Part:BBa_K2951008

LysRS-Hemagglutinin fusion protein

This is a fusion protein of LysRS and Hemagglutinin. LysRS is lysyl tRNA synthase from E.coli str. K-12 substr. MG1655, it is found that it could act as a fusion protein partner and increase the solubility of the other significantly. Hemagglutinin is a surface glycoprotein of influenza viruses, and can be used in immunological experiments as the target antigen. Hemagglutinin is one of the transmembrane proteins that are considered difficult to express as soluble form in E.coli. Combining the two parts together, we call it as “LysRS-HA”. This is also an improvement of a previous part BBa_K1955000. In addition to expressing hemagglutinin in soluble form efficiently, we also added His-tag for further purification, TEV cutting site for removal of LysRS after expression, and a polylinker in order for others to construct a plasmid and applications with other hemagglutinin subtypes or even other proteins that are expressed as insoluble form in E.coli.

Fig.1 The picture shows the construct of this part.

Characterization

This part and the previous part BBa_K1955000 was synthesized by Taihe Biotechnology Co., Ltd and inserted into the pET29a plasmid. First, we transformed LysRS-pET29a 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 plates were inoculated in 10 ml LB-kanamycin (50 μg/ml working concentration) and grew the cells 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. We then tested their OD value and take V ml(V=2/OD) from each tube for centrifugation at 12000rpm. The induction result was checked by SDS-PAGE coomassie blue staining (Fig.2)

Fig.2 SDS-PAGE coomassie blue staining for small scale production of pET29b, LysRS and BBa_K1955000. pET29b was also transformed into BL21 and followed the induction protocol as negative control.

Large scale production

Cultivations and Induction of protein expression

2 colonies from the transformation 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 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 (the result is shown in Fig.3a and 3b.) In Fig. 3a, we could find there was better expression than BBa_K1955000 since the latter result could not be observed by SDS-PAGE(data not shown). In Fig. 3b(I), there was more “16000 G S” group when compared with the “16000G P” group. This result meant that most proteins were dissolved in the supernatant while few proteins deposited in the cell pellet after 16000G centrifugation. However, we infer that BBa_K1955000 does not have good solubility since many proportions of it is in “8700G P”.Their molecular weights are listed below.

Name	Molecular Weight
LysRS-HA	120.63 kDa
BBa_K1955000	64.02 kDa

Fig.3a SDS-PAGE coomassie blue staining for large scale production. T meant the initial sample obtained after sonication; 8700G P and 8700G S meant the pellet and the supernatant obtained after 8700G for 20 min; 8700G S and 16,000G T meant the pellet and the supernatant gotten after 8700G for 20 min.

Fig.3b Western Blotting for large scale production (I)LysRS-HA (II)BBa_K1955000. T meant the initial sample obtained after sonication; 8700G P and 8700G S meant the pellet and the supernatant obtained after 8700G for 20 min; 8700G S and 16,000G T meant the pellet and the supernatant gotten after 8700G for 20 min;16,000G P and 16,000G S meant the pellet and the supernatant obtained after 16,000G for 20 min.

Protein purification and dialysis

After extracting the cell lysates, we used nickel-resin column to purify our target proteins from the cell lysates because all of our proteins were tagged with 6 histidines fusion at their C-terminal ends with the pET29a plasmid. After protein purification, protein dialysis with PBS buffer to remove imidazole in our purified proteins, we did SDS-PAGE gel electrophoresis to ensure our target proteins were purified(Fig.4).

Fig.4 SDS-PAGE of LysRS-HA purification result.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. A band appeared in E2 approximately at 120kDa, which is the correct molecular weight of our target protein.

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.

References

1.Yo Han Jang, Seung HeeCho, Ahyun Son, Yun Ha Lee, Jin hee Lee, Kwang-Hee Lee, Baik Lin Seong, High-yield soluble expression of recombinant influenza virus antigens from Escherichia coli and their potential uses in diagnosis, Journal of Virological Methods,Volume 196, February 2014, Pages 56-64 (https://doi.org/10.1016/j.jviromet.2013.10.035)

Sequence and Features

Assembly Compatibility:

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

  Illegal NotI site found at 275
  Illegal NotI site found at 1256
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 864
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 1845
  
• 1000
  COMPATIBLE WITH RFC[1000]