Efficient production of recombinant cystatin C using a peptide-tag, 4AaCter, that facilitates formation of insoluble protein inclusion bodies in Escherichia coli

Masahiro Hayashi, Shigehisa Iwamoto, Shinya Sato, Shigeo Sudo, Mari Takagi, Hiroshi Sakai, Tohru Hayakawa

Research output: Contribution to journalArticle

16 Citations (Scopus)

Abstract

Cystatin C is a cysteine protease inhibitor produced by a variety of human tissues. The blood concentration of cystatin C depends on the glomerular filtration rate and is an endogenous marker of renal dysfunction. Recombinant cystatin C protein with high immunogenicity is therefore in demand for the diagnostic market. In this study, to establish an efficient production system, a synthetic cystatin C gene was designed and synthesized in accordance with the codon preference of Escherichia coli genes. Recombinant cystatin C was expressed as a fusion with a peptide-tag, 4AaCter, which facilitates formation of protein inclusion bodies in E. coli cells. Fusion with 4AaCter-tag dramatically increased the production level of cystatin C, and highly purified protein was obtained without the need for complicated purification steps. The purity and yield of the final product was estimated as 87 ± 5% and 7.1 ± 1.1 mg/l culture, respectively. The recombinant cystatin C prepared by our method was as reactive against anti-cystatin C antibodies as native human cystatin C. Our results suggest that protein production systems using 4AaCter-tag could be a powerful means of preparing significant amounts of antigen protein.

Original languageEnglish
Pages (from-to)230-234
Number of pages5
JournalProtein Expression and Purification
Volume88
Issue number2
DOIs
Publication statusPublished - 2013

Fingerprint

Cystatin C
Inclusion Bodies
Escherichia coli
Peptides
Proteins
Cysteine Proteinase Inhibitors
Glomerular Filtration Rate
Population Groups
Codon
Genes
Kidney
Antigens

Keywords

  • 4AaCter-tag
  • Bacillus thuringiensis
  • Biomarker for renal dysfunction
  • Cry4Aa toxin
  • Formation of insoluble protein inclusion bodies
  • Production of antigen protein
  • Recombinant cystatin C

ASJC Scopus subject areas

  • Biotechnology

Cite this

Efficient production of recombinant cystatin C using a peptide-tag, 4AaCter, that facilitates formation of insoluble protein inclusion bodies in Escherichia coli. / Hayashi, Masahiro; Iwamoto, Shigehisa; Sato, Shinya; Sudo, Shigeo; Takagi, Mari; Sakai, Hiroshi; Hayakawa, Tohru.

In: Protein Expression and Purification, Vol. 88, No. 2, 2013, p. 230-234.

Research output: Contribution to journalArticle

Hayashi, Masahiro ; Iwamoto, Shigehisa ; Sato, Shinya ; Sudo, Shigeo ; Takagi, Mari ; Sakai, Hiroshi ; Hayakawa, Tohru. / Efficient production of recombinant cystatin C using a peptide-tag, 4AaCter, that facilitates formation of insoluble protein inclusion bodies in Escherichia coli. In: Protein Expression and Purification. 2013 ; Vol. 88, No. 2. pp. 230-234.
@article{e69c36e591d74f84a428342409faaf65,
title = "Efficient production of recombinant cystatin C using a peptide-tag, 4AaCter, that facilitates formation of insoluble protein inclusion bodies in Escherichia coli",
abstract = "Cystatin C is a cysteine protease inhibitor produced by a variety of human tissues. The blood concentration of cystatin C depends on the glomerular filtration rate and is an endogenous marker of renal dysfunction. Recombinant cystatin C protein with high immunogenicity is therefore in demand for the diagnostic market. In this study, to establish an efficient production system, a synthetic cystatin C gene was designed and synthesized in accordance with the codon preference of Escherichia coli genes. Recombinant cystatin C was expressed as a fusion with a peptide-tag, 4AaCter, which facilitates formation of protein inclusion bodies in E. coli cells. Fusion with 4AaCter-tag dramatically increased the production level of cystatin C, and highly purified protein was obtained without the need for complicated purification steps. The purity and yield of the final product was estimated as 87 ± 5{\%} and 7.1 ± 1.1 mg/l culture, respectively. The recombinant cystatin C prepared by our method was as reactive against anti-cystatin C antibodies as native human cystatin C. Our results suggest that protein production systems using 4AaCter-tag could be a powerful means of preparing significant amounts of antigen protein.",
keywords = "4AaCter-tag, Bacillus thuringiensis, Biomarker for renal dysfunction, Cry4Aa toxin, Formation of insoluble protein inclusion bodies, Production of antigen protein, Recombinant cystatin C",
author = "Masahiro Hayashi and Shigehisa Iwamoto and Shinya Sato and Shigeo Sudo and Mari Takagi and Hiroshi Sakai and Tohru Hayakawa",
year = "2013",
doi = "10.1016/j.pep.2013.01.011",
language = "English",
volume = "88",
pages = "230--234",
journal = "Protein Expression and Purification",
issn = "1046-5928",
publisher = "Academic Press Inc.",
number = "2",

}

TY - JOUR

T1 - Efficient production of recombinant cystatin C using a peptide-tag, 4AaCter, that facilitates formation of insoluble protein inclusion bodies in Escherichia coli

AU - Hayashi, Masahiro

AU - Iwamoto, Shigehisa

AU - Sato, Shinya

AU - Sudo, Shigeo

AU - Takagi, Mari

AU - Sakai, Hiroshi

AU - Hayakawa, Tohru

PY - 2013

Y1 - 2013

N2 - Cystatin C is a cysteine protease inhibitor produced by a variety of human tissues. The blood concentration of cystatin C depends on the glomerular filtration rate and is an endogenous marker of renal dysfunction. Recombinant cystatin C protein with high immunogenicity is therefore in demand for the diagnostic market. In this study, to establish an efficient production system, a synthetic cystatin C gene was designed and synthesized in accordance with the codon preference of Escherichia coli genes. Recombinant cystatin C was expressed as a fusion with a peptide-tag, 4AaCter, which facilitates formation of protein inclusion bodies in E. coli cells. Fusion with 4AaCter-tag dramatically increased the production level of cystatin C, and highly purified protein was obtained without the need for complicated purification steps. The purity and yield of the final product was estimated as 87 ± 5% and 7.1 ± 1.1 mg/l culture, respectively. The recombinant cystatin C prepared by our method was as reactive against anti-cystatin C antibodies as native human cystatin C. Our results suggest that protein production systems using 4AaCter-tag could be a powerful means of preparing significant amounts of antigen protein.

AB - Cystatin C is a cysteine protease inhibitor produced by a variety of human tissues. The blood concentration of cystatin C depends on the glomerular filtration rate and is an endogenous marker of renal dysfunction. Recombinant cystatin C protein with high immunogenicity is therefore in demand for the diagnostic market. In this study, to establish an efficient production system, a synthetic cystatin C gene was designed and synthesized in accordance with the codon preference of Escherichia coli genes. Recombinant cystatin C was expressed as a fusion with a peptide-tag, 4AaCter, which facilitates formation of protein inclusion bodies in E. coli cells. Fusion with 4AaCter-tag dramatically increased the production level of cystatin C, and highly purified protein was obtained without the need for complicated purification steps. The purity and yield of the final product was estimated as 87 ± 5% and 7.1 ± 1.1 mg/l culture, respectively. The recombinant cystatin C prepared by our method was as reactive against anti-cystatin C antibodies as native human cystatin C. Our results suggest that protein production systems using 4AaCter-tag could be a powerful means of preparing significant amounts of antigen protein.

KW - 4AaCter-tag

KW - Bacillus thuringiensis

KW - Biomarker for renal dysfunction

KW - Cry4Aa toxin

KW - Formation of insoluble protein inclusion bodies

KW - Production of antigen protein

KW - Recombinant cystatin C

UR - http://www.scopus.com/inward/record.url?scp=84874597890&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84874597890&partnerID=8YFLogxK

U2 - 10.1016/j.pep.2013.01.011

DO - 10.1016/j.pep.2013.01.011

M3 - Article

C2 - 23396100

AN - SCOPUS:84874597890

VL - 88

SP - 230

EP - 234

JO - Protein Expression and Purification

JF - Protein Expression and Purification

SN - 1046-5928

IS - 2

ER -