Original Article

Production and Expression Optimization of Heterologous Serratiopeptidase


Background: Serratiopeptidase is a bacterial metalloprotease, which is useful for the treatment of pain and inflammation. It breaks down fibrin, thins the fluids formed during inflammation and acts as an anti-biofilm agent. Because of medicinally important role of the enzyme, we aimed to study the cloning and the expression optimization of serratiopeptidase.

Methods: The heat-stable serratiopeptidase (5d7w) was selected as the template. Cloning into pET28a expression vector was performed and confirmed by colony PCR and double restriction enzyme digestion. The recombinant protein was expressed in Esherichia coli BL21 and confirmed by SDS-PAGE and Western blot analysis. Different parameters such as expression vector, culture media, post-induction incubation temperature, inducer concentration, and post-induction incubation time were altered to obtain the highest amount of the recombinant protein.

Results: Serratiopeptidase was successfully cloned and expressed under optimized conditions in E. coli which confirmed by western blot analysis. The optimal conditions of expression were determined using pQE30 as vector, cultivating the host bacteria in Terrific Broth (TB) medium, at 37º C, induction by IPTG concentration equal to 0.5 mM, and cells were harvested 4 h after induction.

Conclusion: As serratiopeptidase is a multi-potent enzyme, the expressed recombinant protein can be considered as a valuable agent for pharmaceutical applications in further studies.

1. Hines DA, Saurugger PN, Ihler GM, Ben-edik MJ (1988). Genetic analysis of extra-cellular proteins of Serratia marcescens. J Bacteriol, 170(9):4141-4146.
2. Nakahama K, Yoshimura K, Marumoto R et al (1986). Cloning and sequencing of Serratia protease gene. Nucleic Acids Res, 14(14):5843-55.
3. Isono M, Kazutaka M, Kodama R et al (1970). Method of treating inflammation and composition therefor. Issued 1974-02-12, assigned to Takeda Chemical In-dustries Ltd, US patent 3792160.
4. Yamasaki H, Tsuji H, Saeki K (1967).[ Anti-inflammatory action of a protease, TSP, produced by Serratia]. Nihon Yakurigaku Zasshi, 63:302-14.
5. Mazzone A, Catalani M, Costanzo M et al (1990). Evaluation of Serratia-peptidase in acute or chronic inflammation of otorhi-nolaryngology pathology: a multicentre, double-blind, randomized trial versus placebo. J Int Med Res, 18:379-88.
6. Bhagat S, Agarwal M, Roy V (2013). Serrati-opeptidase: A systematic review of the existing evidence. International Journal of Surgery, 209-217.
7. Rothschild J (1991). Clinical use of serrapep-tase: An alternative to non-steroidal anti-inflammatory agents. The American Chiro-practor, 58:17.
8. Nakamura S, Hashimoto Y, Mikami M et al (2003). Effect of the proteolytic enzyme serrapeptase in patients with chronic air-way disease . Respirology, 8(3):316-20.
9. Badhe RV, Nanda RK, Kulkarni MB et al (2009). Media optimization studies for Serratiopeptidase production from Serra-tia marcescens ATCC 13880. Hindustan Antibiot Bull, 51(1-4):17-23.
10. Delepelaire P, Wandersman C (1991). Char-acterization, localization,and transmem-brane organization of the three proteins PrtD, PrtE,and PrtF necessary for prote-ase secretion by the gram-negative bacte-rium Erwinia chrysanthemi. Mol Microbiol, 5(10):2427-34.
11. Baumann U, Wu S, Flaherty KM, McKay DB (1993). Three dimensional structure of the alkaline protease of Pseudomonas aeruginosa. A two-domain protein with a calcium binding parallel roll motif. EMBO J, 12(9):3357-64.
12. Baumann U, Bauer M, Létoffé S et al (1995). Crystal structure of a complex between Serratia marcescens metalloprotease and an inhibitor from Erwinia chrysanthemi. J Mol Biol, 248(3):653-61.
13. Hege T, Baumann U (2001). Protease C of Erwinia chrysanthemi: the crystal struc-ture and role of amino acids Y228 and E189. J Mol Biol, 314(2):187-93.
14. Wu D, Ran T, Wang W, Xu D (2016). Struc-ture of a thermostable serralysin from Serratia sp. FS14 at 1.1 Å resolution. Acta Crystallogr F Struct Biol Commun, 72(Pt 1):10-5.
15. Redfern, Robert. The Miracle Enzyme. Nat-urally Healthy Publications: Cheshire, UK; 2009; https://www.jandevrieshealth.co.uk/downloads/ebook.pdf
16. Selan L, Papa R, Tilotta M et al (2015). Serra-tiopeptidase: a well-known metalloprote-ase with a new non-proteolytic activity against S. aureus biofilm. BMC Microbiol, 15:207.
17. Valizadeh V, Mirkazemi S, Vaziri B et al (2014). Optimized method for purifica-tion of expressed Plasmodium vivax Duffy Binding Protein-II (PvDBP-II): implication for vivax malaria vaccine de-velopment. Vaccine Research, 1(2):1-6.
18. Rouhani M, Zakeri S, Mehrizi AA, Djadid ND (2015). Comparative analysis of the profiles of IgG subclass-specific re-sponses to Plasmodium falciparum apical membrane antigen-1 and merozoite sur-face protein-1 in naturally exposed indi-viduals living in malaria hypoendemic set-tings, Iran. Malar J, 14:58.
19. Malik A, Alsenaidy AM, Elrobh M et al (2016). Optimization of expression and purification of HSPA6 protein from Camelus dromedarius in E. coli. Saudi J Biol Sci, 23:410-419.
20. Ishii K, Adachi T, Hamamoto H, Sekimizu K (2014). Serratia marcescens suppresses host cellular immunity via the production of an adhesion-inhibitory factor against immune surveillance cells. J Biol Chem, 289(9):5876-88.
IssueVol 49 No 5 (2020) QRcode
SectionOriginal Article(s)
Serratiopeptidase Cloning Expression optimization

Rights and permissions
Creative Commons License This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.
How to Cite
ROUHANI M, VALIZADEH V, MOLASALEHI S, NOROUZIAN D. Production and Expression Optimization of Heterologous Serratiopeptidase. Iran J Public Health. 2020;49(5):931-939.