Abstract
Over the past few decades, Escherichia coli (E. coli) remains the most favorable host among the microbial cell factories for the production of soluble recombinant proteins. Recombinant protein production (RPP) via E. coli is optimized at the level of gene expression (expression level) and the process condition of fermentation (process level). Presently, the reported studies do not give a clear view on the selection of methods employed in the optimization of RPP. Here, we have reviewed various optimization methods and their preferences with respect to the factors at expression and process levels to achieve the optimal levels of soluble RPP. With a greater understanding of these optimization methods, we proposed a stepwise methodology linking the factors from both levels for optimizing the production of soluble recombinant protein in E. coli. The proposed methodology is further explained through five sets of examples demonstrating the optimization of RPP at both expression and process levels.
Key Points • Stepwise methodology of optimizing recombinant protein production is proposed. • In silico tools can facilitate the optimization of gene- and protein-based factors. • Optimization of gene- and protein-based factors aids host-vector selection. • Statistical optimization is preferred for achieving optimal levels of process factors. |
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References
Agostini F, Cirillo D, Livi CM, Delli Ponti R, Tartaglia GG (2014) ccSOL omics: a webserver for solubility prediction of endogenous and heterologous expression in Escherichia coli. Bioinformatics 30:2975–2977. https://doi.org/10.1093/bioinformatics/btu420
Azaman SNA, Ramakrishnan NR, Tan JS, Rahim RA, Abdullah MP, Ariff AB (2010) Optimization of an induction strategy for improving interferon-α2b production in the periplasm of Escherichia coli using response surface methodology. Biotechnol Appl Biochem 56:141–150. https://doi.org/10.1042/BA20100104
Balbás P (2001) Understanding the art of producing protein and nonprotein molecules in Escherichia coli. Mol Biotechnol 19:251–267. https://doi.org/10.1385/MB:19:3:251
Balzer S, Kucharova V, Megerle J, Lale R, Brautaset T, Valla S (2013) A comparative analysis of the properties of regulated promoter systems commonly used for recombinant gene expression in Escherichia coli. Microb Cell Factories 12:26. https://doi.org/10.1186/1475-2859-12-26
Bang HB, Lee YH, Lee YJ, Jeong KJ (2015) High-level production of human papillomavirus (HPV) type 16 l1 in Escherichia coli. J Microbiol Biotechnol 26:356–363. https://doi.org/10.4014/jmb.1511.11010
Basit A, Akhtar MW (2018) Truncation of the processive Cel5A of Thermotoga maritima results in soluble expression and several fold increase in activity. Biotechnol Bioeng 115:1675–1684. https://doi.org/10.1002/bit.26602
Brüsehaber E, Schwiebs A, Schmidt M, Böttcher D, Bornscheuer UT (2010) Production of pig liver esterase in batch fermentation of E. coli Origami. Appl Microbiol Biotechnol 86:1337–1344. https://doi.org/10.1007/s00253-009-2392-y
Capriotti E, Casadio R (2007) K-Fold: a tool for the prediction of the protein folding kinetic order and rate. Bioinformatics 23:385–386. https://doi.org/10.1093/bioinformatics/btl610
Castro-Martínez C, Luna-Suárez S, Paredes-López O (2012) Overexpression of a modified protein from amaranth seed in Escherichia coli and effect of environmental conditions on the protein expression. J Biotechnol 158:59–67. https://doi.org/10.1016/j.jbiotec.2011.12.012
Chang CC, Li C, Webb GI, Tey B, Song J, Ramanan RN (2016) Periscope: quantitative prediction of soluble protein expression in the periplasm of Escherichia coli. Sci Rep 6:21844. https://doi.org/10.1038/srep21844
Chang CC, Song J, Tey BT, Ramanan RN (2014) Bioinformatics approaches for improved recombinant protein production in Escherichia coli: protein solubility prediction. Brief Bioinform 15:953–962. https://doi.org/10.1093/bib/bbt057
Chang CC, Tey BT, Song J, Ramanan RN (2015) Towards more accurate prediction of protein folding rates: a review of the existing web-based bioinformatics approaches. Brief Bioinform 16:314–324. https://doi.org/10.1093/bib/bbu007
Chen Y, Xing X-H, Ye F, Kuang Y, Luo M (2007) Production of MBP–HepA fusion protein in recombinant Escherichia coli by optimization of culture medium. Biochem Eng J 34:114–121. https://doi.org/10.1016/j.bej.2006.11.020
Cheng X, Xiao X, Wu Z, Wang P, Lin W (2013) Swfoldrate: predicting protein folding rates from amino acid sequence with sliding window method. Proteins 81:140–148. https://doi.org/10.1002/prot.24171
Chin JX, Chung BK-SS, Lee D-YY (2014) Codon Optimization OnLine (COOL): a web-based multi-objective optimization platform for synthetic gene design. Bioinformatics 30:2210–2212. https://doi.org/10.1093/bioinformatics/btu192
Chou CP (2007) Engineering cell physiology to enhance recombinant protein production in Escherichia coli. Appl Microbiol Biotechnol 76:521–532. https://doi.org/10.1007/s00253-007-1039-0
Chou K-C, Shen H-B (2009) FoldRate: a web-server for predicting protein folding rates from primary sequence. Open Bioinforma J 3:31–50. https://doi.org/10.2174/1875036200903010031
Chuan YP, Lua LHL, Middelberg APJ (2008) High-level expression of soluble viral structural protein in Escherichia coli. J Biotechnol 134:64–71. https://doi.org/10.1016/j.jbiotec.2007.12.004
Collins JH, Young EM (2018) Genetic engineering of host organisms for pharmaceutical synthesis. Curr Opin Biotechnol 53:191–200. https://doi.org/10.1016/J.COPBIO.2018.02.001
Correa A, Oppezzo P (2011) Tuning different expression parameters to achieve soluble recombinant proteins in E. coli: advantages of high-throughput screening. Biotechnol J 6:715–730. https://doi.org/10.1002/biot.201100025
Daniel E, Onwukwe GU, Wierenga RK, Quaggin SE, Vainio SJ, Krause M (2015) ATGme: open-source web application for rare codon identification and custom DNA sequence optimization. BMC Bioinformatics 16:303. https://doi.org/10.1186/s12859-015-0743-5
Farliahati MR, Ramanan RN, Mohamad R, Puspaningsih NNT, Ariff AB (2010) Enhanced production of xylanase by recombinant Escherichia coli DH5α through optimization of medium composition using response surface methodology. Ann Microbiol 60:279–285. https://doi.org/10.1007/s13213-010-0038-z
Francis DM, Page R (2010) Strategies to optimize protein expression in E. coli. Curr Protoc Protein Sci 61(1):5.24.1–5.24.29. 1–29. https://doi.org/10.1002/0471140864.ps0524s61
Gaspar P, Moura G, Santos MAS, Oliveira JL (2013) mRNA secondary structure optimization using a correlated stem–loop prediction. Nucleic Acids Res 41:e73. https://doi.org/10.1093/nar/gks1473
Goffin P, Dewerchin M, De Rop P, Blais N, Dehottay P (2017) High-yield production of recombinant CRM197, a non-toxic mutant of diphtheria toxin, in the periplasm of Escherichia coli. Biotechnol J 12. https://doi.org/10.1002/biot.201700168
Gould N, Hendy O, Papamichail D (2014) Computational tools and algorithms for designing customized synthetic genes. Front Bioeng Biotechnol 2:41. https://doi.org/10.3389/fbioe.2014.00041
Gromiha MM, Thangakani AM, Selvaraj S (2006) FOLD-RATE: prediction of protein folding rates from amino acid sequence. Nucleic Acids Res 34:W70–W74. https://doi.org/10.1093/nar/gkl043
Grote A, Hiller K, Scheer M, Munch R, Nortemann B, Hempel DC, Jahn D (2005) JCat: a novel tool to adapt codon usage of a target gene to its potential expression host. Nucleic Acids Res 33:W526–W531. https://doi.org/10.1093/nar/gki376
Guimaraes JC, Rocha M, Arkin AP, Cambray G (2014) D-Tailor: automated analysis and design of DNA sequences. Bioinformatics 30:1087–1094. https://doi.org/10.1093/bioinformatics/btt742
Gundinger T, Spadiut O (2017) A comparative approach to recombinantly produce the plant enzyme horseradish peroxidase in Escherichia coli. J Biotechnol 248:15–24. https://doi.org/10.1016/j.jbiotec.2017.03.003
Gupta SK, Shukla P (2016) Advanced technologies for improved expression of recombinant proteins in bacteria: perspectives and applications. Crit Rev Biotechnol 36:1089–1098. https://doi.org/10.3109/07388551.2015.1084264
Gustafsson C, Govindarajan S, Minshull J (2004) Codon bias and heterologous protein expression. Trends Biotechnol 22:346–353. https://doi.org/10.1016/j.tibtech.2004.04.006
Hebditch M, Carballo-Amador MA, Charonis S, Curtis R, Warwicker J (2017) Protein-Sol: a web tool for predicting protein solubility from sequence. Bioinformatics 33:3098–3100. https://doi.org/10.1093/bioinformatics/btx345
Hirose S, Noguchi T (2013) ESPRESSO: a system for estimating protein expression and solubility in protein expression systems. Proteomics 13:1444–1456. https://doi.org/10.1002/pmic.201200175
Hochkoeppler A (2013) Expanding the landscape of recombinant protein production in Escherichia coli. Biotechnol Lett 35:1971–1981. https://doi.org/10.1007/s10529-013-1396-y
Hoover DM, Lubkowski J (2002) DNAWorks: an automated method for designing oligonucleotides for PCR-based gene synthesis. Nucleic Acids Res 30:43e. https://doi.org/10.1093/nar/30.10.e43
Huang C-J, Lin H, Yang X (2012) Industrial production of recombinant therapeutics in Escherichia coli and its recent advancements. J Ind Microbiol Biotechnol 39:383–399. https://doi.org/10.1007/s10295-011-1082-9
Jung S-K, McDonald K (2011) Visual gene developer: a fully programmable bioinformatics software for synthetic gene optimization. BMC Bioinformatics 12:340. https://doi.org/10.1186/1471-2105-12-340
Kaur JJ, Kumar A, Kaur JJ (2018) Strategies for optimization of heterologous protein expression in E. coli: roadblocks and reinforcements. Int J Biol Macromol 106:803–822. https://doi.org/10.1016/J.IJBIOMAC.2017.08.080
Khurana S, Rawi R, Kunji K, Chuang G-Y, Bensmail H, Mall R (2018) DeepSol: a deep learning framework for sequence-based protein solubility prediction. Bioinformatics 34:2605–2613. https://doi.org/10.1093/bioinformatics/bty166
Krogh A, Larsson B, von Heijne G, Sonnhammer EL (2001) Predicting transmembrane protein topology with a hidden markov model: application to complete genomes. J Mol Biol 305:567–580. https://doi.org/10.1006/JMBI.2000.4315
Lebendiker M, Danieli T (2014) Production of prone-to-aggregate proteins. FEBS Lett 588:236–246. https://doi.org/10.1016/j.febslet.2013.10.044
Lee Y-J, Kim H-J, Gao W, Chung C-H, Lee J-W (2012) Statistical optimization for production of carboxymethylcellulase of Bacillus amyloliquefaciens DL-3 by a recombinant Escherichia coli JM109/DL-3 from rice bran using response surface method. Biotechnol Bioprocess Eng 17:227–235. https://doi.org/10.1007/s12257-011-0258-5
Lin GN, Wang Z, Xu D, Cheng J (2010) SeqRate: sequence-based protein folding type classification and rates prediction. BMC Bioinformatics 11:S1. https://doi.org/10.1186/1471-2105-11-S3-S1
Liu M, Feng X, Ding Y, Zhao G, Liu H, Xian M (2015) Metabolic engineering of Escherichia coli to improve recombinant protein production. Appl Microbiol Biotechnol 99:10367–10377. https://doi.org/10.1007/s00253-015-6955-9
Lorenz R, Bernhart SH, Höner Zu Siederdissen C, Tafer H, Flamm C, Stadler PF, Hofacker IL (2011) ViennaRNA Package 2.0. Algorithms Mol Biol 6:26. https://doi.org/10.1186/1748-7188-6-26
Magnan CN, Randall A, Baldi P (2009) SOLpro: accurate sequence-based prediction of protein solubility. Bioinformatics 25:2200–2207. https://doi.org/10.1093/bioinformatics/btp386
Maharjan S, Singh B, Bok JD, Kim JI, Jiang T, Cho CS, Kang SK, Choi YJ (2014) Exploring codon optimization and response surface methodology to express biologically active transmembrane RANKL in E. coli. PLoS One 9:e96259. https://doi.org/10.1371/journal.pone.0096259
Martínez I, Méndez C, Berríos J, Altamirano C, Díaz-Barrera A (2015) Batch production of coenzyme Q10 by recombinant Escherichia coli containing the decaprenyl diphosphate synthase gene from Sphingomonas baekryungensis. J Ind Microbiol Biotechnol 42:1283–1289. https://doi.org/10.1007/s10295-015-1652-3
Menzella HG (2011) Comparison of two codon optimization strategies to enhance recombinant protein production in Escherichia coli. Microb Cell Factories 10:15. https://doi.org/10.1186/1475-2859-10-15
Musil M, Konegger H, Hon J, Bednar D, Damborsky J (2019) Computational design of stable and soluble biocatalysts. ACS Catal 9:1033–1054. https://doi.org/10.1021/acscatal.8b03613
Nelofer R, Ramanan RN, Rahman RNZRA, Basri M, Ariff AB (2012) Comparison of the estimation capabilities of response surface methodology and artificial neural network for the optimization of recombinant lipase production by E. coli BL21. J Ind Microbiol Biotechnol 39:243–254. https://doi.org/10.1007/s10295-011-1019-3
Nelofer R, Ramanan RN, Rahman RNZRA, Basri M, Ariff AB (2011) Sequential optimization of production of a thermostable and organic solvent tolerant lipase by recombinant Escherichia coli. Ann Microbiol 61:535–544. https://doi.org/10.1007/s13213-010-0170-9
Nordström K (2006) Plasmid R1-Replication and its control. Plasmid 55:1–26. https://doi.org/10.1016/j.plasmid.2005.07.002
Oliveira C, Domingues L (2018) Guidelines to reach high-quality purified recombinant proteins. Appl Microbiol Biotechnol 102:81–92. https://doi.org/10.1007/s00253-017-8623-8
Ouyang Z, Liang J (2008) Predicting protein folding rates from geometric contact and amino acid sequence. Protein Sci 17:1256–1263. https://doi.org/10.1110/ps.034660.108
Overton TW (2014) Recombinant protein production in bacterial hosts. Drug Discov Today 19:590–601. https://doi.org/10.1016/j.drudis.2013.11.008
Papaneophytou C (2019) Design of experiments as a tool for optimization in recombinant protein biotechnology: from constructs to crystals. Mol Biotechnol 61:873–891
Papaneophytou CP, Kontopidis G (2014) Statistical approaches to maximize recombinant protein expression in Escherichia coli: a general review. Protein Expr Purif 94:22–32. https://doi.org/10.1016/j.pep.2013.10.016
Papaneophytou CP, Rinotas V, Douni E, Kontopidis G (2013) A statistical approach for optimization of RANKL overexpression in Escherichia coli: purification and characterization of the protein. Protein Expr Purif 90:9–19. https://doi.org/10.1016/j.pep.2013.04.005
Parret AHA, Besir H, Meijers R (2016) Critical reflections on synthetic gene design for recombinant protein expression. Curr Opin Struct Biol 38:155–162. https://doi.org/10.1016/j.sbi.2016.07.004
Pellizza L, Smal C, Rodrigo G, Arán M (2018) Codon usage clusters correlation: towards protein solubility prediction in heterologous expression systems in E. coli. Sci Rep 8:10618. https://doi.org/10.1038/s41598-018-29035-z
Peti W, Page R (2007) Strategies to maximize heterologous protein expression in Escherichia coli with minimal cost. Protein Expr Purif 51:1–10. https://doi.org/10.1016/j.pep.2006.06.024
Puigbo P, Guzman E, Romeu A, Garcia-Vallve S (2007) OPTIMIZER: a web server for optimizing the codon usage of DNA sequences. Nucleic Acids Res 35:W126–W131. https://doi.org/10.1093/nar/gkm219
Quax TEF, Claassens NJ, Söll D, van der Oost J (2015) Codon bias as a means to fine-tune gene expression. Mol Cell 59:149–161. https://doi.org/10.1016/j.molcel.2015.05.035
Richardson SM, Wheelan SJ, Yarrington RM, Boeke JD (2006) GeneDesign: rapid, automated design of multikilobase synthetic genes. Genome Res 16:550–556. https://doi.org/10.1101/gr.4431306
Rosano GL, Ceccarelli EA (2014) Recombinant protein expression in Escherichia coli: advances and challenges. Front Microbiol 5:1–17. https://doi.org/10.3389/fmicb.2014.00172
Rosano GL, Morales ES, Ceccarelli EA (2019) New tools for recombinant protein production in Escherichia coli : a 5-year update. Protein Sci 28:1412–1422. https://doi.org/10.1002/pro.3668
Sevastsyanovich YR, Alfasi SN, Cole JA (2010) Sense and nonsense from a systems biology approach to microbial recombinant protein production. Biotechnol Appl Biochem 55:9–28. https://doi.org/10.1042/BA20090174
Shiloach J, Fass R (2005) Growing E. coli to high cell density – a historical perspective on method development. Biotechnol Adv 23:345–357. https://doi.org/10.1016/j.biotechadv.2005.04.004
Singh K, Piprode V, Mhaske ST, Barhanpurkar-Naik A, Wani MR (2018) IL-3 differentially regulates membrane and soluble RANKL in osteoblasts through metalloproteases and the JAK2/STAT5 pathway and improves the RANKL/OPG ratio in adult mice. J Immunol 200:595–606. https://doi.org/10.4049/jimmunol.1601528
Singh RS, Yadav M (2013) Enhanced production of recombinant aspartase of Aeromonas media NFB-5 in a stirred tank reactor. Bioresour Technol 145:217–223. https://doi.org/10.1016/j.biortech.2012.11.041
Singh V, Haque S, Niwas R, Srivastava A, Pasupuleti M, Tripathi CKM (2017) Strategies for fermentation medium optimization: an in-depth review. Front Microbiol 7:2087. https://doi.org/10.3389/fmicb.2016.02087
Sletta H, Tøndervik A, Hakvåg S, Aune TEV, Nedal A, Aune R, Evensen G, Valla S, Ellingsen TE, Brautaset T (2007) The presence of N-terminal secretion signal sequences leads to strong stimulation of the total expression levels of three tested medically important proteins during high-cell-density cultivations of Escherichia coli. Appl Environ Microbiol 73:906–912. https://doi.org/10.1128/AEM.01804-06
Smialowski P, Doose G, Torkler P, Kaufmann S, Frishman D (2012) PROSO II – a new method for protein solubility prediction. FEBS J 279:2192–2200. https://doi.org/10.1111/j.1742-4658.2012.08603.x
Sohoni SV, Nelapati D, Sathe S, Javadekar-Subhedar V, Gaikaiwari RP, Wangikar PP (2015) Optimization of high cell density fermentation process for recombinant nitrilase production in E. coli. Bioresour Technol 188:202–208. https://doi.org/10.1016/j.biortech.2015.02.038
Song J, Takemoto K, Shen H, Tan H, Gromiha MM, Akutsu T (2010) Prediction of protein folding rates from structural topology and complex network properties. IPSJ Trans Bioinforma 3:40–53. https://doi.org/10.2197/ipsjtbio.3.40
Sørensen HP, Mortensen KK (2005) Advanced genetic strategies for recombinant protein expression in Escherichia coli. J Biotechnol 115:113–128. https://doi.org/10.1016/j.jbiotec.2004.08.004
Srivastava P, Bhattacharaya P, Pandey G, Mukherjee KJ (2005) Overexpression and purification of recombinant human interferon alpha2b in Escherichia coli. Protein Expr Purif 41:313–322. https://doi.org/10.1016/J.PEP.2004.12.018
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. https://doi.org/10.1016/j.ab.2005.11.046
Tabandeh F, Khodabandeh M, Yakhchali B, Habib-Ghomi H, Shariati P (2008) Response surface methodology for optimizing the induction conditions of recombinant interferon beta during high cell density culture. Chem Eng Sci 63:2477–2483. https://doi.org/10.1016/j.ces.2008.02.003
Tan JS, Ramanan RN, Azaman SNA, Ling TC, Shuhaimi M, Ariff AB (2010) Enhanced interferon-α2b production in periplasmic space of Escherichia coli through medium optimization using response surface method. Open Biotechnol J 3:117–124. https://doi.org/10.2174/1874070700903010117
Tan JS, Ramanan RN, Ling TC, Shuhaimi M, Ariff AB (2011) Enhanced production of periplasmic interferon alpha-2b by Escherichia coli using ion-exchange resin for in situ removal of acetate in the culture. Biochem Eng J 58–59:124–132. https://doi.org/10.1016/J.BEJ.2011.08.018
Terpe K (2003) Overview of tag protein fusions: from molecular and biochemical fundamentals to commercial systems. Appl Microbiol Biotechnol 60:523–533. https://doi.org/10.1007/s00253-002-1158-6
Tong Y, Yang H, Xin Y, Zhang L, Wang W (2015) Novel integration strategy coupling codon and fermentation optimization for efficiently enhancing sarcosine oxidase (SOX) production in recombinant Escherichia coli. World J Microbiol Biotechnol 31:707–716. https://doi.org/10.1007/s11274-014-1795-9
Tripathi NK, Shukla J, Biswal KC, Lakshmana Rao PV (2010) Development of a simple fed-batch process for the high-yield production of recombinant Japanese encephalitis virus protein. Appl Microbiol Biotechnol 86:1795–1803. https://doi.org/10.1007/s00253-010-2488-4
Uhoraningoga A, Kinsella GK, Henehan GT, Ryan BJ (2018) The Goldilocks approach: a review of employing design of experiments in prokaryotic recombinant protein production. Bioengineering 5:89. https://doi.org/10.3390/bioengineering5040089
Volontè F, Marinelli F, Gastaldo L, Sacchi S, Pilone MS, Pollegioni L, Molla G (2008) Optimization of glutaryl-7-aminocephalosporanic acid acylase expression in E. coli. Protein Expr Purif 61:131–137. https://doi.org/10.1016/j.pep.2008.05.010
Volontè F, Piubelli L, Pollegioni L (2011) Optimizing HIV-1 protease production in Escherichia coli as fusion protein. Microb Cell Factories 10:53. https://doi.org/10.1186/1475-2859-10-53
Volontè F, Pollegioni L, Molla G, Frattini L, Marinelli F, Piubelli L (2010) Production of recombinant cholesterol oxidase containing covalently bound FAD in Escherichia coli. BMC Biotechnol 10:33. https://doi.org/10.1186/1472-6750-10-33
Vuillemin M, Malbert Y, Laguerre S, Remaud-Siméon M, Moulis C (2014) Optimizing the production of an α-(1→2) branching sucrase in Escherichia coli using statistical design. Appl Microbiol Biotechnol 98:5173–5184. https://doi.org/10.1007/s00253-014-5627-5
Wen WS, Hsieh MC, Wang SSS (2011) High-level expression and purification of human γD-crystallin in Escherichia coli. J Taiwan Inst Chem Eng 42:547–555. https://doi.org/10.1016/j.jtice.2010.10.002
Yari K, Fatemi SSA, Tavallaei M (2010) Optimization of the BoNT/A-Hc expression in recombinant Escherichia coli using the Taguchi statistical method. Biotechnol Appl Biochem 56:35–42. https://doi.org/10.1042/BA20090315
Yari K, Fatemi SSA, Tavallaei M (2012) High level expression of recombinant BoNT/A-Hc by high cell density cultivation of Escherichia coli. Bioprocess Biosyst Eng 35:407–414. https://doi.org/10.1007/s00449-011-0579-y
Zamani M, Berenjian A, Hemmati S, Nezafat N, Ghoshoon MB, Dabbagh F, Mohkam M, Ghasemi Y (2015) Cloning, expression, and purification of a synthetic human growth hormone in Escherichia coli using response surface methodology. Mol Biotechnol 57:241–250. https://doi.org/10.1007/s12033-014-9818-1
Zaslona H, Trusek-Holownia A, Radosinski L, Hennig J (2015) Optimization and kinetic characterization of recombinant 1,3-β-glucanase production in Escherichia coli K-12 strain BL21/pETSD10 - a bioreactor scale study. Lett Appl Microbiol 61:36–43. https://doi.org/10.1111/lam.12419
Zhang JD, Li AT, Xu JH (2010) Improved expression of recombinant cytochrome P450 monooxygenase in Escherichia coli for asymmetric oxidation of sulfides. Bioprocess Biosyst Eng 33:1043–1049. https://doi.org/10.1007/s00449-010-0429-3
Zhang W, Lu J, Zhang S, Liu L, Pang X, Lv J (2018) Development an effective system to expression recombinant protein in E. coli via comparison and optimization of signal peptides: expression of Pseudomonas fluorescens BJ-10 thermostable lipase as case study. Microb Cell Factories 17:50. https://doi.org/10.1186/s12934-018-0894-y
Zhou Y, Lu Z, Wang X, Selvaraj JN, Zhang G (2018) Genetic engineering modification and fermentation optimization for extracellular production of recombinant proteins using Escherichia coli. Appl Microbiol Biotechnol 102:1545–1556. https://doi.org/10.1007/s00253-017-8700-z
Zuker M (2003) Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res 31:3406–3415. https://doi.org/10.1093/nar/gkg595
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The authors acknowledge Monash University Malaysia for providing the research support needed for this work.
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This work was supported by the Fundamental Research Grant Scheme (FRGS) (FRGS/1/2016/TK02/MUSM/02/3) of Malaysia.
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RR conceived the work. KP and RR performed the literature search, data analysis, and wrote the manuscript. CO, LK, and BT critically revised the work. All authors read and approved the manuscript.
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Packiam, K.A.R., Ramanan, R.N., Ooi, C.W. et al. Stepwise optimization of recombinant protein production in Escherichia coli utilizing computational and experimental approaches. Appl Microbiol Biotechnol 104, 3253–3266 (2020). https://doi.org/10.1007/s00253-020-10454-w
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DOI: https://doi.org/10.1007/s00253-020-10454-w