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Original article
Bainan Liu
Zunyi Medical University
Corresponding Author
ORCiD: https://orcid.org/0000-0002-5855-137X
License:
This work is licensed under a CC BY 4.0 License. Read Full License
iLRP (immature laminin receptor protein) is a tumor associated antigen over-expressed on the surface of most human cancer cells and plays an important role in the process of tumorigenesis and development. It has strong auto-immunogenicity in patients and is a good target protein for tumor immunotherapy. To find a practical and an efficient method for the production of recombinant iLRP, three expression vectors based on pET-30a(+) from pET expression systems were constructed. The first one is to add a 6xHis-Tag to the N-terminal of natural iLRP, which is called as pET-His-iLRP. The second one is to include an S-Tag between the C-terminal of 6xHis-Tag and the N-terminal of natural iLRP, which is called as pET-His-S-iLRP. The third one only contains the 6xHis-Tag in front of the N-terminal of iLRP, but the natural iLRP gene was first optimized according to the bacterial genome, and the constructed vector was named as pET-His-Opt-iLRP. Then the expression of three vectors in Escherichia coli (E. coli) BL21 (DE3) was analyzed. Results demonstrated that the iLRP expression was the highest in pET-His-Opt-iLRP, followed by pET-His-S-iLRP, and pET-His-iLRP expressed the lowest iLRP. This implied that the vector combining the codon-optimized iLRP gene with a 6xHis-Tag had the best effect on the expression of iLRP, and the expression in vectors with the natural iLRP gene depends on its leader sequences in the expression frame. It can be consequently concluded that the heterologous expression of human iLRP in a bacterial host could be greatly increased by genetically modifying gene constituents and using proper protein-tags, which laying the foundation on the future researches on its applications.
Keywords
Immature laminin receptor protein, Prokaryotic expression vector, Recombinant expression, Protein tag
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This is a preprint. It has not completed peer review.
Original article
Bainan Liu
Zunyi Medical University
Corresponding Author
ORCiD: https://orcid.org/0000-0002-5855-137X
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
History
CURRENT STATUS: Posted
Version 1
Posted 22 Jan, 2020
No community comments so far
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
License:
This work is licensed under a CC BY 4.0 License. Read Full License
iLRP (immature laminin receptor protein) is a tumor associated antigen over-expressed on the surface of most human cancer cells and plays an important role in the process of tumorigenesis and development. It has strong auto-immunogenicity in patients and is a good target protein for tumor immunotherapy. To find a practical and an efficient method for the production of recombinant iLRP, three expression vectors based on pET-30a(+) from pET expression systems were constructed. The first one is to add a 6xHis-Tag to the N-terminal of natural iLRP, which is called as pET-His-iLRP. The second one is to include an S-Tag between the C-terminal of 6xHis-Tag and the N-terminal of natural iLRP, which is called as pET-His-S-iLRP. The third one only contains the 6xHis-Tag in front of the N-terminal of iLRP, but the natural iLRP gene was first optimized according to the bacterial genome, and the constructed vector was named as pET-His-Opt-iLRP. Then the expression of three vectors in Escherichia coli (E. coli) BL21 (DE3) was analyzed. Results demonstrated that the iLRP expression was the highest in pET-His-Opt-iLRP, followed by pET-His-S-iLRP, and pET-His-iLRP expressed the lowest iLRP. This implied that the vector combining the codon-optimized iLRP gene with a 6xHis-Tag had the best effect on the expression of iLRP, and the expression in vectors with the natural iLRP gene depends on its leader sequences in the expression frame. It can be consequently concluded that the heterologous expression of human iLRP in a bacterial host could be greatly increased by genetically modifying gene constituents and using proper protein-tags, which laying the foundation on the future researches on its applications.
Figure 1
Figure 2
Figure 3
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