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中国生物工程杂志

China Biotechnology
China Biotechnology
China Biotechnology  2019, Vol. 39 Issue (3): 21-28    DOI: 10.13523/j.cb.20190304
    
Efficient Heterologous Expression, Purification and Activity Analysis of Fusion Protein NusA-hRI
Da-wei FU(),Ying-ying SUN,wei XU
Key Laboratory for Food Science and Engineering, Harbin University of Commerce, Harbin 150076, China
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Abstract  

Human ribonuclease inhibitor (hRI) is an acidic pulping protein that regulates ribonuclease activity. By constructing a recombinant expression vector containing SUMO, IF2, GST, NusA, MsyB, Trx and MBP fusion tags, E.coli BL21 (DE3) was used as a host strain for auto-induction (AI) expression, thereby the expression level of hRI is improved. The expression of hRI was analyzed by MagNi magnetic bead purification and electrophoresis, and the protein with higher purity was obtained by RNase/Sepharose affinity chromatography.The concentration of the fusion protein obtained after purification was 2 960.513mg/L, which was compared with other companies, and its enzyme activity was about 50U/μl, which was successfully used for RNA protection. Purely provide a theoretic-al basis for the application of NusA-hRI.

Key words： Human      ribonuclease      inhibitor      Fusion      tags      Auto-induction      Magnetic      bead      me-thod      Affinity      chromatography     
Received: 30 September 2018      Published: 12 April 2019
ZTFLH:  Q816  
Corresponding Authors: Da-wei FU     E-mail: fudaweinovo@163.com
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Da-wei FU
Ying-ying SUN
wei XU
Cite this article:

Da-wei FU,Ying-ying SUN,wei XU. Efficient Heterologous Expression, Purification and Activity Analysis of Fusion Protein NusA-hRI. China Biotechnology, 2019, 39(3): 21-28.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.20190304     OR     https://manu60.magtech.com.cn/biotech/Y2019/V39/I3/21

水平 因素
A/温度
(℃)		 B/pH C/接种量
(‰)		 D/瓶装量
(ml/250)
1 15 6 1 25
2 20 7 2 50
3 25 8 5 75
Table 1 Orthogonal test factor level table
Fig.1 PCR amplification of hRI gene M:DNA marker; 1: hRI gene PCR product
Fig.2 Detection of the recombinant expression vector by restriction enzyme digestion M: DNA molecular marker; 1: PCR amplified hRI gene; 29: BamH I and Not I digested recombinant vectors pNBE I to VIII
Fig.3 Detection of the recombinant expression vector by PCR M: DNA molecular marker; 14: recombinant plasmid pNBE I-hRI; 58: recombinant plasmid pNBE II-hRI; 912: recombinant plasmid pNBE IIII-hRI; 1316: recombinant plasmid pNBE IV-hRI; 1720: recombinant plasmid pNBE V-hRI; 2124: recombinant plasmid pNBE VI-hRI; 2528: recombinant plasmid pNBE VII-hRI; 2932: recombinant plasmid pNBE VIII-hRI
Fig.4 Analysis of hRI I induced expression of 12% polyacrylamide gel M: protein molecular marker; 18: self-inducible recombinant strains of vector pNBE I-VIII, respectively
Fig. 5 Analysis of soluble expression of hRI by 12% polyacrylamide gel M: protein molecular marker; 14: precipitation, supernatant, purification penetration and elution of recombinant plasmid pNBE I; 58: precipitation and supernatant of recombinant plasmid pNBE II , Penetration and elution after purification; 912: Precipitation, supernatant, and penetration and elution of recombinant lysate of pNBE II; 1316: Precipitation and supernatant of recombinant lysate of pNBE III , Penetration and elution after purification; 1720: Precipitation, supernatant, and penetration and elution of recombinant lysate of pNBE IV; 2124: Precipitation and supernatant of recombinant lysate of pNBE V , Penetration and elution after purification; 25-28: Precipitation, supernatant, and penetration and elution of recombinant lysate of pNBE VI; 2629: Precipitation and supernatant of recombinant lysate of pNBE VII , Penetration and elution after purification; 3032: Precipitation, supernatant, and penetration and elution of recombinant cleavage of pNBE VIII
因素 水平 菌体密度
(OD )		 蛋白表达量
(%)		 蛋白产量
(mg/L)
温度 16 1.56 30.12 305.272
Temperature 20 1.82 38.37 437.113
(℃) 25 2.03 37.13 418.720
30 2.17 35.88 407.335
pH 5 2.42 49.17 534.322
6 2.56 51.22 541.270
7 2.50 53.70 546.339
8 2.63 52.91 540.433
9 2.33 49.03 531.701
接种量 1 2.92 46.42 535.133
Inoculum size 2 3.03 50.07 542.011
(‰) 5 3.37 55.97 539.228
10 3.52 43.31 526.970
20 3.66 34.88 462.034
瓶装量 25 2.99 52.55 562.020
Bottled volume 50 3.28 57.14 587.243
(ml/250 ml) 75 3.11 56.98 577.312
100 3.08 54.77 543.098
150 2.97 49.13 506.771
Table 2 Design and results of single factor experiment on culture conditions
试验序号 A/温度(℃) B/pH C/接种量(‰) D/装瓶量
(ml/250 ml)		 蛋白产量(mg/L)
1 1 1 1 1 518.335±0.011
2 1 2 2 2 546.431±0.002
3 1 3 3 3 512.785±0.003
4 2 1 2 3 583.213±0.005
5 2 2 3 1 687.236±0.009
6 2 3 1 2 654.112±0.002
7 3 1 3 2 623.621±0.003
8 3 2 1 3 503.901±0.007
9 3 3 2 1 584.097±0.002
蛋白产量Proteinyield(mg/L) K1 1577.55 1725.17 1676.35 1789.67
K2 1924.56 1737.57 1713.74 1824.16
K3 1711.62 1750.99 1823.64 1599.90
极差R 347.01 25.82 147.29 224.26
Table 3 L9 (34) orthogonal design table and interpretation of results
因素 离差平方和 自由度 F比 P值 显著性
温度 20414.941 2 183.564 0.0054 **
pH 111.214 2 1.000
接种量 3907.999 2 35.139 0.0277 *
瓶装量 9721.893 2 87.416 0.0113 *
误差 111.214 2
Table 4 Variance analysis table of orthogonal test
Fig.6 12% polyacrylamide gel analysis of hRI purified by magnetic beads M: protein molecular marker; 1: precipitate after bacterial cell lysis; 2: supernatant after bacterial cell lysis; 3: purified breakthrough peak; 47: purified hybrid protein rinsing; 812: first to first The hRI obtained by 5 batches eluted
Fig.7 Purification of RNase/Sepharose affinity chromatography
Fig.8 detection of hRI enzyme activity M: DNA molecular marker; 1: Extracted plant leaf RNA; 26: Add RI of M company with volume of 0.4, 0.6, 0.8, 1.0, 1.2 μL, respectively; 710: Add volume of 0.4, 0.6, 0.8 , hRI of 1.0
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