Ubiquitin-like prokaryotic MoaD as a fusion tag for expression of heterologous proteins in Escherichia coli.
BACKGROUND
Eukaryotic ubiquitin and SUMO are frequently used as tags to enhance the fusion protein expression in microbial host. They increase the solubility and stability, and protect the peptides from proteolytic degradation due to their stable and highly conserved structures. Few of prokaryotic ubiquitin-like proteins was used as fusion tags except ThiS, which enhances the fusion expression, however, reduces the solubility and stability of the expressed peptides in E. coli. Hence, we investigated if MoaD, a conserved small sulfur carrier in prokaryotes with the similar structure of ubiquitin, could also be used as fusion tag in heterologous expression in E. coli.


RESULTS
Fusion of MoaD to either end of EGFP enhanced the expression yield of EGFP with a similar efficacy of ThiS. However, the major parts of the fusion proteins were expressed in the aggregated form, which was associated with the retarded folding of EGFP, similar to ThiS fusions. Fusion of MoaD to insulin chain A or B did not boost their expression as efficiently as ThiS tag did, probably due to a less efficient aggregation of products. Interestingly, fusion of MoaD to the murine ribonuclease inhibitor enhanced protein expression by completely protecting the protein from intracellular degradation in contrast to ThiS fusion, which enhanced degradation of this unstable protein when expressed in E. coli.


CONCLUSIONS
Prokaryotic ubiquitin-like protein MoaD can act as a fusion tag to promote the fusion expression with varying mechanisms, which enriches the arsenal of fusion tags in the category of insoluble expression.
Sujuan
S
Yuan
Xin
X
Wang
Jian
J
Xu
Zheng
Z
Yan
Chinese Academy of Medical Sciences & Peking Union Medical College, Institute of Materia Medica, Beijing Key Laboratory of New Drug Mechanisms and Pharmacological Evaluation Study, Beijing, People's Republic of China. wangnan@imm.ac.cn.
Nan
N
Wang
D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D051379:Mice; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis*; D014452:Ubiquitins / Q000378:metabolism*
D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D011993:Recombinant Fusion Proteins; D014452:Ubiquitins; C485184:enhanced green fluorescent protein; C411655:thiS protein, E coli; D049452:Green Fluorescent Proteins