Email this article A novel approach to enhance the performance of kallikrein 6 enzyme using Pichia pastoris GS115 as a host
, Hamid Bakherad, Navid Mogharrab , Mohammad Rabbani Abstract
Background and purpose: Enzyme engineering is the process of raising enzyme efficiency and activity by altering amino acid sequences. Kallikrein 6 (KLK6) enzyme is a secreted serine protease involved in a variety of physiological and pathological activities. The increased expression of KLK6 plays a key role in various diseases. Instability and spontaneous activation and deactivation are major challenges in the study of this enzyme. This study aimed to create a stable pro-KLK6 enzyme by enzyme engineering, designing a specific cleavage site for enterokinase, and using Pichia pastoris GS115 as a host cell. Then, recombinant pro-KLK6 was used to introduce a novel inhibitor for it.
Experimental approach: An engineered pro-KLK6 gene was cloned into the pPICZα A expression vector. Then, it was expressed in P. pastoris GS115 and purified by Ni-NTA chromatography. An inactive engineered pro-KLK6 gene was cleaved by enterokinase and converted to an active KLK6. The KLK6 enzyme activity and its kinetic parameters were measured using N-benzoyl-L-arginine ethyl ester (BAEE) substrates.
Findings/Results: The secretory form of the pro-KLK6 was expressed at about 11 mg/L in P. pastoris (GS115). Before activation with enterokinase, pro-KLK6 was inactive and did not activate spontaneously. The kinetic parameters, including Km and Vmax, were estimated at 113.59 µM and 0.432 μM/s, respectively.
Conclusion and implications: A stable pro-KLK6 enzyme was produced using P. pastoris (GS115) as the host cell and a specific cleavage site for enterokinase. Additionally, this study assessed the kinetic parameters of the KLK6 enzyme using the BAEE substrate for the first time.
Highlights
Mohammad Rabbani : PubMed , Google Scholar
Keywords
References
Plapp BV. Site-directed mutagenesis: a tool for studying enzyme catalysis. Methods Enzymol. 1995;249:91-119. DOI: 10.1016/0076-6879(95)49032-9.
Mandeep G, Gupta G.K, Shukla P. Enzyme engineering techniques for biotechnological applications. In: Shukla P, editor. Microbial enzymes and biotechniques. Singapore: Springer; 2020. pp. 235-249.DOI: 10.1007/978-981-15-6895-4.
da Silva Amatto IV, da Rosa-Garzon NG, de Oliveira Simões FA, Santiago F, da Silva Leite NP, Martins JR, et al. Enzyme engineering and its industrial applications. Biotechnol Appl Biochem. 2022;69(2):389-409. DOI: 10.1002/bab.2117.
Yousef GM, Diamandis EP. Human kallikreins: common structural features, sequence analysis and evolution. Curr Genomics. 2003;4(2):147-165.DOI: 10.2174/1389202033350074.
Stefanini ACB, da Cunha BR, Henrique T, Tajara EH. Involvement of kallikrein-related peptidases in normal and pathologic processes. Dis Markers. 2015;2015:946572,1-17. DOI: 10.1155/2015/946572.
Diamandis EP. Prostate-specific antigen: its usefulness in clinical medicine. Trends Endocrinol Metab. 1998;9(8):310-316. DOI: 10.1016/s1043-2760(98)00082-4.
Diamandis EP, Yousef GM, Luo LY, Magklara A, Obiezu CV. The new human kallikrein gene family: implications in carcinogenesis. Trends Endocrinol Metab. 2000;11(12):54-60, DOI: 10.1016/s1043-2760(99)00225-8.
Anisowicz SG, Stenman G, Mok SC, Sager R. A novel protease homolog differentially expressed in breast and ovarian cancer. Mol Med. 1996;2(5):624-636. PMID: 8898378.
Bernett ML, Blaber SI, Scarisbrick IA, Dhanarajan P, Thompson SM, Blaber M. Crystal structure and biochemical characterization of human kallikrein 6 reveals that atrypsin-like kallikrein is expressed in the central nervous system. J Biol Chem. 2002;277(27):24562-24570. DOI: 10.1074/jbc.M202392200.
Bayani J, Diamandis EP. The physiology and pathobiology of human kallikrein-related peptidase 6 (KLK6). Clin Chem Lab Med. 2011;50(2):211‐233.
DOI: 10.1515/CCLM.2011.750.
Vakrakou A, Devetzi M, Papachristopoulou G, Malachias A, Scorilas A, Xynopoulos D, et al. Kallikrein-related peptidase 6 (KLK6) expression in the progression of colon adenoma to carcinoma. Biol Chem. 2014;395(9):1105-1117. DOI: 10.1515/hsz-2014-0166.
Kolin DL, Keiyan Sy, Rotondo F, Bassily MN, Kovacs K, Brezden-Masley C, et al. Prognostic significance of human tissue kallikrein-related peptidases 6 and 10 in gastric cancer. Biol Chem. 2014;395(9):1087-1093. DOI: 10.1515/hsz-2014-0143.
Mella C, Figueroa CD, Otth C, Ehrenfeld P. Involvement of kallikrein-related peptidases in nervous system disorders. Front Cell Neurosci. 2020;14:166,1-13. DOI: 10.3389/fncel.2020.00166.
Komatsu N, Suga Y, Saijoh K, Liu AC, Khan S, Mizuno Y, et al. Elevated human tissue kallikrein levels in the stratum corneum and serum of peeling skin syndrome-type B patients suggests an over-desquamation of corneocytes. J Invest Dermato. 2005;126(10):2338-2342. DOI: 10.1038/sj.jid.5700379.
Yousef EP, Diamandis EP. Human tissue kallikreins: a new enzymatic cascade pathway? Biol Chem. 2002;383(7-8):1045-1057. DOI: 10.1515/BC.2002.113.
Bayés A, Tsetsenis T, Ventura S, Vendrell J, Aviles FX. Human kallikrein 6 activity is regulated via an autoproteolytic mechanism of activation/inactivation. Biol Chem. 2004;385(6):517-524. DOI: 10.1515/BC.2004.061.
Guerfal M, Ryckaert S, Jacobs PP, Ameloot P, Van Craenenbroeck K, Derycke R, et al. The HAC1 gene from Pichia pastoris: characterization and effect of its overexpression on the production of secreted, surface displayed, and membrane proteins. Microb Cell Fact. 2010;9:49,1-12. DOI: 10.1186/1475-2859-9-49.
Becker DM, Guarente L. High-efficiency transformation of yeast by electroporation. Methods Enzymol. 1991;194:182-187. DOI: 10.1016/0076-6879(91)94015-5.
Sananes A, Cohen I, Shahar A, Hockla A, De Vita E, Miller AK, et al. A potent, proteolysis-resistant inhibitor of kallikrein-related peptidase 6 (KLK6) for cancer therapy, developed by combinatorial engineering. J Biol Chem. 2018;293(33):12663-12680. DOI: 10.1074/jbc.RA117.000871.
Blaber SI, Yoon H, Scarisbrick IA, Juliano MA, Blaber M. The autolytic regulation of human kallikrein-related peptidase 6. Biochemistry. 2007;46(17):5209-5217. DOI: 10.1021/bi6025006.
Gomis-Rüth FX, Bayés A, Sotiropoulou G, Pampalakis G, Tsetsenis T, Villegas V, et al. The structure of human pro-kallikrein 6 reveals a novel activation mechanism for the kallikrein family. J Biol Chem. 2002;277(30):27273-27281. DOI: 10.1074/jbc.M201534200.
Azadi S, Sadjady SK, Mortazavi SA, Naghdi N, Mahboubi A, Solaimanian R. Bioprocess and downstream optimization of recombinant human growth hormone in Pichia pastoris. Res Pharm Sci. 2018;13(3):222-238. DOI: 10.4103/1735-5362.228953.
De Schutter K, Lin YC, Tiels P, Van Hecke A, Glinka S, Weber-Lehmann J, et al. Genome sequence of the recombinant protein production host Pichia pastoris. Nat Biotechnol. 2009;27(6):561-566. DOI: 10.1038/nbt.1544.
Inan M, Meagher MM. Non-repressing carbon sources for alcohol oxidase (AOX1) promoter of Pichia pastoris. J Biosci Bioeng. 2001;92(6):585-589. DOI: 10.1263/jbb.92.585.
Cregg JM, Vedvick TS, Raschke WC. Recent advances in the expression of foreign genes in Pichia pastoris. Biotechnology (N Y). 1993;11(8):905-910. DOI: 10.1038/nbt0893-905.
Brierley RA, Bussineau C, Kosson R, Melton A, Siegel RS. Fermentation development of recombinant Pichia pastoris expressing the heterologous gene: bovine lysozyme. Ann NY Acad Sci. 1990;589: 350-362. DOI: 10.1111/j.1749-6632.1990.tb24257.x.
Hartner FS, Glieder A. Regulation of methanol utilisation pathway genes in yeasts. Microb Cell Fact. 2006;5:39,1-21. DOI: 10.1186/1475-2859-5-39.
Howles CM. Genetic engineering of human FSH (Gonal-F). Hum Reprod Update. 1996;2(2): 172-191. DOI: 10.1093/humupd/2.2.172.
Guerfal M, Ryckaert S, Jacobs PP, Ameloot P, Van Craenenbroeck K, Derycke R, et al. The HAC1 gene from Pichia pastoris: characterization and effect of its overexpression on the production of secreted, surface displayed, and membrane proteins. Microb Cell Fact. 2010;9:49,1-12, DOI: 10.1186/1475-2859-9-49.
Blaber SI, Scarisbrick IA, Bernett MJ, Dhanarajan P, Seavy MA, Jin Y, et al. Enzymatic properties of rat myelencephalon-specific protease. Biochemistry. 2002;41(4):1165-1173. DOI: 10.1021/bi015781a.
Magklara A, Mellati AA, Wasney GA, Little SP, Sotiropoulou G, Becker GW, et al. Characterization of the enzymatic activity of human kallikrein 6: autoactivation, substrate specificity, and regulation by inhibitors. Biochem Biophys Res Commun. 2003;307(4):948-955. DOI: 10.1016/s0006-291x(03)01271-3.
Kalinska M, Meyer-Hoffert U, Kantyka T, Potempa J. Kallikreins - The melting pot of activity and function. Biochimie. 2016;122:270-282. DOI: 10.1016/j.biochi.2015.09.023.
Kenneth A, Menten ML, Johnson KA, Goody RS. The original Michaelis constant: translation of the 1913 Michaelis-Menten paper. Biochemistry. 2011;50(39):8264-8269. DOI: 10.1021/bi201284u.
Hans L, Dean B. The Determination of Enzyme Dissociation Constants. Journal of the American Chemical Society. 1934(3):658-66. DOI: 10.1021/ja01318a036.
Bernett MJ, Blaber SI, Scarisbrick IA, Dhanarajan P, Thompson SM, Blaber M. Crystal structure and biochemical characterization of human kallikrein 6 reveals that a trypsin-like kallikrein is expressed in the central nervous system. J Biol Chem. 2002;227(27):24562-24570. DOI: 10.1074/jbc.m202392200.
Magklara A, Mellati AA, Wasney GA, Little SP, Sotiropoulou G, Becker GW, et al. Characterization of the enzymatic activity of human kallikrein 6: autoactivation, substrate specificity, and regulation by inhibitors. Biochem Biophys Res Commun. 2003;307(4):948-955. DOI: 10.1016/s0006-291x(03)01271-3
Refbacks
- There are currently no refbacks.
This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly.