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 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 addition of isopropyl-β-D-thiogalactopyranoside (IPTG) to expression culture, since IPTG induces T7 RNA polymerase promoter leading to expression of gene of interest in plasmid.
Small scale production
Cultivations and Induction of protein expression
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 uninduced control. Both were shaked 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.
Large scale production
Cultivations and Induction of protein expression
After transformation, 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 are 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 gel electrophoresis 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
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 at 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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE 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 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 828
- 1000COMPATIBLE WITH RFC[1000]
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