Part:BBa_K2951002

Influenza A virus nucleocapsid protein codon optimized for E.coli

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 lower 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) and it was further codon-optimized for E.coli to reach higher expression in comparison to part BBa_K2951000.

• 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 the plasmid.

Small scale production

Cultivations and Induction of protein expression

Random 1 of the plated transformation colony 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 not added to the other tube as the uninduced 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 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.) IPTG was further added and the following incubation was 2.5 hrs.

Protein Solubility Analysis

To further characterize the solubility of this part, we sonicated the culture and did 8700G and 16,000G centrifugation. We then did SDS-PAGE for coomassie brilliant blue staining and western blot to detect the content of our protein (Fig.1). Most of the production is found in S and S1, indicating that the expressed proteins are highly soluble. The dark band also indicates that both of them are the main products of E.coli expression.

Fig.1 SDS PAGE for NPA large scale production, the framed band is our target NPA protein(approximately 55kDa).M:Marker; T:Total, cell lysis; P:precipitate, the precipitation after 8700G centrifuge; S: soluble, the supernatant after 8700G centrifuge; T1: Total1, identically the supernatant after 16,000G; P1:the precipitation after 16,000G centrifuge; S1:the supernatant after 16,000G centrifuge.

Protein Purification and Dialysis

As seen in Fig1., many other non-target protein existed in the solution. Hence, purification was taken out in the next step. After extracting the cell lysates, 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 coomassie brilliant blue staining and western blotting to ensure our target protein was purified(Fig.2a and b). An obvious band appeared from E1~E8 as framed at 55kDa. Indicating that the proteins on the correct site are our target NPA protein.

Fig.2a Purification result: 2a.SDS PAGE 2b.Western Blotting 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 a dialysis tube by adding gradient concentrations of imidazole(150M, 50M, 0M) and 0.01M PBS buffer, and by 30mins of 6000rpm centrifugation, this sorts out the imidazole and preserves the protein on membrane.

Expression efficiency improved by codon optimization

This part, which we named “NP A optimized”, is a codon optimization version of BBa_K2951000, using the preferred codon for E.coli, which we named “NP A original”. We did small scale production as described in the section above. We then ran SDS-PAGE coomassie blue staining and western blotting to examine the difference of expression degree between two parts(Fig.3).

Fig.3 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 BglII site found at 762
  Illegal BglII site found at 1017
  Illegal BglII site found at 1362
  Illegal BamHI site found at 477
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 828
  
• 1000
  COMPATIBLE WITH RFC[1000]