Difference between revisions of "Part:BBa K2951001"

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Influenza B virus nucleoprotein

Influenza B virus (B/Colorado/16/2017) nucleoprotein (NP) gene, complete cds

Usage and biology

 The nucleoprotein 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 is from an influenza B virus strain (B/Colorado/16/2017) gene. Part BBa_K2951003was further codon optimized based on this part for E.coli to reach higher expression.

• Influenza B virus nucleoprotein is abbreviated as “NPB” 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 western blotting to detect the content of our protein.

Fig.1 Western blotting for NPB large scale production, the band approximately at 63kDa is our target NPB protein(63.3kDa). 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

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. 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 vesion of BBa_K2951003, using the preferred codon for E.coli, named “NPA-original” here to distinguish between the optimized one. We did small scale production as described at 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 B optimized and NP B original. NP B optimized already shown stronger signal than NP B original even being 10x diluted, which indicates that it has much larger expression than the latter.

Sequence and Features