Abstract
Recent studies have revealed the potential of B-type natriuretic peptide (BNP) as a good prognostic marker for patients with heart failure. Antibodies against BNP are expected to be usefully employed in the diagnosis of heart failures. We established a more efficient method to produce functional anti-BNP, single chain variable fragment (scFv) using a eukaryotic expression system of Pichia pastoris. Although analysis of the N-terminal (NT) sequence of the expressed anti-BNP scFv indicated that the two Ste13 sites of the secreted anti-BNP scFv were not cleaved, the specificity of anti-BNP scFv was not affected significantly. The binding activity of anti-BNP scFv against other antigens, against four other antigens, NT probrain peptide (NT-pro BNP), atrial natriuretic peptide (ANP), carcinoembryonic antigen (CEA) and human serum albumin (HSA), was only one thousandth that of the original BNP antigen, which clearly demonstrated the specific binding of recombinant scFv toward BNP. The anti-BNP, scFv-based, electrochemical immunoassay exhibited excellent analytical performance with a detection limit of 1 fg/ml and a wide linear detection range from 1 to 10,000 fg/ml. The optimum culture conditions to obtain the maximum concentration of recombinant scFv were a pH range of 5.0–7.0 and an incubation temperature of 20°C. This anti-BNP scFv expressed in P. pastoris has the potential for promising applications in the diagnosis of heart failure.
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References
Bach H, Mazor Y, Shaky S, Shoham-Lev A, Berdichevsky Y, Gutnick DL, Benhar I (2001) Escherichia coli maltose-binding protein as a molecular chaperone for recombinant intracellular cytoplasmic single-chain antibodies. J Mol Biol 312:79–93
Batra G, Gurramkonda C, Nemani SK, Jain SK, Swaminathan S, Khanna N (2010) Optimization of conditions for secretion of dengue virus type 2 envelope domain III using Pichia pastoris. J Biosci Bioeng 110:408–414
Brophy SE, Tyner JD, Tu BX, Pinkus MS, Shin JW (2007) Human BNP immunospecific antibodies. US Patent 2007/0207152 A1
Cardarelli R, Lumicao TG Jr (2003) B-type natriuretic peptide: a review of its diagnostic, prognostic, and therapeutic monitoring value in heart failure for primary care physicians. J Am Board Fam Pract 16:327–333
Chang HJ, Choi SW, Chun HS (2008) Expression of functional single-chain variable domain fragment antibody (scFv) against mycotoxin zearalenone in Pichia pastoris. Biotechnol Lett 30:1801–1806
Chauhan AK, Arora D, Khanna N (1999) A novel feeding strategy for enhanced protein production by fed-batch fermentation in recombinant Pichia pastoris. Process Biochem 34:139–145
Crepin VF, Faulds CB, Connerton IF (2003) Production and characterization of the Talaromyces stipitatus feruloyl esterase FAEC in Pichia pastoris: identification of the nucleophilic serine. Protein Expr Purif 29(2):176–184
Dragosits M, Stadlmann J, Albiol J, Baumann K, Maurer M, Gasser B, Sauer M, Altmann F, Ferrer P, Mattanovich D (2009) The effect of temperature on the proteome of recombinant Pichia pastoris. J Proteome Res 8:1380–1392
Ecamilla-Treviño LL, Viader-Salvad’o JM, Barrera-Saldaña HA, Guerrero-Olazar’an M (2000) Biosynthesis and secretion of recombinant human growth hormone in Pichia pastoris. Biotechnol Lett 22:109–114
Eurwilaichitr L, Roytrakul S, Suprasongsin C, Manitchotpisit P, Panyim S (2002) Glutamic acid and alanine spacer is not necessary for removal of MF α-1 signal sequence fused to the human growth hormone produced from Pichia pastoris. World J Microbiol Biotechnol 18:493–498
Ghosalkar A, Sahai V, Srivastava A (2008) Secretory expression of interferon-alpha 2b in recombinant Pichia pastoris using three different secretion signals. Protein Expr Purif 60:103–109
Goda S, Takano K, Yamagata Y, Katakura Y, Yutani K (2000) Effect of extra N-terminal residues on the stability and folding of human lysozyme expressed in Pichia pastoris. Protein Eng 13(4):299–307
Grantham JA, Borgeson DD, Burnett JC (1997) BNP: pathophysiological and potential therapeutic roles in acute congestive heart failure. Am J Physiol 92:1077–1083
Hammerer-Lercher A, Halfinger B, Sarg B, Mair J, Puschendorf B, Griesmacher A, Guzman NA, Lindner HH (2008) Analysis of circulating forms of proBNP and NT-proBNP in patients with severe heart failure. Clin Chem 54:858–865
Hayden MS, Gilliland LK, Ledbetter JA (1997) Antibody engineering. Curr Opin Immunol 9:201–212
Ionescu RE, Jaffrezic-Renault N, Bouffier L, Gondran C, Cosnier S, Pinacho DG, Marcoc MP, S’anchez-Baeza FJ, Healy T, Martelet C (2007) Impedimetric immunosensor for the specific label free detection of ciprofloxacin antibiotic. Biosens Bioelectron 23:549–555
Kim TR, Goto Y, Hirota N, Kuwata K, Denton H, Wu SY, Sawyer L, Batt CA (1997) High-level expression of bovine β-lactoglobulin in Pichia pastoris and characterization of its physical properties. Protein Eng 10(11):1339–1345
Kurita R, Yokota Y, Sato Y, Mizutani F, Niwa O (2006) On-chip enzyme immunoassay of a cardiac marker using a microfluidic device combined with a portable surface plasmon resonance system. Anal Chem 78:5525–5531
Li Z, Xiong F, Lin Q, d’Anjou M, Daugulis AJ, Yang DSC, Hew CL (2001) Low-temperature increases the yield of biologically active herring antifreeze protein in Pichia pastoris. Protein Expr Purif 21:438–445
Li T, Cheng J, Hu B, Liu Y, Qian G, Liu F (2008) Construction, production, and characterization of recombinant scFv antibodies against methamidophos expressed in Pichia pastoris. World J Microbiol Biotechnol 24:867–874
Lin J, Ju H (2005) Electrochemical and chemiluminescent immunosensors for tumor markers. Biosens Bioelectron 20:1461–1470
Longenecker KL, Ruan Q, Fry EH, Saldana SC, Brophy SE, Richardson PL, Tetin SY (2009) Crystal structure and thermodynamic analysis of diagnostic mAb 106.3 complexed with BNP 5–13 (C10A). Proteins 76:536–547
Macauley-Patrick S, Fazenda ML, McNeil B, Harvey LM (2005) Heterologous protein production using the Pichia pastoris expression system. Yeast 22:249–270
Maeng BH, Nam DH, Kim YH (2011) Coexpression of molecular chaperones to enhance functional expression of anti-BNP scFv in the cytoplasm of Escherichia coli for the detection of B-type natriuretic peptide. World J Microbiol Biotechnol 27:1391–1398
McDonnell B, Hearty S, Leonard P, O’Kennedy R (2009) Cardiac biomarkers and the case for point-of-care testing. Clinical Biochem 42:549–561
Morrison LK, Harrison A, Krishnaswamy P, Kazanegra R, Clopton P, Maisel A (2002) Utility of a rapid B-natriuretic peptide assay in differentiating congestive heart failure from lung disease in patients presenting with dyspnea. J Am Coll Cardio 39(2):202–209
Mueller C, Scholer A, Laule-Kilian K, Martina B, Schindler C, Buser P, Pfisterer M, Perruchoud AP (2004) Use of B-type natriuretic peptide in the evaluation and management of acute dyspnea. N Engl J Med 350:647–654
Mukoyama M, Nakao K, Hosoda K, Suga S, Saito Y, Ogawa Y, Shirakami G, Jougasaki M, Obata K, Yasue H, Kambayashi Y, Inouye K, Imura H (1991) Brain natriuretic peptide as a novel cardiac hormone in humans evidence for an exquisite dual natriuretic peptide system, atrial natriuretic peptide and brain natriuretic peptide. J Clin Invest 87(4):1402–1412
Palecek E, Kizek R, Havran L, Billova S, Fojta M (2002) Electrochemical enzyme-linked immunoassay in a DNA hybridization sensor. Anal Chim Acta 469:73–83
Polevoda B, Sherman F (2003) N-terminal acetyltransferases and sequence requirements for N-terminal acetylation of eukaryotic proteins. J Mol Biol 325:595–622
Seferian KR, Tamm NN, Semenov AG, Mukharyamova KS, Tolstaya AA, Koshkina EV, Kara AN, Krasnoselsky MI, Apple FS, Esakova TV, Filatov VL, Katrukha AG (2007) The brain natriuretic peptide (BNP) precursor is the major immunoreactive form of BNP in patients with heart failure. Clinical Chem 53:866–873
Shi X, Karkut T, Chamankhah M, Alting-Mees M, Hemmingsen SM, Hegedus D (2003) Optimal conditions for the expression of a single-chain antibody (scFv) gene in Pichia pastoris. Protein Expr Purif 28:321–330
Sreekrishna K, Brankamp RG, Kropp KE, Blankenship DT, Tsay J, Smith PL, Wierschke JD, Subramaniam A, Birkenberger LA (1997) Strategies for optimal synthesis and secretion of heterologous proteins in the methylotrophic yeast Pichia pastoris. Gene 190:55–62
Tanfous NGB, Kallel H, Jarboui MA, Fathallah DM (2006) Expression in Pichia pastoris of a recombinant scFv form of MAb 107, an anti human CD11b integrin antibody. Enzyme Microb Technol 38:636–642
Vedvick T, Buckholz RG, Engel M, Urcan M, Kinney J, Provow S, Siegel RS, Thill GP (1990) High-level secretion of biologically active aprotinin from the yeast Pichia pastoris. J Ind Microbiol 7:197–202
Wang J, Ibanez A, Chatrathi MP, Escarpa A (2001) Electrochemical enzyme immunoassays on microchip platforms. Anal Chem 73:5323–5327
Wang J, Xu D, Erdem A, Polsky R, Salazar MA (2002) Genomagnetic electrochemical assays of DNA hybridization. Talanta 56:931–938
Wang J, Ibanez A, Chatrathi MP (2003) On-chip integration of enzyme and immunoassays: simultaneous measurements of insulin and glucose. J Am Chem Soc 125:8444–8445
Wellcome G (1995) Advances in the use of Pichia pastoris for high-level gene expression. Curr Opin Biotechnol 6:527–533
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We are grateful for the financial support of i-SENS, the Ministry of Knowledge Economy (No. 70004324) and Kwangwoon University Research Grant 2011.
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Maeng, B.H., Choi, J., Sa, Y.S. et al. Functional expression of recombinant anti-BNP scFv in methylotrophic yeast Pichia pastoris and application as a recognition molecule in electrochemical sensors. World J Microbiol Biotechnol 28, 1027–1034 (2012). https://doi.org/10.1007/s11274-011-0901-5
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DOI: https://doi.org/10.1007/s11274-011-0901-5