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.
Subject(s)
Bacterial Proteins/genetics , Cystatin C/genetics , Endotoxins/genetics , Escherichia coli/genetics , Hemolysin Proteins/genetics , Inclusion Bodies/genetics , Recombinant Fusion Proteins/genetics , Amino Acid Sequence , Antibodies/immunology , Bacillus/chemistry , Bacillus/genetics , Bacillus thuringiensis Toxins , Bacterial Proteins/chemistry , Bacterial Proteins/isolation & purification , Base Sequence , Cystatin C/chemistry , Cystatin C/immunology , Cystatin C/isolation & purification , Endotoxins/chemistry , Endotoxins/isolation & purification , Escherichia coli/chemistry , Gene Expression , Hemolysin Proteins/chemistry , Hemolysin Proteins/isolation & purification , Humans , Inclusion Bodies/chemistry , Molecular Sequence Data , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/immunology , Recombinant Fusion Proteins/isolation & purification , SolubilityABSTRACT
Numerous proteins cannot be sufficiently prepared by ordinary recombinant DNA techniques because they are unstable or have deleterious effects on the host cell. One idea to prepare such proteins is to produce them as protein inclusions. Here we developed a novel system to effectively prepare proteins by using peptide tags derived from the insecticidal Cry toxin of a soil bacterium, Bacillus thuringiensis. Fusion with this peptide tag, designated 4AaCter, facilitates the formation of protein inclusions of glutathione S-transferase in Escherichia coli without losing the enzyme activity. Application of 4AaCter to the production of syphilis antigens TpN15, TpN17 and TpN47 from Treponema pallidum yielded excellent results, including a dramatic increase in the production level, simplification of the product purification and high reactivity with syphilis antibody. The use of 4AaCter may provide an innovational strategy for the efficient production of proteins.
