Abstract
Experimental design and Response Surface Methodology (RSM) were used to optimize the production of ∆N123-GBD-CD2, an α-(1 → 2) branching sucrase previously reported as mainly produced in inclusion bodies. The ∆N123-GBD-CD2 encoding gene was cloned into two expression vectors in fusion with 6xHis tag or Strep tag II encoding sequences at 5′ and 3′ ends of the gene and expressed in five Escherichia coli strains. Three host-vector combinations were first selected on the basis of the amount of soluble enzyme produced. RSM with Box-Behnken design was used to optimize the expression conditions in an auto-inducible medium. Five factors were considered, i.e. culture duration, temperature and the concentrations of glycerol, lactose inducer and glucose repressor. The design consisted of three blocks of 45 assays performed in deep well microplates. The regression models were built and fitted well to the experimental data (R 2 coefficient >94 %). The best response (production level of soluble enzyme) was obtained with E. coli BL21 Star DE3 cells transformed with the pET-55 vector. Using the predicted optimal conditions, 5,740 U L−1 of culture of soluble enzyme was produced in microtiter plates and more than 12,000 U L−1 of culture in Erlenmeyer flask, which represents a 165-fold increase compared to the production levels previously reported.
Similar content being viewed by others
References
Baneyx F (1999) Recombinant protein expression in Escherichia coli. Curr Opin Biotechnol 10:411–421. doi:10.1016/S0958-1669(99)00003-8
Baş D, Boyacı İH (2007) Modeling and optimization I: usability of response surface methodology. J Food Eng 78:836–845. doi:10.1016/j.jfoodeng.2005.11.024
Blommel PG, Becker KJ, Duvnjak P, Fox BG (2008) Enhanced bacterial protein expression during auto-induction obtained by alteration of Lac repressor dosage and medium composition. Biotechnol Prog 23:585–598. doi:10.1021/bp070011x
Bolanos-Garcia VM, Davies OR (2006) Structural analysis and classification of native proteins from E. coli commonly co-purified by immobilised metal affinity chromatography. Acta Biochim Biophys Sin 1760:1304–1313. doi:10.1016/j.bbagen.2006.03.027
Box GEP, Behnken DW (1960) Simplex-sum designs: a class of second order rotatable designs derivable from those of first order. Ann Math Stat 31:838–864. doi:10.1214/aoms/1177705661
Brison Y, Fabre E, Moulis C, Portais J-C, Monsan P, Remaud-Siméon M (2009) Synthesis of dextrans with controlled amounts of α-1,2 linkages using the transglucosidase GBD–CD2. Appl Microbiol Biotechnol 86:545–554. doi:10.1007/s00253-009-2241-z
Brison Y, Pijning T, Malbert Y, Fabre E, Mourey L, Morel S, Potocki-Veronese G, Monsan P, Tranier S, Remaud-Simeon M, Dijkstra BW (2012) Functional and structural characterization of a-1,2 branching sucrase derived from DSR-E glucansucrase. J Biol Chem 287:7915–7924. doi:10.1074/jbc.M111.305078
Diaz Ricci JC, Hernández ME (2000) Plasmid effects on Escherichia coli metabolism. Crit Rev Biotechnol 20:79–108. doi:10.1080/07388550008984167
Dümmler A, Lawrence A-M, de Marco A (2005) Simplified screening for the detection of soluble fusion constructs expressed in E. coli using a modular set of vectors. Microb Cell Factories 4:34. doi:10.1186/1475-2859-4-34
Emond S, Potocki-Veronese G, Mondon P, Bouayadi K, Kharrat H, Monsan P, Remaud-Simeon M (2007) Optimized and automated protocols for high-throughput screening of amylosucrase libraries. J Biomol Screen 12:715–723. doi:10.1177/1087057107301978
Emond S, Mondeil S, Jaziri K, André I, Monsan P, Remaud-Siméon M, Potocki-Véronèse G (2008) Cloning, purification and characterization of a thermostable amylosucrase from Deinococcus geothermalis. FEMS Microbiol Lett 285:25–32. doi:10.1111/j.1574-6968.2008.01204.x
Fabre E, Bozonnet S, Arcache A, Willemot R-M, Vignon M, Monsan P, Remaud-Simeon M (2004) Role of the two catalytic domains of DSR-E dextransucrase and their involvement in the formation of highly alpha-1,2 branched dextran. J Bacteriol 187:296–303. doi:10.1128/JB.187.1.296-303.2005
Feller G, Bussy OL, Gerday C (1998) Expression of psychrophilic genes in mesophilic hosts: assessment of the folding state of a recombinant α-amylase. Appl Environ Microbiol 64:1163–1165
Gordon E, Horsefield R, Swarts HGP, de Pont JJHHM, Neutze R, Snijder A (2008) Effective high-throughput overproduction of membrane proteins in Escherichia coli. Protein Expr Purif 62:1–8. doi:10.1016/j.pep.2008.07.005
Jana S, Deb JK (2005) Strategies for efficient production of heterologous proteins in Escherichia coli. Appl Microbiol Biotechnol 67:289–298. doi:10.1007/s00253-004-1814-0
Kimata K, Yamaguchi M, Saito Y, Hata H, Miyake K, Yamane T, Nakagawa Y, Yano A, Ito K, Kawarasaki Y (2012) High cell-density expression system: a novel method for extracellular production of difficult-to-express proteins. J Biosci Bioeng 113:154–159. doi:10.1016/j.jbiosc.2011.10.007
Ko Y-F, Bentley WE, Weigand WA (1995) The effect of cellular energetics on foreign protein production. Appl Biochem Biotechnol 50:145–159. doi:10.1007/BF02783451
Koehn J, Hunt I (2009) High-throughput protein production (HTPP): a review of enabling technologies to expedite protein production. In: Doyle SA (ed) High throughput protein expression and purification. Humana Press, Totowa, NJ, pp 1–18
Lin ECC (1976) Glycerol dissimilation and its regulation in bacteria. Annu Rev Microbiol 30:535–578. doi:10.1146/annurev.mi.30.100176.002535
Lombard V, Golaconda Ramulu H, Drula E, Coutinho PM, Henrissat B (2013) The carbohydrate-active enzymes database (CAZy) in 2013. Nucleic Acids Res 42:D490–D495. doi:10.1093/nar/gkt1178
Miller GL (1959) Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 31:426–428. doi:10.1021/ac60147a030
Nawani NN, Kapadnis BP (2005) Optimization of chitinase production using statistics based experimental designs. Process Biochem 40:651–660. doi:10.1016/j.procbio.2004.01.048
Noguère C, Larsson AM, Guyot J-C, Bignon C (2012) Fractional factorial approach combining 4 Escherichia coli strains, 3 culture media, 3 expression temperatures and 5 N-terminal fusion tags for screening the soluble expression of recombinant proteins. Protein Expr Purif 84:204–213. doi:10.1016/j.pep.2012.05.011
Papaneophytou CP, Kontopidis GA (2012) Optimization of TNF-α overexpression in Escherichia coli using response surface methodology: purification of the protein and oligomerization studies. Protein Expr Purif 86:35–44. doi:10.1016/j.pep.2012.09.002
Peroutka RJ III, Orcutt SJ, Strickler JE, Butt TR (2011) SUMO fusion technology for enhanced protein expression and purification in prokaryotes and eukaryotes. In: Xu M-Q (ed) Evans, TC. Heterologous gene expression in E. coli. Humana Press, Totowa, NJ, pp 15–30
Peti W, Page R (2007) Strategies to maximize heterologous protein expression in Escherichia coli with minimal cost. Protein Expr Purif 51:1–10. doi:10.1016/j.pep.2006.06.024
Sarbini SR, Kolida S, Naeye T, Einerhand A, Brison Y, Remaud-Simeon M, Monsan P, Gibson GR, Rastall RA (2011) In vitro fermentation of linear and alpha-1,2-branched dextrans by the human fecal microbiota. Appl Environ Microbiol 77:5307–5315. doi:10.1128/AEM.02568-10
Sarbini SR, Kolida S, Naeye T, Einerhand AW, Gibson GR, Rastall RA (2013) The prebiotic effect of α-1,2 branched, low molecular weight dextran in the batch and continuous faecal fermentation system. J Funct Foods 5:1938–1946. doi:10.1016/j.jff.2013.09.015
Serino M, Luche E, Gres S, Baylac A, Berge M, Cenac C, Waget A, Klopp P, Iacovoni J, Klopp C, Mariette J, Bouchez O, Lluch J, Ouarne F, Monsan P, Valet P, Roques C, Amar J, Bouloumie A, Theodorou V, Burcelin R (2011) Metabolic adaptation to a high-fat diet is associated with a change in the gut microbiota. Gut 61:543–553. doi:10.1136/gutjnl-2011-301012
Song JM, An YJ, Kang MH, Lee Y-H, Cha S-S (2012) Cultivation at 6–10°C is an effective strategy to overcome the insolubility of recombinant proteins in Escherichia coli. Protein Expr Purif 82:297–301. doi:10.1016/j.pep.2012.01.020
Studier FW (2005) Protein production by auto-induction in high-density shaking cultures. Protein Expr Purif 41:207–234. doi:10.1016/j.pep.2005.01.016
Studier FW (2009) High density growth of T7 expression strains with auto-induction option. Patent No. US 7560264 B2
Swalley SE, Fulghum JR, Chambers SP (2006) Screening factors effecting a response in soluble protein expression: formalized approach using design of experiments. Anal Biochem 351:122–127. doi:10.1016/j.ab.2005.11.046
Terpe K (2003) Overview of tag protein fusions: from molecular and biochemical fundamentals to commercial systems. Appl Microbiol Biotechnol 60:523–533. doi:10.1007/s00253-002-1158-6
Tyler RC, Sreenath HK, Singh S, Aceti DJ, Bingman CA, Markley JL, Fox BG (2005) Auto-induction medium for the production of [U-15 N]- and [U-13C, U-15 N]-labeled proteins for NMR screening and structure determination. Protein Expr Purif 40:268–278. doi:10.1016/j.pep.2004.12.024
Upadhyay AK, Murmu A, Singh A, Panda AK (2012) Kinetics of inclusion body formation and its correlation with the characteristics of protein aggregates in Escherichia coli. PLoS ONE 7:e33951. doi:10.1371/journal.pone.0033951
Villaverde A, Carrió MM (2003) Protein aggregation in recombinant bacteria: biological role of inclusion bodies. Biotechnol Lett 25:1385–1395. doi:10.1023/A:1025024104862
Waugh DS (2005) Making the most of affinity tags. Trends Biotechnol 23:316–320. doi:10.1016/j.tibtech.2005.03.012
Acknowledgments
We thankfully acknowledge the assistance of S. Bozonnet and S. Pizzut-Serin in using the ICEO facility. This work was supported by the French National Research Agency (ANR-10-ALIA-0003 Oenopolys 2011-2013).
Author information
Authors and Affiliations
Corresponding author
Additional information
Marlène Vuillemin and Yannick Malbert contributed equally to this work.
Electronic supplementary material
Below is the link to the electronic supplementary material.
ESM 1
(PDF 791 kb)
Rights and permissions
About this article
Cite this article
Vuillemin, M., Malbert, Y., Laguerre, S. et al. Optimizing the production of an α-(1→2) branching sucrase in Escherichia coli using statistical design. Appl Microbiol Biotechnol 98, 5173–5184 (2014). https://doi.org/10.1007/s00253-014-5627-5
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00253-014-5627-5