Part:BBa_K2951000

Influenza A virus nucleocapsid protein

Usage and Biology

The nucleocapsid protein is a protein that binds the negative-sense RNA segmented genome of the influenza virus. In addition, NP is relatively conserved across all known influenza strains and has a low sequence drift rate than the glycoproteins. Thus it serves as an ideal antigen for detection. The sequence of this part originates from an influenza A virus strain (A/Michigan/297/2017(H1N1). Part BBa_K2951002 is the codon-optimized version of it, intended to improve its expression efficiency.

• Influenza A virus nucleocapsid protein is abbreviated as “NPA” for convenience in the descriptions below.

Characterization

This part was synthesized by Taihe Biotechnology Co., Ltd and inserted into the pET29a plasmid. First, we transformed NPA into E. coli BL21 (DE3) strain to express our proteins. Our expression system is inducible with the addition of isopropyl-β-D-thiogalactopyranoside (IPTG) to expression culture since IPTG induces T7 RNA polymerase promoter leading to expression of the gene of interest in a plasmid.

Small scale production

Cultivations and Induction of protein expression

After transformation, 1 colony from the transformation colony plates were inoculated in 10 ml LB-kanamycin (50 μg/ml working concentration) and grew at +37 °C, 150rpm for 12-18hrs. 1cc was taken out to centrifuge and added 20ml of LB-kanamycin (50 μg/ml) to make the OD value~0.1. After incubation for another 2 hrs, it reached the OD595 value 0.4~0.7. (If the OD value exceeds 0.7, the culture would be diluted again.) When finished growing the cells, the 20ml was split into two, and one of them was induced to express the gene of interest by adding a final concentration of 0.5 mM IPTG in the cultures and the other tube as the non-induced control. Both were shaken at +37 °C for 2.5hrs. The OD value of each tube was then tested and taken V ml(V=2/OD) from each tube for centrifugation at 12000rpm to examine the expression result for the same amount of induced and non-induced colony.

Large scale production

Cultivations and Induction of protein expression

2 colonies from the plates were inoculated in two tubes of 15 ml LB-kanamycin (50 μg/ml working concentration) and grew the cells at +37 °C, 150rpm for 12-18hrs. 8cc was taken out from each tube to centrifuge and diluted with LB-kanamycin (50 μg/ml) to 200ml to make the OD value~0.1. After incubation for another 2 hrs, it reached the OD595 value 0.4~0.7. (if the OD value exceeds 0.7, the culture would be diluted again.) A final concentration of 0.5 mM IPTG was further added and the following incubation was 2.5 hrs.

Protein Purification and Dialysis

After sonicating and extracting the cell lysates, the nickel-resin column was utilized to purify our target proteins from the cell lysates because all of the proteins were tagged with 6 histidines fusion at their C-terminal ends due to fusion with pET29a plasmid. After protein purification, we did SDS-PAGE gel electrophoresis and western blotting to ensure our target protein was purified (Fig.1). A band appeared from E2-E4 at 55kDa. Indicating that the proteins on the correct site are our target NPA protein.

Fig.1 SDS PAGE coomassie blue staining of purification result M: marker; L: lysis, S1 form protein expression; FT: flow through, the protein that didn’t bound to resin gel; W: washing, including proteins bound to resin gel without His-tag, E: elution buffer

The high concentration of imidazole contained in the protein after purification could cause protein self-degeneration or aggregation. Thus, we dialyzed and concentrated the purified protein with dialysis tube by adding PBS buffer.

Expression efficiency improved by codon optimization

This part is the original version of BBa_K2951008, using the preferred codon for E.coli, named “NPA-original” here to distinguish between the optimized one. We did small scale production as described in the section above. We then did western blotting to examine the difference of expression degree between two parts(Fig.2).

Fig.2 This is the western blotting result of the expression from NP A optimized and NP A original. NP A optimized already shown stronger signal than NP A original even being 10x diluted, which indicates that it has much larger expression than the latter.

Sequence and Features

Assembly Compatibility:

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

  Illegal BamHI site found at 477
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 238