Part:BBa_K2989008

DNLS-NS5B

Nonstructural protein 5B (NS5B) is a viral protein found in the hepatitis C virus (HCV). It's a kind of RNA-dependent RNA polymerase which means it is an enzyme that replicates RNA using RNA as a template. You can use it to generate double-stranded RNAs or substitutional hybridization in situ in the nucleus.

Usage

At the C-terminus of the original NS5B, we added a nuclear localization sequence with a repeat sequence to ensure that the protein can enter the nucleus efficiently so that it can be further designed in transcriptional regulation.

A: NS5B (no NLS)
B: DNLS-NS5B (after adding NLS)
We transfected NS5B and DNLS-NS5B with NLS (nuclear localization sequence) into HepG2 cells respectively and detected them by immunofluorescence. The results are shown in figures. NLS-free NS5B was mainly expressed in the cytoplasm, while the NS5B protein with NLS (nuclear localization sequence) was obviously colocalized with the nucleus, which proved that NS5B entered the nucleus successfully.

Biology

NS5B is essentially a kind of RdRP in non-structural proteins and has no functional similarities in human cells. With a typical "right hand" polymerase shape, the finger, palm and thumb subdomains surround a complete circular active site. The subdomain of the finger interacts with the nucleoside triphosphate while binding to the template according to the base pairing principle. The palm domain is the catalytic center for the nucleotide transfer reaction. The thumb subdomain plays an important role in the localization of RNA replication and elongation.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 492
  Illegal BglII site found at 1340
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 537