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Protein & Peptide Letters

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ISSN (Print): 0929-8665
ISSN (Online): 1875-5305

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Research Article

Expression, Purification and Characterization of Recombinant Human Coagulation Factor XIIIa in Pichia Pastoris

Author(s): Linyan Cheng, Ting Zhang, Yuchang Fei, Hao Shen, Hui Huang, Jin Chen, Bin Xu* and Jian Xu*

Volume 28, Issue 1, 2021

Published on: 25 June, 2020

Page: [55 - 62] Pages: 8

DOI: 10.2174/0929866527666200625203240

open access plus

Abstract

Background: Coagulation factor XIIIa(FXIIIa) plays a critical role in the final stage of blood coagulation. It is extremely important in wound healing, tissue repairing and promoting cell adhesion. The deficiency of the coagulation factor can cause hemorrhage and slow wound healing.

Objective: In this study, recombinant pPICZαC-FXIIIa was expressed in Pichia pastoris, purified as well as its biological activity was determined.

Methods: The FXIIIa fragment obtained from the human placenta was inserted into pPICZαC to obtain pPICZαC-FXIIIa, which was transformed into X33 after linearization, and FXIIIa inserted into Pichia pastoris X33 was screened for methanol induction. The expressed product was identified by western blotting, then the supernatant was purified by affinity chromatography, and the purified product was determined by plasma coagulation experiment.

Results: Polymerase Chain Reaction(PCR) showed that the FXIIIa fragment of 2250 bp was inserted successfully into pPICZαC. The expression and purification products of the same molecular weight as target protein(about 83 kDa) were obtained, which solidified significantly when reacted with plasma.

Conclusion: The expression and purification products were successful, with sufficient biological activity, which can be used as a candidate FXIIIa hemostatic agent in genetic engineering.

Keywords: pPICZαC-FXIIIa, Pichia pastoris, secretory expression, protein purification, biological activity, hemostatic agent.

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Graphical Abstract

[1]
Shi, D.Y.; Wang, S.J. Advances of coagulation factor XIII. Chin. Med. J. (Engl.), 2017, 130(2), 219-223.
[PMID: 28091415]
[2]
I, KOMáROMI.; Bagoly, Z.; Muszbek, L. Factor XIII: novel structural and functional aspects. J. Thromb. Haemost., 2011, 9, 9-20.
[http://dx.doi.org/10.1111/j.1538-7836.2010.04070.x]
[3]
Lorand, L. Factor XIII: structure, activation, and interactions with fibrinogen and fibrin. Ann. N. Y. Acad. Sci., 2001, 936, 291-311.
[http://dx.doi.org/10.1111/j.1749-6632.2001.tb03516.x] [PMID: 11460485]
[4]
Matthew, Tak.; Sheng, Natalis, R.; Dawn, G.; Michelle, S. Acquired factor XIII deficiency: A review. Transfus. Apheresis Sci., 2018, 57, 724-730.
[http://dx.doi.org/10.1016/j.transci.2018.10.013]
[5]
Muszbek, L.; Katona, É. Diagnosis and management of congenital and acquired FXIII deficiencies. Semin. Thromb. Hemost., 2016, 42(4), 429-439.
[http://dx.doi.org/10.1055/s-0036-1572326] [PMID: 27071048]
[6]
Naderi, M.; Dorgalaleh, A.; Alizadeh, S.; Tabibian, S.; Hosseini, S.; Shamsizadeh, M.; Bamedi, T. Clinical manifestations and management of life-threatening bleeding in the largest group of patients with severe factor XIII deficiency. Int. J. Hematol., 2014, 100(5), 443-449.
[http://dx.doi.org/10.1007/s12185-014-1664-1] [PMID: 25230816]
[7]
Gemmati, D.; Vigliano, M.; Burini, F.; Mari, R.; El Mohsein, H.H.; Parmeggiani, F.; Serino, M.L. Coagulation factor XIIIA (F13A1): novel perspectives in treatment and pharmacogenetics. Curr. Pharm. Des., 2016, 22(11), 1449-1459.
[http://dx.doi.org/10.2174/1381612822666151210122954] [PMID: 26654441]
[8]
Kasahara, K.; Kaneda, M.; Miki, T.; Iida, K.; Sekino-Suzuki, N.; Kawashima, I.; Suzuki, H.; Shimonaka, M.; Arai, M.; Ohno-Iwashita, Y.; Kojima, S.; Abe, M.; Kobayashi, T.; Okazaki, T.; Souri, M.; Ichinose, A.; Yamamoto, N. Clot retraction is mediated by factor XIII-dependent fibrin-αIIbβ3-myosin axis in platelet sphingomyelin-rich membrane rafts. Blood, 2013, 122(19), 3340-3348.
[http://dx.doi.org/10.1182/blood-2013-04-491290] [PMID: 24002447]
[9]
Kulkarni, S.; Jackson, S.P. Platelet factor XIII and calpain negatively regulate integrin alphaIIbbeta3 adhesive function and thrombus growth. J. Biol. Chem., 2004, 279(29), 30697-30706.
[http://dx.doi.org/10.1074/jbc.M403559200] [PMID: 15131115]
[10]
Mitchell, J.L.; Lionikiene, A.S.; Fraser, S.R.; Whyte, C.S.; Booth, N.A.; Mutch, N.J. Functional factor XIII-A is exposed on the stimulated platelet surface. Blood, 2014, 124(26), 3982-3990.
[http://dx.doi.org/10.1182/blood-2014-06-583070] [PMID: 25331118]
[11]
Weidner, M.; Taupp, M.; Hallam, S.J. Expression of recombinant proteins in the methylotrophic yeast Pichia pastoris. J. Vis. Exp., 2010, 36(36), 330-339.
[http://dx.doi.org/10.3791/1862] [PMID: 20186119]
[12]
Fischer, J.E.; Glieder, A. Current advances in engineering tools for Pichia pastoris. Curr. Opin. Biotechnol., 2019, 59, 175-181.
[http://dx.doi.org/10.1016/j.copbio.2019.06.002] [PMID: 31470258]
[13]
Zahrl, R.J.; Peña, David, A.; Mattanovich, D; Gasser, B Systems biotechnology for protein production in Pichia pastoris. FEMS Yeast Res., 2017, 17.
[14]
Aeschlimann, D.; Paulsson, M. Transglutaminases: protein cross-linking enzymes in tissues and body fluids. Thromb. Haemost., 1994, 71(4), 402-415.
[http://dx.doi.org/10.1055/s-0038-1642451] [PMID: 7914385]
[15]
Gao, J.; Hooker, B.S.; Anderson, D.B. Expression of functional human coagulation factor XIII A-domain in plant cell suspensions and whole plants. Protein. Expr. Purif.,, 2004, 37, 89-96.
[16]
Peng, B.; Xue, G.; Xu, D.; Feng, Z.; Chen, J.; Huang, M.; Lu, H.; Gong, L. Expression and purification of recombinant serine protease domain of human coagulation factor XII in Pichia pastoris. Biosci. Biotechnol. Biochem., 2019, 83(10), 1815-1821.
[http://dx.doi.org/10.1080/09168451.2019.1621151] [PMID: 31117893]
[17]
Kazemzadeh-Narbat, M.; Annabi, N.; Khademhosseini, A. Surgical sealants and high strength adhesives. Mater. Today, 2015, 18, 176-177.
[http://dx.doi.org/10.1016/j.mattod.2015.02.012]
[18]
Rawnaq, T.; M, Doebert.; J, Beek.; P, Schwaerzler. Viable Caesarean scar pregnancy managed by sonographically guided application of a fibrin sealant. Ultrasound Obstet. Gynecol., 2016, 48, 665-667.
[http://dx.doi.org/10.1002/uog.15870] [PMID: 26822929]
[19]
Scognamiglio, F.; Travan, A.; Rustighi, I.; Tarchi, P.; Palmisano, S.; Marsich, E.; Borgogna, M.; Donati, I.; de Manzini, N.; Paoletti, S. Adhesive and sealant interfaces for general surgery applications. J. Biomed. Mater. Res. B Appl. Biomater., 2016, 104(3), 626-639.
[http://dx.doi.org/10.1002/jbm.b.33409] [PMID: 25891348]
[20]
Randelli, F.; D’Anchise, R.; Ragone, V.; Serrao, L.; Cabitza, P.; Randelli, P. Is the newest fibrin sealant an effective strategy to reduce blood loss after total knee arthroplasty? A randomized controlled study. J. Arthroplasty, 2014, 29(8), 1516-1520.
[http://dx.doi.org/10.1016/j.arth.2014.02.024] [PMID: 24674732]
[21]
Jain, R.; Wairkar, S. Recent developments and clinical applications of surgical glues: an overview. Int. J. Biol. Macromol., 2019, 137, 95-106.
[http://dx.doi.org/10.1016/j.ijbiomac.2019.06.208] [PMID: 31255625]
[22]
Orosz, Z.Z.; Katona, É.; Facskó, A.; Módis, L.; Muszbek, L.; Berta, A. Factor XIII subunits in human tears; their highly elevated levels following penetrating keratoplasty. Clin. Chim. Acta, 2011, 412(3-4), 271-276.
[http://dx.doi.org/10.1016/j.cca.2010.10.017] [PMID: 20974119]
[23]
Tsujimoto, I.; Moriya, K.; Sakai, K.; Dickneite, G.; Sakai, T. Critical role of factor XIII in the initial stages of carbon tetrachloride-induced adult liver remodeling. Am. J. Pathol., 2011, 179(6), 3011-3019.
[http://dx.doi.org/10.1016/j.ajpath.2011.08.037] [PMID: 22019897]
[24]
Sugitani, K.; Ogai, K.; Hitomi, K.; Nakamura-Yonehara, K.; Shintani, T.; Noda, M.; Koriyama, Y.; Tanii, H.; Matsukawa, T.; Kato, S. A distinct effect of transient and sustained upregulation of cellular factor XIII in the goldfish retina and optic nerve on optic nerve regeneration. Neurochem. Int., 2012, 61(3), 423-432.
[http://dx.doi.org/10.1016/j.neuint.2012.06.004] [PMID: 22709671]
[25]
Yan, MTS.; Rydz, N.; Goodyear, D.; Sholzberg, M. Acquired factor XIII deficiency: a review. Transfus. apher. sci, 2018, 57, 724-730.
[http://dx.doi.org/10.1016/j.transci.2018.10.013]
[26]
Pitkänen, H.; Jouppila, A.; Mowinckel, M-C.; Lemponen, M.; Patiwael, S.; Sandset, P.M.; Lassila, R.; Brinkman, H.J.M. Enhanced thrombin generation and reduced tissue factor pathway inhibitor and intact protein S in processed solvent detergent plasma. Thromb. Res., 2015, 135, 167-174.
[http://dx.doi.org/10.1016/j.thromres.2014.10.020] [PMID: 25466844]
[27]
Macauley-Patrick, S.; Fazenda, M.L.; McNeil, B.; Harvey, L.M. Heterologous protein production using the Pichia pastoris expression system. Yeast, 2005, 22(4), 249-270.
[http://dx.doi.org/10.1002/yea.1208] [PMID: 15704221]
[28]
Shan, J.; Baguinon, M.; Zheng, L.; Krishnamoorthi, R. Expression, refolding, and activation of the catalytic domain of human blood coagulation factor XII. Protein Expr. Purif., 2003, 27(1), 143-149.
[http://dx.doi.org/10.1016/S1046-5928(02)00608-3] [PMID: 12509996]
[29]
A R, S.R.; Satheeshkumar, P.K.; Vijayalakshmi, M.A. Expression, purification, and partial in vitro characterization of biologically active human coagulation factor VIII light chain (A3-C1-C2) in Pichia pastoris. Appl. Biochem. Biotechnol., 2013, 171(1), 10-19.
[http://dx.doi.org/10.1007/s12010-013-0338-4] [PMID: 23813406]
[30]
Cheng, E.; Jinzenji, D.; Lorthiois, A.P.A.A.; de Carvalho, R.R.; Tanaka-Azevedo, A.M.; Raw, I.; Martins, E.A.L. Purification of coagulation factor VIII using chromatographic methods. Direct chromatography of plasma in anion exchange resins. Biotechnol. Lett., 2010, 32(9), 1207-1214.
[http://dx.doi.org/10.1007/s10529-010-0282-0] [PMID: 20431912]
[31]
Rodrigues, E.S.; Verinaud, C.I.; Oliveira, D.S.; Raw, I.; Lopes, A.P.; Martins, E.A.; Cheng, E. Purification of coagulation factor VIII by immobilized metal affinity chromatography. Biotechnol. Appl. Biochem., 2015, 62(3), 343-348.
[http://dx.doi.org/10.1002/bab.1276] [PMID: 25082654]
[32]
Nikolajsen, C.L.; Dyrlund, T.F.; Poulsen, E.T.; Enghild, J.J.; Scavenius, C. Coagulation factor XIIIa substrates in human plasma: identification and incorporation into the clot. J. Biol. Chem., 2014, 289(10), 6526-6534.
[http://dx.doi.org/10.1074/jbc.M113.517904] [PMID: 24443567]
[33]
Xi, X.; Li, X.; Wu, F.; Guan, X.; Jin, L.; Guo, Y.; Song, W.; Du, B. Expression, purification and characterization of active untagged recombinant human leukemia inhibitory factor from E.coli. Protein Expr. Purif., 2017, 134, 139-146.
[http://dx.doi.org/10.1016/j.pep.2017.03.020] [PMID: 28347752]

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