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.
authors: Graduate School of Natural Science and Technology, Okayama University, 3-1-1 Tsushima-naka, Kita-ku, Okayama 700-8530, Japan.
authors: Masahiro
authors: M
authors: Hayashi
authors: Shigehisa
authors: S
authors: Iwamoto
authors: Shinya
authors: S
authors: Sato
authors: Shigeo
authors: S
authors: Sudo
authors: Mari
authors: M
authors: Takagi
authors: Hiroshi
authors: H
authors: Sakai
authors: Tohru
authors: T
authors: Hayakawa
mesh_terms: D000595:Amino Acid Sequence; D000906:Antibodies / Q000276:immunology; D001407:Bacillus / Q000737:chemistry / Q000235:genetics; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D001483:Base Sequence; D055316:Cystatin C / Q000737:chemistry / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D004731:Endotoxins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics*; D015870:Gene Expression; D006460:Hemolysin Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D006801:Humans; D002479:Inclusion Bodies / Q000737:chemistry / Q000235:genetics*; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D012995:Solubility
chemical_list: D000906:Antibodies; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D055316:Cystatin C; D004731:Endotoxins; D006460:Hemolysin Proteins; D011993:Recombinant Fusion Proteins; C030956:insecticidal crystal protein, Bacillus Thuringiensis