Integrated refolding techniques for Schistosoma japonicum MTH1 overexpressed as inclusion bodies in Escherichia coli.
The full-length cDNA of MTH1in Schistosoma japonicum was previously isolated. However, insoluble protein expression in Escherichia coli is the biggest bottleneck limiting biological and biophysical studies. Protein aggregation could not be significantly prevented using solubilization or refolding techniques, and denatured MTH1 protein could not be refolded to the native monomer form. Hence, integrating several refolding techniques within the protein refolding process of MTH1, a large amount of active MTH1 was obtained for protein crystallization. We primarily utilized the two-step-denaturing and refolding method and the protein refolding screening technique, as well as the continuous dialysis method. First, we identified the refolding buffer composition that allowed for successful refolding to overcome protein precipitation. Next, we used the two-step-denaturing and refolding method and the continuous dialysis method to suppress protein aggregation. In the end, we obtained 15 mg of active MTH1 monomer with 95% purity from 0.5l medium. Integrated refolding techniques proved to be excellent for obtaining the native monomer of S. japonicum MTH1 from inclusion bodies, paving the way for future biological and biophysical studies.
State Key Laboratory of Bioreactor Engineering, School of Biotechnology, East China University of Science and Technology, Shanghai 200237, China. semifyy@163.com
Yanye
Y
Feng
Lu
L
Liu
Jipeng
J
Wang
Jian
J
Liu
Wei
W
Hu
Xiaoning
X
Wang
Zhong
Z
Yang
D000818:Animals; D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D015801:Helminth Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D002479:Inclusion Bodies / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011489:Protein Denaturation; D058849:Protein Refolding; D011755:Pyrophosphatases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012549:Schistosoma japonicum / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012995:Solubility; D015854:Up-Regulation; D000096964:Nudix Hydrolases
D018076:DNA, Complementary; D015801:Helminth Proteins; D011755:Pyrophosphatases; D011994:Recombinant Proteins; D000096964:Nudix Hydrolases