Thermostable blunt-end ligase and methods of use
Fusion proteins having thermostable blunt-end ligase activity are provided. Blunt-end ligases are useful for DNA amplification, sequencing, production of recombinant DNA and recombinant fusion proteins, and other purposes. These thermostable blunt-end DNA ligases are useful in ligation schemes which include, e.g., an incubation at about 60-65° C. or higher, or as high as about 94° C., or at other temperatures. The ligases disclosed herein may enable high temperature blunt-end ligation without need for molecular crowding agents, and so may be useful for many nucleic acid ligation-amplification schemes, e.g., ones which operate at a uniform temperature (e.g., at about 60° C. or higher), including ones which require temperature cycling, e.g., from about 94° C. to about 60° C. (or higher) for one, two, three, or more cycles. The thermostable blunt-end DNA ligases disclosed herein enable higher specificity target amplification, for example, by permitting temperature denaturation of double-stranded DNA templates as well as specific primer binding.
1. A thermostable blunt-end DNA ligase suitable for use in a blunt-ended DNA ligation reaction performed at about 60° C. or higher, comprising the amino acid sequence:
(SEQ ID NO: 8)
mghhhhhhhhhhssghiegrasadgpylqileqpkqrgfrfryvcegpshg
glpgasseknkksypqvkicnyvgpakvivqlvtngknihlhahslvgkhc
edgictvtagpkdmvvgfanlgilhvtkkkvfetlearmteacirgynpgl
lvhpdlaylqaegggdrqlgdrekelirqaalqqtkemdlsvvrlmftafl
pdstgsftrrlepvvsdaiydskapnasnlkivrmdrtagcvtggeeiyll
cdkvqkddiqirfyeeeenggvwegfgdfsptdvhrqfaivfktpkykdin
itkpasvfvqlrrksdletsepkpflyypeikdkeevqrkrqkgssgtsgg
gsgggmtleearkrvnelrdliryhnyryyvladpeisdaeydrllrelke
leerfpelkspdsptlqvgarpleatfrpvrhptrmysldnafnldelkaf
eerieralgrkgpfaytvehkvdglsvnlyyeegvlvygatrgdgevgeev
tqnlltiptiprrlkgvperlevrgevympieaflrlneeleergerifkn
prnaaagslrqkdpritakrglratfyalglgleeveregvatqfallhwl
kekgfpvehgyaravgaegveavyqdwlkkrralpfeadgvvvkldelalw
relgytaraprfaiaykfpaeeketrlldvvfqvgrtgrvtpvgilepvfl
egsevsrvtlhnesyieeldirigdwvlvhkaggvipevlrvlkerrtgee
rpirwpetcpecghrllkegkvhrcpnplcpakrfeairhfasrkamdiqg
lgeklierllekglvkdvadlyrlrkedlvglermgeksaqnllrqieesk
krglerllyalglpgvgevlarnlaarfgnmdrlleasleelleveevgel
tarailetlkdpafrdlvrrlkeagvemeakekggealkgltfvitgelsr
preevkallrrlgakvtdsvsrktsylvvgenpgsklekaralgvptltee
elyrlleartgkkaeelv.
2. An article of manufacture, comprising the thermostable blunt-end DNA ligase of claim 1 and a container.
3. The article of manufacture of claim 2 , further comprising a buffer.
4. The thermostable blunt-end DNA ligase of claim 1 , wherein said thermostable blunt-end DNA ligase is suitable for use in a blunt-ended DNA ligation reaction performed at about 75° C.
5. The thermostable blunt-end DNA ligase of claim 1 , wherein said thermostable blunt-end DNA ligase is capable of making concatamers upon multiple ligation events in a blunt-ended DNA ligation reaction performed at about 60° C. or higher.
6. The thermostable blunt-end DNA ligase of claim 1 , wherein said thermostable blunt-end DNA ligase is suitable for use in a nucleic acid amplification scheme which operates at a uniform temperature of about 60° C. or higher.
7. The thermostable blunt-end DNA ligase of claim 1 , wherein said thermostable blunt-end DNA ligase is suitable for use in a nucleic acid amplification scheme which comprises temperature cycling.
8. The thermostable blunt-end DNA ligase of claim 7 , wherein said nucleic acid amplification scheme comprises temperature cycling to temperatures of about 60° C. or higher.
9. The thermostable blunt-end DNA ligase of claim 7 , wherein said nucleic acid amplification scheme comprises temperature cycling from about 94° C. to about 60° C.
10. The thermostable blunt-end DNA ligase of claim 9 , wherein said nucleic acid amplification scheme comprises two or more cycles of temperature cycling.
11. The thermostable blunt-end DNA ligase of claim 9 , wherein said nucleic acid amplification scheme comprises three or more cycles of temperature cycling.
12. The thermostable blunt-end DNA ligase of claim 1 , wherein said fusion protein comprises a component selected from a peptide linker, an N-terminal addition, a C-terminal addition, a tag peptide, a D-amino acid, and a peptide mimetic.
13. The thermostable blunt-end DNA ligase of claim 1 , wherein said fusion protein comprises a component selected from a sugar and a polymer.
14. The thermostable blunt-end DNA ligase of claim 1 , consisting of the amino acid sequence:
(SEQ ID NO: 8)
mghhhhhhhhhhssghiegrasadgpylqileqpkqrgfrfryvcegpshgglpgasseknkksypqvkicnyvgpakvivqlvtngknihlh
ahslvgkhcedgictvtagpkdmvvgfanlgilhvtkkkvfetlearmteacirgynpgllvhpdlaylqaegggdrqlgdrekelirqaalqqtke
mdlsvvrlmftaflpdstgsftrrlepvvsdaiydskapnasnlkivrmdrtagcvtggeeiyllcdkvqkddiqirfyeeeenggvwegfgdfspt
dvhrqfaivfktpkykdinitkpasvfvqlrrksdletsepkpflyypeikdkeevqrkrqkgssgtsgggsgggmtleearkrvnelrdliryhnyr
yyvladpeisdaeydrllrelkeleerfpelkspdsptlqvgarpleatfrpvrhptrmysldnafnldelkafeerieralgrkgpfaytvehkvdgls
vnlyyeegylvygatrgdgevgeevtqnlltiptiprrlkgyperlevrgevympieaflrlneeleergerifknprnaaagslrqkdpritakrglra
tfyalglgleeveregvatqfallhwlkekgfpvehgyaravgaegveavyqdwlkkrralpfeadgvvvkldelalwrelgytaraprfaiaykf
paeeketrlldvvfqvgrtgrvtpvgilepvflegsevsrvtlhnesyieeldirigdwvlvhkaggvipevlrvlkerrtgeerpirwpetcpecghrl
lkegkvhrcpnplcpakrfeairhfasrkamdiqglgekliellekglvkdvadlyrlrkedlvglermgeksaqnllrqieeskkrglerllyalgl
pgvgevlarnlaarfgnmdrlleasleelleveevgeltarailetlkdpafrdlvrrlkeagvemeakekggealkgltfvitgelsrpreevkallrrl
gakvtdsysrktsylvvgenpgsklekaralgyptlteeelyrlleartgkkaeelv.
15. The thermostable blunt-end DNA ligase of claim 1 , consisting of an amino acid having the amino acid of SEQ ID NO: 8 linked to a sugar.
16. The article of manufacture of claim 2 , wherein said container comprises a sterile access port.
17. The article of manufacture of claim 2 , wherein said thermostable blunt-end DNA ligase comprises a sugar.