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中国生物工程杂志

China Biotechnology
China Biotechnology
China Biotechnology  2018, Vol. 38 Issue (2): 54-60    DOI: 10.13523/j.cb.20180208
Orginal Article     
Studies on the Protein Purification Ability of an ELP30-Tag in Prokaryotic Expression System
Yuan-qiao CHEN,Ding-pei LONG,Xiao-xue DOU,Run QI,Ai-chun ZHAO()
State Key Laboratory of Silkworm Genome Biology, College of Biotechnology, Southwest University,Chongqing 400716, China
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Abstract  

Objective： Exploration of the purification efficiency of proteins in prokaryotic expression system(Escherichia coli) by using an elastin-like protein tag(ELP30-tag) with small molecular weight.Methods: The ELP30-tag gene was synthesized and inserted into the pET-28a(+) vector,two intein genes (intein1 & intein2) and an enhanced green fluorescent protein (eGFP) gene were cloned and applied to construct four prokaryotic expression vectors: pET-ELP30, pET-ELP30-eGFP, pET-ELP30-intein1-eGFP and pET-eGFP-intein2-ELP30. All the recombinant plasmids were transferred into E. coli BL21(DE3)and induced by IPTG, respectively. Recombinant proteins ELP30, ELP30-eGFP,ELP30-intein1-eGFP and eGFP-intein2-ELP30 were purified by inverse transition cycling (ITC), and then the cleavage reaction of intein1 and intein2 were induced by adjusting the pH value of the solution or adding DL-Dithiothreitol (DTT), respectively, and last the pure eGFPs were separated by one more ITC reaction.Results: The recombinant proteins ELP30, ELP30-eGFP and eGFP-intein2-ELP30 were purified by using the designed ELP30-tag; the cleavage reaction of inteins from the recombinant proteins ELP30-intein1-eGFP and eGFP-intein2-ELP30, which could be successfully induced,and then the eGFPs were released into the solution but not separated. This lays some foundations for the application and optimization of the ELP-tags with small molecular weight.

Key words： Elastin-like protein(ELP)      Intein      Enhanced green fluorescent protein (eGFP)      Prokaryotic expression      Protein purification     
Received: 05 July 2017      Published: 21 March 2018
ZTFLH:  Q819  
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Yuan-qiao CHEN
Ding-pei LONG
Xiao-xue DOU
Run QI
Ai-chun ZHAO
Cite this article:

Yuan-qiao CHEN,Ding-pei LONG,Xiao-xue DOU,Run QI,Ai-chun ZHAO. Studies on the Protein Purification Ability of an ELP30-Tag in Prokaryotic Expression System. China Biotechnology, 2018, 38(2): 54-60.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.20180208     OR     https://manu60.magtech.com.cn/biotech/Y2018/V38/I2/54

引物名称 序列(5’-3’)
F1 ACATgcatgcAAGGAGATGGCGCC
R1 CCGctcgagggatccgcatgcggtaccgtcgacCATTATATCTCCTTCTTAAA
eGFP-F1 GGggtaccAACAACAACAACAACAACAACAACAACAACATGGTGAGCAAGGGCGAGGA
eGFP-R1 CGggatccCTTGTACAGCTCGTCCATGC
eGFP-F2 GCtctagaAATAATTTTGTTTAACTTTAAGAAGGAGATATACCATGGTGAGCAAGGGCGAGGA
eGFP-R2 GCATGGACGAGCTGTACAAGTGCATCACGGGAGATGCACT
intein2-F AGTGCATCTCCCGTGATGCACTTGTACAGCTCGTCCATGC
intein2-R CCGctcgagGTTGTTGTTGTTGTTGTTGTTGTTGTTGTTAGCGTGGCTGACGAACCCGT
Table 1 Primers for fragments amplification
Fig.1 Construction of expression vectors(a)The expression cassettes of designed expression plasmids (b) Identification of recombinant plasmids by SphⅠ & Xho I 1: pET-28a’; 2: pET-ELP30; 3: pET-ELP30-eGFP;4: pET-ELP30-intein1-eGFP;5: pET-eGFP-intein2-ELP30
Fig.2 Purification of ELP30 and ELP30-eGFPM: Protein maker; 1: Uninduced cell extract; 2: Purification of ELP30 via ELP30-tag;3: Purification of ELP30-eGFP via ELP30-tag; 4: Purification of ELP30-eGFP via His-tag
Fig.3 Detection of fluorescence activity of recombinant proteinsA: PBS(used as negative control); B: ELP30-eGFP;C: ELP30-intein1-eGFP; D: eGFP-intein2-ELP30. White scale bar represents 1 mm
Fig.4 Purification and cleavage of ELP30-intein1-eGFPM: Protein maker; 1: Uninduced cell extract; 2:Supernatant of lysate of ELP30-intein1-eGFP, 3: ELP30-intein1-eGFP purification via ELP30-tag; 4: Cleavage products of ELP30-intein1-eGFP induced by pH shift; 5: Eluate gained by another ITC operation after the cleavage
Fig.5 Purification and cleavage of eGFP-intein2-ELP30M: Protein maker; 1: Uninduced cell extract; 2: Supernatant of lysate of eGFP-intein2-ELP30, 3: eGFP-intein2-ELP30purification via ELP30-tag; 4: Cleavage products of eGFP-intein2-ELP30 induced by DTT; 5: Eluate gained by another ITC operation after the cleavage
重组蛋白 重组蛋白回收率 蛋白浓度
(mg/ml,
ITC-3)
ITC-1 ITC-2 ITC-3
ELP30 93% 70% 61% 0.18
ELP30-eGFP 96% 84% 65% 0.44
eGFP-intein2-ELP30 95% 88% 72% 0.29
Table 2 Recovery rate analysis of recombinant proteins
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