Team:CSMU Taiwan/Improve
Abstract
This year, we aimed to develop a rapid screening kit for influenza. We enabled the kit to become a tool to further obtain more detailed epidemiology statistics for better measures of prevention. Thus, one of the aspects of our project was to distinguish between the subtypes of influenza with Hemagglutinin (HA). To select aptamers against HA, expression of a certain amount was needed. However, the lack of general technical platform for the timely supply of soluble and highly purified influenza HA presents a bottleneck for the subsequent analysis for the effective control of the viral disease.
For this reason, we were contributing to the iGEM community with BBa_K2951008 by improving an existing part: BBa_K1955000, codon optimizing it and also using the Escherichia coli (E. coli) lysyl tRNA synthetase (LysRS) as a fusion partner. In addition to express hemagglutinin in soluble form efficiently, we also added His-tag, TEV cutting site and polylinker in order to construct a plasmid for further purification, aptamer selection, and applications for other hemagglutinin subtypes. Detailed descriptions can be seen in the following section
Sequence selection and Codon Optimization
The previous part (BBa_K1955000) was documented to be acquired from NCBI with no further indication. The sequence of HA strain(A/WSN 1933/TS61(H1N1)) is based on NCBI(CY010788.1), being 7 bp difference to the previous part(Fig.1).
To express the target protein, we chosed to use BL21 E.coli strain with a pET29a vector. In order to improve the expression efficiency of our chassis, we first codon optimized the sequence and checked for illegal cutting sites using ATGme for E.coli.
Fig.1Sequence alignment of NCBI(CY010788.1) AND BBa_K1955000 protein coding part. The 7bp difference is indicated by color yellow.
Improved Solubility
First, we analyzed the transmembrane domain of HA using TMHMM Server (Fig.2) to delete its coding sequence and the four hydrophobic amino acids’ in front, which is a total of 35 a.a. .
Fig.2Transmembrane domain of analyzed by TMHMM
Second, we added a novel fusion partner to promote its folding and solubility--Escherichia coli (E. coli) lysyl tRNA synthetase (LysRS), whose coding sequence was obtained from NCBI (NC_000913.3). The sequence is also optimized and checked for illegal restriction sites.
TEV Cutting site
Considering that our expressed HA will be used as a target protein for further aptamer selection with SELEX, the removal of the protein generated from LysRS is needed. Thus, we added a TEV protease cutting site (GAAAACCTGTATTTTCAGGGC) obtained from BBa_K1319016 between the coding sequence of LysRS and HA.
Further Applications
Adding the histidine tag creates the opportunity to use a simpler method of purification, as well as increase selectivity for the desired protein, which is necessary for the next step of SELEX after expression.
Furthermore, with a view to construct a plasmid as a ready-made vector for high yield soluble different HA subtypes, we designed a polylinker(Fig.3) between LysRS and HA.
Experimental Data
The previous part BBa_K1955000 and this part BBa_K2951008 as an improvement, was inserted into pET29a vector. They were transformed into E.coli BL21(DE3) strain to express our proteins. pET29b was also transformed into BL21 and followed the induction protocol as negative control.Our expression system is inducible with addition of IPTG to expression culture. Small and large scale production was first done to confirm that we have successfully express the target protein needed in our project-hemagglutinin(Fig.3).
Fig.3SDS-PAGE coomassie blue staining for small scale production of pET29a, LysRS-HA and BBa_K1955000.
Protein solubility analysis
To further characterize the solubility of this part, we then sonicated the culture and did 8700G and 16,000G centrifugation. In Fig.4, we could find that there was better expression than BBa_K1955000 since the latter result could not be observed by SDS-PAGE. To be sure that we did expressed BBa_K1955000, further western blotting results were examined(Fig.5). In Fig.5(I), there was more “16000G 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 infered that BBa_K1955000 did not have good solubility since many proportions of it is in “8700G P”(Fig.5(II)).
Fig.4 SDS-PAGE coomassie blue staining for large scale production. T:the initial sample obtained after sonication; 8700G P and 8700G S:the pellet and the supernatant obtained after 8700G for 20 min; 8700G S and 16,000G T:the pellet and the supernatant gotten after 8700G for 20 min.
Fig.5 Western Blotting for large scale production (I)LysRS-HA (II)BBa_K1955000.
Protein purification and dialysis
We used nickel-resin column to purify our target proteins from the cell lysates, protein dialysis with PBS buffer to remove imidazole in our purified proteins and SDS-PAGE gel electrophoresis to ensure our target proteins were purified.
Fig.6 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.
For detailed experiment conditions and method, please check out our documentation on Part’s main pageBBa_K2951008!
Reference
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)