Difference between revisions of "Part:BBa K1955000"

Latest revision as of 01:50, 22 October 2019

pSB1C3-Hemagglutinin

The coding sequence of Hemagglutinin (HA) from H1N1 acquired from NCBI. HA is a glycoprotein found on the surface of influenza viruses and can be used in immunological experiments as the target antigen.

In order to prove the HA(H1N1) sequence which we designed can express protein correctly, we use BL21 competent cell to express the HA sequence and detect the protein by Western blot analysis.

(1) Transform pSB1C3-J04500-HA into BL21 to express desired gene and validate the normal function of J04500 by GFP.

J04500 is a LacI inducible promoter, so it can be induced by IPTG to activate the promoter for transcription. We picked two colonies of pSB1C3-J04500-HA and pSB1A2-J04500-GFP and incubated in 4ml LB broth containing antibiotics at 37℃, 250rpm for overnight. Inoculate 4ml fresh LB supplemented with antibiotics with 20μl of overnight cultures. Shake cultures at 37℃, 250rpm for 3hr. Split cultures into two (2ml each) and add 2μl 1M IPTG to one of the tube to reach 1mM IPTG for induction. The other tube will be the uninduced control. Shake both tubes for another 3hrs at 37℃, 250rpm.

For the pSB1C3-J04500-HA, their induction can be checked by Coomassie blue or Western blot analysis. Transfer cultures from both tubes to two Eppendorf tubes and centrifuge bacteria at max speed for 3min. Discard the supernatant, and resuspend each pellet in 100μl 1x SDS sample buffer by pipetting. Fit cap-guards onto Eppendorf tubes and boil the samples at 100℃ for 10min. Run SDS-PAGE to check the expression of desired protein.

Conduct the Western blot analysis, we successfully recognize the HA protein (approximately 62.3kd) in our two colonies (JH1 and JH2) when induced by IPTG (Fig. 1A).

To check the feasibility of J04500, we also built up a construct containing J04500 and GFP. After induction of 3hrs, the induced bacteria turn fluorescent green. In contrast, the uninduced bacteria did not appear to have any color change. (Fig. 1B)



Fig. 1. Protein expression of pSB1C3-HA and pSB1A2-GFP.

The day before induction, two different colonies were picked up from each construct into 4ml LB broth at 37℃, 250rpm for overnight. Inoculated 4ml fresh LB broth with 20μl of overnight cultured at 37℃, 250rpm for 3hrs. Splited into two tubes(each 2ml), and added 2μl 1M IPTG to one tube as the induced group, the other tube without IPTG was the uninduced group. IPTG induction for 3~4hrs.

(A) After IPTG induction, centrifuged bacteria at max speed. Discarded the supernatant, lysed the cell with 100μl 1x SDS sample buffer. Western blotting to check the desired gene expression. In fig. 5A, His-tag HA serves as positive control to validate that the antibody can work normally. We also transform pUC-19 into BL21 and follow the induction protocol as the negative control.

(B) IPTG induction of pSB1A2-J04500-GFP to verify the LacI inducible promoter. The GFP fluorescence can be observed easily by bare eye.

The biobrick parts, including 5’HYG, 3’UTR, HA and OVA, were synthesized directly by IDT. After receiving the synthesized parts, we used EcoRI and PstI to digest the parts and pSB1C3 backbone, then ligated and transformed the DNA samples into DH5a competent cells. According to the digestion and colony PCR results of the colony, all the parts were inserted into the pSB1C3 vector with the right length of DNA sequences, 5’HYG is 1446 bp, HA is 1700 bp, OVA is 2098 bp and 3’UTR is 774 bp.


(Fig. 1) pSB1C3-3’UTR, pSB1C3-5’HYG, pSB1C3-OVA checked by colony PCR and enzyme digestion

(A),(C) The pSB1C3-3’UTR and pSB1C3-OVA were transformed and the colonies were picked to perform colony PCR. The forward primer sequence was 5’- GAATTCGCGGCCGCTTCTAGAG-3’, which was in the prefix site. And the reverse primer sequence was 5’-CTGCAGCGGCCGCTACTAGTA-3’, which was in the suffix site. The PCR reaction was performed with Taq polymerase, and screened in 0.8％ agarose gel by electrophoresis. As the results, a 700~800 bp sequence was proliferated in pSB1C3-3’UTR, and a 2000~2500 bp sequence was proliferated from pSB1C3-OVA. (B) The pSB1C3-5’HYG was transformed and the colonies were picked and amplified in LB broth. pSB1C3-5’HYG plasmid was purified by miniprep, and digested with EcoRI and PstI for 4 hrs, then screened in 0.8％ agarose gel by electrophoresis. The results showed a 2000 bp band of pSB1C3 and the 1500 bp 5’HYG.


(Fig. 2) The basic part checked by PCR

We used pSB1C3-5’HYG, pSB1C3-3’UTR, pSB1C3-HA, pSB1C3-OVA as template, to check the length of the inserts. The PCR reaction was performed with Taq polymerase, and screened in 0.8％ agarose gel by electrophoresis.

• Group: CSMU_Taiwan2019
• Author: YU-RU,LIN
• Summary: Fusion with LysRS, linker design, and codon optimization for improved solubility and efficiency production.

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.

Characterizaiton

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 pET29a, LysRS and BBa_K1955000. pET29b is 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.) IPTG was further added and the following incubation was 2 hrs.

Protein solubility analysis

To further characterize the solubility of this part, we then 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.

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.

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

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)