Increasing the Expression Level of Soluble Tumor Necrosis Factor Type I Based on Optimization of Secondary Structure of mRNA 5' Terminal TIR

doi:10.13523/j.cb.20180308

China Biotechnology

2018, Vol. 38

Issue (3): 62-69 DOI: 10.13523/j.cb.20180308

Orginal Article

Increasing the Expression Level of Soluble Tumor Necrosis Factor Type I Based on Optimization of Secondary Structure of mRNA 5' Terminal TIR

Jiao-rong QIN,Zhao ZHAO,Xin-mei LUO,Chun-yang LI(

)

Chengdu Institute of Biological Products Co. Ltd, Chengdu 610023,China

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Abstract Objective:

To increase the expression level of soluble tumor necrosis factor type-I receptor(sTNFαRI)in E.coli BL21(DE3)by optimizing the secondary structure of PET11b-sTNFaRI translation initiation region(TIR).

Methods:

The free energy and nucleotide position entroy of the secondary structure of the translational initiation region was analyzed as the first step, and the primers were designed to mutate the codons of TIR of the PET11b-sTNFαRI in order to exposure of ribosome binding site and start codon to the outside of hairpin structure, in addition to mutating the pET11b ribosome binding site from GAAGGAGA to GAAGAA in order to facilitate assembly of the translational complex and initiation of translation. The optimized sequence of 5' terminal TIR was cloned into PET11b vector and transformed into E.coli BL21(DE3). The positive transformants were induced by IPTG and analyzed by SDS-PAGE and Western blot.

Results:

SDS-PAGE and Western blot analysis showed that the expression of Recombinant sTNFαRI was increased by 50%~60%, after optimizing the secondary structure of 5' terminal TIR of PET11b-sTNFαRI.

Conclusion:

The optimization of the secondary structure of the translation initiation region(TIR)mRNA of recombinant vector can effectively increase the expression level of the target protein, which is of great value for further industrialized production.

Key words： sTNFαRI mRNA secondary structure Translation initiation region Free energy

Received: 07 December 2017 Published: 04 April 2018

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Cite this article:

Jiao-rong QIN, Zhao ZHAO, Xin-mei LUO, Chun-yang LI. Increasing the Expression Level of Soluble Tumor Necrosis Factor Type I Based on Optimization of Secondary Structure of mRNA 5' Terminal TIR. China Biotechnology, 2018, 38(3): 62-69.

URL:

https://manu60.magtech.com.cn/biotech/10.13523/j.cb.20180308 OR https://manu60.magtech.com.cn/biotech/Y2018/V38/I3/62

Table 1 Comparision of sTNFα 5'TIR sequence before and after mutation

Fig.1 Prediction of 5'TIR secondary structure and total free energy of sTNFαRI
(a)Rbs1 (b)Rbs2 (c)Rbs3 (d)Rbs4 (e)Ori-sTNFαRI (f)Probability

Fig.2 Prediction of relative free energy and position entroy

Table 2 Primers of mutant PCR

Fig.3 Electrophoretogram of sTNFαRI gene amplified by PCR
M:DNA marker II;1:rbs1+TNFαRI;2:rbs2+TNFαRI;3:rbs3+TNFαRI;4:rbs4+TNFαRI;5: Negative control;6: Positive control

Fig.4 Identification of recombinant plasmid by double digestion
M1: DNA marker IV; 1-4:Recombinant plasmid pET11b-TNFa RI-rbs1/2/3/4 digest by XbaI+BamHI; M2: DNA marker II

Fig.5 Analysis expression level of sTNFαRI by (a)SDS-PAGE and Western blot(b)
M:Marker; 1-4:Expression level of sTNFαRI by pET11b-rbs1/2/3/4-sTNFα RI/BL21(DE3);5: pET11b-TNFa RI/BL21(DE3)(no reduce);6: Expression level of sTNFαRI by pET11b-TNFa RI/BL21(DE3)(before mutation)

Fig.6 Image Lab 4.0 software analysis
Line 2-5: Analysis of expression level of pET11b-rbs1/2/3/4-sTNFα RI/BL21(DE3)induced by IPTG;Line 6: pET11b-TNFa RI/BL21(DE3)(no reduce);Line 7: pET11b-TNFa RI/BL21(DE3)induced by IPTG (before mutation)

Fig.7 Comparision of expression level of sTNFαRI


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