Recombinant Expression and Purification of Human Cystatin C Biomarker in Escherichia coli for Prediagnostic Renal Disorders

Document Type : Original Article


1 Applied Biotechnology Research Center, Systems Biology and Poisonings Institute, Baqiyatallah University of Medical Sciences, Tehran, Iran

2 Department of Biology, Faculty of Science, University of Guilan, Rasht, Iran

3 Applied Microbiology Research Center, Systems Biology and Poisonings Institute, Baqiyatallah University of Medical Sciences, Tehran, Iran

4 Molecular Biology Research Center, Systems Biology and Poisonings Institute, Baqiyatallah University of Medical Sciences, Tehran, Iran


Introduction: Chronic kidney disease is a worldwide health problem and Glomerular filtration rate (GFR) is the most frequently used criteria in the assessment of rental function. Cystatin C as a member of type 2 cystatin superfamily and cysteine-protease inhibitor is found in high concentrations in all biological fluids. This study is aimed to study cystatin C as a potent biomarker for clinical measurement of renal disorders and other diseases.
Materials and Methods: In this study, the human cystatin C construct was analyzed by bioinformatics software. It was cloned and expressed to produce an appropriate antigen for anti-cystatin C (anti-Cys C) obtained from mice Balb/C as a crucial point in the improvement of an enzyme-linked immunosorbent assay (ELISA) method. Serum samples were given from 32 hospitalized patients with renal failure and cardiovascular disease and non-hospitalized patients were tested by ELISA method using anti-Cys C obtained from mice Balb/C.
Results: Our findings indicated 0.36-2.4 mg/L as the best conclusion for antigen cystatin C in patients’ sera and 1/50 dilution for anti-Cys C obtained from mice Balb/C and showed a relationship between patients with high creatinine and high concentration of cystatin C. In case of five cardiovascular disease patients with normal upper limit of Creatinine we obtained cystatin C lower than kidney failure and raising of cystatin C in 6 patients with increased TSH were seen.
Conclusions: Polyclonal anti-Cys C antibodies were obtained through the immunization of Balb/C mice can be employed as an anti-Cys C in ELISA for diagnosis of some renal dysfunction.


Levey AS, Coresh J, Balk E, et al. National Kidney Foundation practice guidelines for chronic kidney disease: evaluation, classification, and stratification. Ann Intern Med. 2003;139(2):137- 147.
Myers GL, Miller WG, Coresh J, et al. Recommendations for improving serum creatinine measurement: a report from the Laboratory Working Group of the National Kidney Disease Education Program. Clin Chem. 2006;52(1):5-18. doi:10.1373/ clinchem.2005.0525144
Rule AD, Larson TS, Bergstralh EJ, Slezak JM, Jacobsen SJ, Cosio FG. Using serum creatinine to estimate glomerular filtration rate: accuracy in good health and in chronic kidney disease. Ann Intern Med. 2004;141(12):929-937.
Inker LA, Schmid CH, Tighiouart H, et al. Estimating glomerular filtration rate from serum creatinine and cystatin C. N Engl J Med. 2012;367(1):20-29. doi:10.1056/NEJMoa1114248
Jung K, Jung M. Cystatin C: a promising marker of glomerular filtration rate to replace creatinine. Nephron. 1995;70(3):370-371. doi:10.1159/000188621
Zhang Z, Lu B, Sheng X, Jin N. Cystatin C in prediction of acute kidney injury: a systemic review and meta-analysis. Am J Kidney Dis. 2011;58(3):356-365. doi:10.1053/j.ajkd.2011.02.389
Mousa M, El-Saeid M, Hamdy M. Evaluation of Cystatin C, Fibronectin and Alpha-Feto Protein as Biochemical Markers in Patients with Liver Diseases. J Am Sci. 2012;8(7):770-779.
Bengtsson E, To F, Hakansson K, et al. Lack of the cysteine protease inhibitor cystatin C promotes atherosclerosis in apolipoprotein E-deficient mice. Arterioscler Thromb Vasc Biol. 2005;25(10):2151- 2156. doi:10.1161/01.ATV.0000179600.34086.7d
Gashenko EA, Lebedeva VA, Brak IV, Tsykalenko EA, Vinokurova GV, Korolenko TA. Evaluation of serum procathepsin B, cystatin B and cystatin C as possible biomarkers of ovarian cancer. Int J Circumpolar Health. 2013;72. doi:10.3402/ijch.v72i0.21215
Wilson ME, Boumaza I, Bowser R. Measurement of cystatin C functional activity in the cerebrospinal fluid of amyotrophic lateral sclerosis and control subjects. Fluids Barriers CNS. 2013;10(1):15. doi:10.1186/2045-8118-10-15
Andersen TB, Jodal L, Boegsted M, et al. GFR prediction from cystatin C and creatinine in children: effect of including body cell mass. Am J Kidney Dis. 2012;59(1):50-57. doi:10.1053/j. ajkd.2011.09.013
Brguljan PM, Cimerman N. Human cystatin C. Turk J Biochem. 2007;32(3):95-103.
Turk B, Turk D, Salvesen GS. Regulating cysteine protease activity: essential role of protease inhibitors as guardians and regulators. Curr Pharm Des. 2002;8(18):1623-1637.
Dousdampanis P, Trigka K, Fourtounas C. Diagnosis and management of chronic kidney disease in the elderly: a field of ongoing debate. Aging Dis. 2012;3(5):360-372.
Hayashi M, Iwamoto S, Sato S, et al. Efficient production of recombinant cystatin C using a peptide-tag, 4AaCter, that facilitates formation of insoluble protein inclusion bodies in Escherichia coli. Protein Expr Purif. 2013;88(2):230-234. doi:10.1016/j. pep.2013.01.011
Shi GP, Sukhova GK, Grubb A, et al. Cystatin C deficiency in human atherosclerosis and aortic aneurysms. J Clin Invest. 1999;104(9):1191-1197. doi:10.1172/jci7709
Chen ZK, Ge CJ, Hu SJ. [The relationship of cystatin C and cardiovascular diseases]. Sheng Li Ke Xue Jin Zhan. 2003;34(3):269-271.
Piwowar A, Knapik-Kordecka M, Buczynska H, Warwas M. Plasma cystatin C concentration in non-insulin-dependent diabetes mellitus: relation with nephropathy. Arch Immunol Ther Exp (Warsz). 1999;47(5):327-331.
Wang F, Pan W, Wang H, Zhou Y, Wang S, Pan S. The impacts of thyroid function on the diagnostic accuracy of cystatin C to detect acute kidney injury in ICU patients: a prospective, observational study. Crit Care. 2014;18(1):R9. doi:10.1186/cc13186
Chew JS, Saleem M, Florkowski CM, George PM. Cystatin C--a paradigm of evidence based laboratory medicine. Clin Biochem Rev. 2008;29(2):47-62.
Ristiniemi N. Quantification and Clinical Relevance of Cystatin C. Turku, Finland: Annales Universitatis Turkuensis; 2014.
Abdollah Z, Karami A, Amani J, Khodi S, Gheybi E. In silico study of Cystatin C protein to Develop an ELISA kit using Computational tools and servers. Bull Environ Pharmacol Life Sci. 2015;4(7):87- 94.
Heiat M, Aghamollaei H, Hoseini SM, Abbasi Larki R, Yari K. Optimization of plasmid electrotransformation into Escherichia coli using Taguchi statistical method. Afr J Biotechnol. 2012;11(30):7603-7608. doi:10.5897/ajb11.4078
Amani J, Salmanian AH, Rafati S, Mousavi SL. Immunogenic properties of chimeric protein from espA, eae and tir genes of Escherichia coli O157:H7. Vaccine. 2010;28(42):6923-6929. doi:10.1016/j.vaccine.2010.07.061
Assarehzadegan MA, Sankian M, Jabbari F, Tehrani M, Varasteh A. Expression of the recombinant major allergen of Salsola kali pollen (Sal k 1) and comparison with its low-immunoglobulin E-binding mutant. Allergol Int. 2010;59(2):213-222. doi:10.2332/ allergolint.09-OA-0155
Gheybi E, Amani J, Salmanian AH, Mashayekhi F, Khodi S. Designing a recombinant chimeric construct contain MUC1 and HER2 extracellular domain for prediagnostic breast cancer. Tumour Biol. 2014;35(11):11489-11497. doi:10.1007/s13277- 014-2483-y
Abrahamson M. Human cysteine proteinase inhibitors. Isolation, physiological importance, inhibitory mechanism, gene structure and relation to hereditary cerebral hemorrhage. Scand J Clin Lab Invest Suppl. 1988;191:21-31.
Saitoh E, Sabatini LM, Eddy RL, et al. The human cystatin C gene (CST3) is a member of the cystatin gene family which is localized on chromosome 20. Biochem Biophys Res Commun. 1989;162(3):1324-1331.
Filler G, Bokenkamp A, Hofmann W, Le Bricon T, Martinez-Bru C, Grubb A. Cystatin C as a marker of GFR--history, indications, and future research. Clin Biochem. 2005;38(1):1-8. doi:10.1016/j. clinbiochem.2004.09.025
Jiang R, Xu C, Zhou X, Wang T, Yao G. Detection of cystatin C biomarker for clinical measurement of renal disease by developed ELISA diagnostic kits. J Transl Med. 2014;12:205. doi:10.1186/1479-5876-12-205
Dierkes J, Westphal S, Luley C. Serum homocysteine increases after therapy with fenofibrate or bezafibrate. Lancet. 1999;354(9174):219-220. doi:10.1016/s0140-6736(99)02153-4
Cornwall GA, Hsia N. A new subgroup of the family 2 cystatins. Mol Cell Endocrinol. 2003;200(1-2):1-8. doi:10.1016/S0303- 7207(02)00408-2