Soluble expression of human leukemia inhibitory factor with protein disulfide isomerase in Escherichia coli and its simple purification

A. Song Jung, Bon Kyung Koo, Seon Ha Chong, Kyunhoo Kim, Dong Kyu Choi, Thu Trang Thi Vu, Minh Tan Nguyen, Boram Jeong, Han Bong Ryu, Injune Kim, Yeon Jin Jang, Robert Charles Robinson, Han Choe

Research output: Contribution to journalArticle

13 Citations (Scopus)

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.

Original languageEnglish
Article numbere83781
JournalPloS one
Volume8
Issue number12
DOIs
Publication statusPublished - Dec 16 2013
Externally publishedYes

Fingerprint

protein disulfide-isomerase
Protein Disulfide-Isomerases
Escherichia coli
Purification
biomedical research
embryonic stem cells
Embryonic Stem Cells
Stem cells
Biomedical Research
human LIF protein
leukemia inhibitory factor
Prokaryotic Cells
Zygote
Clinical Medicine
disulfide bonds
Eukaryotic Cells
endotoxins
prokaryotic cells
Endotoxins
Disulfides

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Agricultural and Biological Sciences(all)

Cite this

Soluble expression of human leukemia inhibitory factor with protein disulfide isomerase in Escherichia coli and its simple purification. / Jung, A. Song; Koo, Bon Kyung; Chong, Seon Ha; Kim, Kyunhoo; Choi, Dong Kyu; Vu, Thu Trang Thi; Nguyen, Minh Tan; Jeong, Boram; Ryu, Han Bong; Kim, Injune; Jang, Yeon Jin; Robinson, Robert Charles; Choe, Han.

In: PloS one, Vol. 8, No. 12, e83781, 16.12.2013.

Research output: Contribution to journalArticle

Jung, AS, Koo, BK, Chong, SH, Kim, K, Choi, DK, Vu, TTT, Nguyen, MT, Jeong, B, Ryu, HB, Kim, I, Jang, YJ, Robinson, RC & Choe, H 2013, 'Soluble expression of human leukemia inhibitory factor with protein disulfide isomerase in Escherichia coli and its simple purification', PloS one, vol. 8, no. 12, e83781. https://doi.org/10.1371/journal.pone.0083781
Jung, A. Song ; Koo, Bon Kyung ; Chong, Seon Ha ; Kim, Kyunhoo ; Choi, Dong Kyu ; Vu, Thu Trang Thi ; Nguyen, Minh Tan ; Jeong, Boram ; Ryu, Han Bong ; Kim, Injune ; Jang, Yeon Jin ; Robinson, Robert Charles ; Choe, Han. / Soluble expression of human leukemia inhibitory factor with protein disulfide isomerase in Escherichia coli and its simple purification. In: PloS one. 2013 ; Vol. 8, No. 12.
@article{6429e997bb4547f6a9cbabd2a7cce7c0,
title = "Soluble expression of human leukemia inhibitory factor with protein disulfide isomerase in Escherichia coli and its simple purification",
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.",
author = "Jung, {A. Song} and Koo, {Bon Kyung} and Chong, {Seon Ha} and Kyunhoo Kim and Choi, {Dong Kyu} and Vu, {Thu Trang Thi} and Nguyen, {Minh Tan} and Boram Jeong and Ryu, {Han Bong} and Injune Kim and Jang, {Yeon Jin} and Robinson, {Robert Charles} and Han Choe",
year = "2013",
month = "12",
day = "16",
doi = "10.1371/journal.pone.0083781",
language = "English",
volume = "8",
journal = "PLoS One",
issn = "1932-6203",
publisher = "Public Library of Science",
number = "12",

}

TY - JOUR

T1 - Soluble expression of human leukemia inhibitory factor with protein disulfide isomerase in Escherichia coli and its simple purification

AU - Jung, A. Song

AU - Koo, Bon Kyung

AU - Chong, Seon Ha

AU - Kim, Kyunhoo

AU - Choi, Dong Kyu

AU - Vu, Thu Trang Thi

AU - Nguyen, Minh Tan

AU - Jeong, Boram

AU - Ryu, Han Bong

AU - Kim, Injune

AU - Jang, Yeon Jin

AU - Robinson, Robert Charles

AU - Choe, Han

PY - 2013/12/16

Y1 - 2013/12/16

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=84892666852&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84892666852&partnerID=8YFLogxK

U2 - 10.1371/journal.pone.0083781

DO - 10.1371/journal.pone.0083781

M3 - Article

C2 - 24358310

AN - SCOPUS:84892666852

VL - 8

JO - PLoS One

JF - PLoS One

SN - 1932-6203

IS - 12

M1 - e83781

ER -