Please use this identifier to cite or link to this item: https://doi.org/10.1371/journal.pone.0083781
Title: Soluble expression of human leukemia inhibitory factor with protein disulfide isomerase in Escherichia coli and its simple purification
Authors: Jung A.S.
Koo B.-K.
Chong S.-H.
Kim K.
Choi D.K.
Vu T.T.T.
Nguyen M.T.
Jeong B.
Ryu H.-B.
Kim I.
Jang Y.J.
Robinson R.C. 
Choe H.
Keywords: endotoxin
glycine
hybrid protein
leukemia inhibitory factor
protein disulfide isomerase
proteinase
TEV protease
unclassified drug
amino terminal sequence
animal cell
article
controlled study
disulfide bond
embryonic stem cell
Escherichia coli
low temperature
mass spectrometry
mouse
nonhuman
pluripotent stem cell
protein analysis
protein cleavage
protein domain
protein expression
protein modification
protein purification
solubility
Amino Acid Sequence
Escherichia coli
Gene Expression
Gene Order
Humans
Leukemia Inhibitory Factor
Mass Spectrometry
Molecular Sequence Data
Plasmids
Protein Disulfide-Isomerases
Protein Stability
Recombinant Fusion Proteins
Solubility
Issue Date: 2013
Citation: Jung A.S., Koo B.-K., Chong S.-H., Kim K., Choi D.K., Vu T.T.T., Nguyen M.T., Jeong B., Ryu H.-B., Kim I., Jang Y.J., Robinson R.C., Choe H. (2013). Soluble expression of human leukemia inhibitory factor with protein disulfide isomerase in Escherichia coli and its simple purification. PLoS ONE 8 (12) : e83781. ScholarBank@NUS Repository. https://doi.org/10.1371/journal.pone.0083781
Rights: Attribution 4.0 International
Abstract: Human leukemia inhibitory factor (hLIF) is a multifunctional cytokine that is essential for maintaining the pluripotency of embryonic stem cells. hLIF may be also be useful in aiding fertility through its effects on increasing the implantation rate of fertilized eggs. Thus these applications in biomedical research and clinical medicine create a high demand for bioactive hLIF. However, production of active hLIF is problematic since eukaryotic cells demonstrate limited expression and prokaryotic cells produce insoluble protein. Here, we have adopted a hybrid protein disulfide isomerase design to increase the solubility of hLIF in Escherichia coli. Low temperature expression of hLIF fused to the b'a' domain of protein disulfide isomerase (PDIb'a') increased the soluble expression in comparison to controls. A simple purification protocol for bioactive hLIF was established that includes removal of the PDIb'a' domain by cleavage by TEV protease. The resulting hLIF, which contains one extra glycine residue at the N-terminus, was highly pure and demonstrated endotoxin levels below 0.05 EU/μg. The presence of an intramolecular disulfide bond was identified using mass spectroscopy. This purified hLIF effectively maintained the pluripotency of a murine embryonic stem cell line. Thus we have developed an effective method to produce a pure bioactive version of hLIF in E. coli for use in biomedical research. © 2013 Song et al.
Source Title: PLoS ONE
URI: https://scholarbank.nus.edu.sg/handle/10635/161443
ISSN: 1932-6203
DOI: 10.1371/journal.pone.0083781
Rights: Attribution 4.0 International
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