Bacterial expression and in vitro folding of the β-subunit of human chorionic gonadotropin (hCG β) and functional assembly of recombinant hCG β with hCG α

Jeffrey R. Huth, Sheila E. Norton, Oksana Lockridge, Toshihiko Shikone, Aaron J W Hsueh, Raymond W. Ruddon

Research output: Contribution to journalArticle

Abstract

A bacterial expression system for the β-subunit of hCG (hCG β) has been developed to produce suitable amounts of this protein for structural and biological studies. To produce hCG β in Escherichia coli, the nucleotide sequence that encodes the amino acid leader sequence was removed from the hCG β complementary DNA, and the gene was cloned into a pET expression vector. After induction of protein synthesis in host bacteria, recombinant hCG β (rhCG β) accumulated in inclusion bodies in an unfolded state. The inclusion bodies were purified from induced cultures of E. coli, solubilized in urea, and fractionated by reverse phase HPLC. In this way, 6-7 mg unfolded hCG β were recovered from 1 liter culture, rhCG β was folded in the presence of 6.4 mM cysteamine and 3.6 mM cystamine at pH 8.7 at a final concentration of 0.02 mg/ml protein. The folded protein assembled with urinary α hCG and the purified rhCG β/urinary α dimer bound to and activated the human LH/CG receptor permanently expressed in a cell line, indicating that it was a functional hormone. The rhCG β/urinary α dimer also stimulated in vivo ovulation in rats, thus confirming the biological activity of bacterially expressed hCG β. Because E. coli lacks the ability to glycosylate proteins, these activity results indicate that the N-linked and O-linked oligosaccharides of hCG β are not required for protein folding, subunit assembly, or full biological activity. The success of producing hCG β in bacteria and of folding it in vitro implies that the β-subunits of the other members of the glycoprotein hormone family, LH, FSH, and TSH, can also be produced in this manner. This may facilitate structural studies of these hormones as well as lead to the production of recombinant hormones for biological studies and clinical use.

Original languageEnglish (US)
Pages (from-to)911-918
Number of pages8
JournalEndocrinology
Volume135
Issue number3
DOIs
StatePublished - 1994

Fingerprint

Chorionic Gonadotropin
Hormones
Inclusion Bodies
Escherichia coli
Proteins
Cystamine
In Vitro Techniques
Bacteria
Cysteamine
LH Receptors
Protein Folding
Protein Subunits
Ovulation
Oligosaccharides
Urea
Amino Acid Sequence
Glycoproteins
Complementary DNA
High Pressure Liquid Chromatography
Cell Line

ASJC Scopus subject areas

  • Endocrinology

Cite this

Bacterial expression and in vitro folding of the β-subunit of human chorionic gonadotropin (hCG β) and functional assembly of recombinant hCG β with hCG α. / Huth, Jeffrey R.; Norton, Sheila E.; Lockridge, Oksana; Shikone, Toshihiko; Hsueh, Aaron J W; Ruddon, Raymond W.

In: Endocrinology, Vol. 135, No. 3, 1994, p. 911-918.

Research output: Contribution to journalArticle

Huth, Jeffrey R. ; Norton, Sheila E. ; Lockridge, Oksana ; Shikone, Toshihiko ; Hsueh, Aaron J W ; Ruddon, Raymond W. / Bacterial expression and in vitro folding of the β-subunit of human chorionic gonadotropin (hCG β) and functional assembly of recombinant hCG β with hCG α. In: Endocrinology. 1994 ; Vol. 135, No. 3. pp. 911-918.
@article{55ac886f109045b490d8490d734af849,
title = "Bacterial expression and in vitro folding of the β-subunit of human chorionic gonadotropin (hCG β) and functional assembly of recombinant hCG β with hCG α",
abstract = "A bacterial expression system for the β-subunit of hCG (hCG β) has been developed to produce suitable amounts of this protein for structural and biological studies. To produce hCG β in Escherichia coli, the nucleotide sequence that encodes the amino acid leader sequence was removed from the hCG β complementary DNA, and the gene was cloned into a pET expression vector. After induction of protein synthesis in host bacteria, recombinant hCG β (rhCG β) accumulated in inclusion bodies in an unfolded state. The inclusion bodies were purified from induced cultures of E. coli, solubilized in urea, and fractionated by reverse phase HPLC. In this way, 6-7 mg unfolded hCG β were recovered from 1 liter culture, rhCG β was folded in the presence of 6.4 mM cysteamine and 3.6 mM cystamine at pH 8.7 at a final concentration of 0.02 mg/ml protein. The folded protein assembled with urinary α hCG and the purified rhCG β/urinary α dimer bound to and activated the human LH/CG receptor permanently expressed in a cell line, indicating that it was a functional hormone. The rhCG β/urinary α dimer also stimulated in vivo ovulation in rats, thus confirming the biological activity of bacterially expressed hCG β. Because E. coli lacks the ability to glycosylate proteins, these activity results indicate that the N-linked and O-linked oligosaccharides of hCG β are not required for protein folding, subunit assembly, or full biological activity. The success of producing hCG β in bacteria and of folding it in vitro implies that the β-subunits of the other members of the glycoprotein hormone family, LH, FSH, and TSH, can also be produced in this manner. This may facilitate structural studies of these hormones as well as lead to the production of recombinant hormones for biological studies and clinical use.",
author = "Huth, {Jeffrey R.} and Norton, {Sheila E.} and Oksana Lockridge and Toshihiko Shikone and Hsueh, {Aaron J W} and Ruddon, {Raymond W.}",
year = "1994",
doi = "10.1210/endo.135.3.8070386",
language = "English (US)",
volume = "135",
pages = "911--918",
journal = "Endocrinology",
issn = "0013-7227",
publisher = "The Endocrine Society",
number = "3",

}

TY - JOUR

T1 - Bacterial expression and in vitro folding of the β-subunit of human chorionic gonadotropin (hCG β) and functional assembly of recombinant hCG β with hCG α

AU - Huth, Jeffrey R.

AU - Norton, Sheila E.

AU - Lockridge, Oksana

AU - Shikone, Toshihiko

AU - Hsueh, Aaron J W

AU - Ruddon, Raymond W.

PY - 1994

Y1 - 1994

N2 - A bacterial expression system for the β-subunit of hCG (hCG β) has been developed to produce suitable amounts of this protein for structural and biological studies. To produce hCG β in Escherichia coli, the nucleotide sequence that encodes the amino acid leader sequence was removed from the hCG β complementary DNA, and the gene was cloned into a pET expression vector. After induction of protein synthesis in host bacteria, recombinant hCG β (rhCG β) accumulated in inclusion bodies in an unfolded state. The inclusion bodies were purified from induced cultures of E. coli, solubilized in urea, and fractionated by reverse phase HPLC. In this way, 6-7 mg unfolded hCG β were recovered from 1 liter culture, rhCG β was folded in the presence of 6.4 mM cysteamine and 3.6 mM cystamine at pH 8.7 at a final concentration of 0.02 mg/ml protein. The folded protein assembled with urinary α hCG and the purified rhCG β/urinary α dimer bound to and activated the human LH/CG receptor permanently expressed in a cell line, indicating that it was a functional hormone. The rhCG β/urinary α dimer also stimulated in vivo ovulation in rats, thus confirming the biological activity of bacterially expressed hCG β. Because E. coli lacks the ability to glycosylate proteins, these activity results indicate that the N-linked and O-linked oligosaccharides of hCG β are not required for protein folding, subunit assembly, or full biological activity. The success of producing hCG β in bacteria and of folding it in vitro implies that the β-subunits of the other members of the glycoprotein hormone family, LH, FSH, and TSH, can also be produced in this manner. This may facilitate structural studies of these hormones as well as lead to the production of recombinant hormones for biological studies and clinical use.

AB - A bacterial expression system for the β-subunit of hCG (hCG β) has been developed to produce suitable amounts of this protein for structural and biological studies. To produce hCG β in Escherichia coli, the nucleotide sequence that encodes the amino acid leader sequence was removed from the hCG β complementary DNA, and the gene was cloned into a pET expression vector. After induction of protein synthesis in host bacteria, recombinant hCG β (rhCG β) accumulated in inclusion bodies in an unfolded state. The inclusion bodies were purified from induced cultures of E. coli, solubilized in urea, and fractionated by reverse phase HPLC. In this way, 6-7 mg unfolded hCG β were recovered from 1 liter culture, rhCG β was folded in the presence of 6.4 mM cysteamine and 3.6 mM cystamine at pH 8.7 at a final concentration of 0.02 mg/ml protein. The folded protein assembled with urinary α hCG and the purified rhCG β/urinary α dimer bound to and activated the human LH/CG receptor permanently expressed in a cell line, indicating that it was a functional hormone. The rhCG β/urinary α dimer also stimulated in vivo ovulation in rats, thus confirming the biological activity of bacterially expressed hCG β. Because E. coli lacks the ability to glycosylate proteins, these activity results indicate that the N-linked and O-linked oligosaccharides of hCG β are not required for protein folding, subunit assembly, or full biological activity. The success of producing hCG β in bacteria and of folding it in vitro implies that the β-subunits of the other members of the glycoprotein hormone family, LH, FSH, and TSH, can also be produced in this manner. This may facilitate structural studies of these hormones as well as lead to the production of recombinant hormones for biological studies and clinical use.

UR - http://www.scopus.com/inward/record.url?scp=84995842082&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84995842082&partnerID=8YFLogxK

U2 - 10.1210/endo.135.3.8070386

DO - 10.1210/endo.135.3.8070386

M3 - Article

C2 - 8070386

VL - 135

SP - 911

EP - 918

JO - Endocrinology

JF - Endocrinology

SN - 0013-7227

IS - 3

ER -