» ÁøÇàÇÏ°í LC/MS·Î capping È¿À²À» (Cap 0 ºñÀ²)À» ÃøÁ¤Çß´Ù. ±× °á°ú FCE°¡ VCEº¸´Ù ³ÐÀº ¿Âµµ ¹üÀ§¿¡¼­ ³ôÀº capping È¿À²À» º¸¿´´Ù.
¿ëµµ
- Cap 0 ±¸Á¶¸¦ °®´Â mRNA ÇÕ¼º
- mRNA Cap 2-O-Methyltransferase¿Í º´¿ëÇÏ¿© Cap 1 ±¸Á¶¸¦ °®´Â mRNA ÇÕ¼º
³»¿ë

±¸¼ºÇ°

¿ë·®

Faustovirus Capping Enzyme (S17) (25 U/¥ìl)

500 U

10¡¿Capping Buffer 2

100 §¡

S-adenosylmethionine (SAM) (32 mM)

100 §¡

GTP (10 mM)

50 §¡

º¸Á¸: -20 ¡É
³óµµ: 25 U/§¡
±â¿ø
Escherichia coli carrying a plasmid containing the gene for Faustovirus capping enzyme (S17)
ÀϹÝÀûÀÎ ¼ºÁú
- ºÐÀÚ·®: ¾à 100 kDa
È°¼ºÀÇ Á¤ÀÇ
37 ¡É¿¡¼­ 30ºÐ µ¿¾È 5 pmolÀÇ 100 nt Àü»çü¸¦ Cap 0 RNA·Î Àüȯ½ÃÅ°´Â È¿¼ÒÀÇ ¾çÀ» 1UÀ¸·Î ÇÑ´Ù.
È°¼º ÃøÁ¤¿ë ¹ÝÀÀ¾×ÀÇ Á¶¼º

1 x

 

Capping Buffer 2

25 §¡

 

GTP

20 ¥ìCi/§¢

 

[3H]SAM

2.5 §¶/50 §¡

 

100 nt RNA

»ç¿ë ¿¹
1. Uncapped RNA (5¡¯-triphosphate RNA)¿¡¼­ Cap 0/Cap 1 RNA ÇÕ¼º [2 step reaction]

RNA transcript (50 §¶)*1

 

38 §¡

10x Capping Buffer 2

 

5 §¡

GTP (10 mM)

 

2.5 §¡

SAM (2 mM, dilute 32 mM stock to 2 mM)*2

 

2.5 §¡

Faustovirus Capping Enzyme (S17)*3

 

2 §¡

Total

 

50 §¡ *4

37¡É¿¡¼­ 30ºÐ incubation*3
À̾ mRNA Cap 2-O-Methyltransferase (Code 2470A)·Î Cap 1 RNA ÇÕ¼º °¡´É

2. Uncapped RNA (5¡¯-triphosphate RNA)¿¡¼­ Cap 1 RNA ÇÕ¼º [1 step reaction]

RNA transcript (50 §¶)*1

 

34 §¡

10x Capping Buffer 2

 

5 §¡

GTP (10 mM)

 

2.5 §¡

SAM (4 mM, dilute 32 mM stock to 4 mM)*2

 

2.5 §¡

Faustovirus Capping Enzyme (S17)*3

 

2 §¡

mRNA Cap 2¡¯-O-Methyltransferase (50 U/§¡)*3

 

4 §¡

Total

 

50 §¡ *4

37¡É¿¡¼­ 30ºÐ incubation*3

*1 RNA (5¡¯-triphosphate RNA)ÀÇ 5¡¯¸»´ÜÀÌ º¹ÀâÇÑ 2Â÷ ±¸Á¶¸¦ °¡Áø °æ¿ì ¹ÝÀÀ Àü ¿­·Î º¯¼º½ÃÅ°´Â °ÍÀ¸·Î capping È¿À²ÀÌ ³ô¾ÆÁú ¼ö ÀÖ´Ù.
<¿­ º¯¼º>
    (1) Á¤Á¦µÈ RNA 50 §¶À» RNase free water·Î 38 §¡ (Cap 0 RNA ÇÕ¼º) ȤÀº 34 §¡ (Cap 1 RNA ÇÕ¼º)·Î Èñ¼®ÇÑ´Ù.
    (2) 65¡É¿¡¼­ 5~10ºÐ ¿­º¯¼º ÈÄ 5ºÐµ¿¾È ¾óÀ½¿¡¼­ incubationÇÑ´Ù.
*2 SAMÀº ºÒ¾ÈÁ¤ÇÑ ¹°ÁúÀ̱⠶§¹®¿¡ ¹ÝÀÀ Àü ÇÊ¿äÇÑ ¾çÀ» ¹Ì¸®32mM stock solution¿¡¼­ RNase free water·Î Èñ¼®ÇÏ¿© »ç¿ëÇÑ´Ù. Èñ¼®µÈ ½Ã¾àÀº ¹ÝÀÀ Àü±îÁö ¾óÀ½ À§¿¡¼­ º¸°üÇÑ´Ù.
*3 RNAÀÇ capping È¿À²ÀÌ ³·Àº °æ¿ì È¿¼ÒÀÇ »ç¿ë·®À» ´Ã¸®°Å³ª ¹ÝÀÀ ½Ã°£À» ´Ã¸°´Ù.
*4 ¹ÝÀÀ¾×ÀÇ ºÎÇÇ´Â ÇÊ¿ä¿¡ µû¶ó ½ºÄÉÀϾ÷ °¡´ÉÇÏ´Ù.
*5 mRNA Cap 2-O-Methyltransferase (Code 2470A)¿¡ Æ÷ÇÔµÈ ¹öÆÛ°¡ ¾Æ´Ï¶ó Faustovirus Capping Enzyme (S17)¿¡ ÃÖÀûÈ­µÈ ¹öÆÛ (10¡¿Capping Buffer 2)¸¦ »ç¿ëÇØ¾ß ÇÑ´Ù.
°ü·ÃÁ¦Ç°
- °íÇ°Áú °í¼öÀ² in vitro transcription ½Ã½ºÅÛ: Takara IVTpro¢â mRNA Synthesis System (Code 6141)
- dsRNA È¥ÀÔÀÌ Àû¾î ÀÓ»óºÐ¾ß ¿¬±¸°³¹ß¿¡ ÀûÇÕ: PrimeCap¢â T7 RNA Polymerase (low dsRNA) (Code 2560A)
- Capping enzyme (cap-0 RNA -> cap-1 RNA): mRNA Cap 2-O-Methyltransferase (Code 2470A)
- IVTÀü vectorÀÇ ¼±ÇüÈ­¿¡ »ç¿ëÇÏ´Â Type IIS Á¦ÇÑÈ¿¼Ò: BspQ I (Code 1227A)
- BspQ I Àνļ­¿­ÀÌ ÀÖ´Â IVT template vector: Template Vector (BspQ I) for T7 mRNA Synthesis (Code 6146)
- mRNA ¹ßÇöÀ» ³ôÀÌ´Â Boost½Ã¾àÀÌ Æ÷ÇÔµÈ transfection reagent: TransIT¢ç-mRNA Transfection Kit (Code MIR 2250)

Keyword : FCE

in vitro transcription
>
°íÇ°Áú °í¼öÀ² in vitro transcriptionÀ» À§ÇÑ ..
Takara IVTpro¢â mRNA Synthesis System
>
dsRNA »ý¼ºÀÌ Àû¾î ÀÓ»óºÐ¾ßÀÇ ¿¬±¸°³¹ß¿¡ ÀûÇÕ..
PrimeCap¢â T7 RNA Polymerase (low dsRNA)
>
Poly(A) ÀÌÈÄ Ãß°¡¼­¿­ÀÌ ¾ø´Â scarless mRNA ..
Template Vector (BspQ I) for T7 mRNA Synthesis
>
RNAÀÇ 5¡¯-¸»´Ü¿¡ Cap (Cap 0)À» ºÎ°¡ÇÏ´Â È¿¼Ò
Vaccinia Capping Enzyme
>
RNAÀÇ 5¡¯ ¸»´ÜÀ» Cap 0 ¡æ Cap 1·Î º¯È¯ÇÏ´Â ..
mRNA Cap 2-O-Methyltransferase
>
´Ù¾çÇÑ ¿Âµµ¿¡¼­ ¹ÝÀÀÇÒ ¼ö ÀÖ´Â Capping enzym..
Faustovirus Capping Enzyme (S17)
>
Poly(A) ¹× ¹Ýº¹ ¼­¿­À» Æ÷ÇÔÇÏ´Â À¯ÀüÀÚÀÇ Å¬..
Takara Stable Competent Cells
>
RNA ÇÕ¼º ¼öÀ²ÀÌ Çâ»óµÈ
T7 RNA Polymerase ver.2.0
>
Àú³óµµ DTT¿¡¼­µµ ¾ÈÁ¤ÀûÀÎ RNase inhibitor
Recombinant RNase Inhibitor ver.2.0
>
DNA, RNA ÇÕ¼º ¹ÝÀÀÀ» ÃËÁø
Pyrophosphatase (inorganic)
>
IVT Àû¿ë¿¡ ÃÖÀû, Type IIS Á¦ÇÑÈ¿¼Ò
BspQ I
>
´Ù¾çÇÑ Å©±âÀÇ ssRNA »çÀÌÁî È®ÀÎ
0.5-10 kb ssRNA Ladder Marker
>
BspQ IÀ» »ç¿ëÇÑ °í¼öÀ² IVT ½Ã½ºÅÛ
(BspQ I) IVTpro¢â ½Ã½ºÅÛ
>
Low dsRNA Polymerase¸¦ »ç¿ëÇÑ °í¼öÀ² IVT ½Ã..
(PrimeCap¢â T7) IVTpro¢â ½Ã½ºÅÛ