China Biotechnology

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

China Biotechnology, 2018, 38(2): 54-60. DOI: 10.13523/j.cb.20180208

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

Other figure/table from this article

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
Table 2 Recovery rate analysis of recombinant proteins