Abstract
At present, isopropyl β-D-thiogalactopyranoside (IPTG) is the universal inducer for expressing recombinant proteins under the lac operator/repressor system. In this study, we propose an autoinduction (IPTG-independent) system for recombinant proteins using E. coli as the expression host. We applied this bacterial host for autoinduction to the expression of recombinant proteins, including green fluorescence protein (GFP) and an artificial extracellular matrix protein (aECM-CS5-ELF). The host harbors a mutant Ala294Gly/Thr251Gly phenylalanyl-tRNA synthetase (PheRS**) with an enlarged binding pocket that is expressed under the control of the T7 promoter. Using this system, we demonstrate marked overexpression of the biosynthesized GFP and aECM-CS5-ELF from a 1-L culture containing glucose (5 g/L) and galactose (20 g/L) as the carbon sources, with GFP and aECM-CS5-ELF yields 2.3- and 8.1-fold higher, respectively, than that from an IPTG-induced culture. This unique trial is intended to stimulate novel overexpression strategies based on autoinduction.
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References
Jacob F, Monod J. Genetic regulatory mechanisms in the synthesis of proteins. J Mol Biol. 1961;3:318–56.
Takasu A, Makino T, Hirabayashi T. Synthesis of new S-glycodendrimer toward activation of Lac operon transcription for protein. Biosynth J Polym Sci Pol Chem. 2009;47:310–4.
Takasu A, Kojima H. Synthesis and ring-opening polymerizations of novel S-glycooxazolines. J Polym Sci Pol Chem. 2010;48:5953–60.
Katagiri K, Takasu A, Higuchi M. Synthesis of glycopolymer containing cell-penetrating peptides as inducers of recombinant protein expression under the control of lactose operator/repressor systems. Biomacromolecules. 2016;17:1902–8.
Du EY, Ziaee F, Wang L, Nordon RE, Thordarson P. The correlations between structure, rheology, and cell growth in peptide-based multicomponent hydrogels. Polym J. 2020;52:947–57.
Sisak AAM, Louis F, Matsusaki M. In vitro fabrication and application of engineered vascular hydrogels. Polym J. 2020;52:871–81.
van Hest JCM, Tirrell DA. Protein-based materials, toward a new level of structural control. Chem Commun. 2001;19:1897–904.
Kiick KL, van Hest JCM, Tirrell DA. Expanding the scope of protein biosynthesis by altering the methionyl-tRNA synthetase activity of a bacterial expression host. Angew Chem Int Ed. 2000;39:2148–52.
Wang P, Tang Y, Tirrell DA. Incorporation of trifluoroisoleucine into proteins in vivo. J Am Chem Soc. 2003;125:6900–6.
Kwon I, Kirshenbaum K, Tirrell DA. Breaking the degeneracy of the genetic code. J Am Chem Soc. 2003;125:7512–3.
Mock M, Michon T, Tirrell DA. Stereoselective incorporation of unsaturated amino acids into proteins in vivo. Abstr Pap Am Chem S. 2003;225:U543–3.
Kirshenbaum K, Carrico IS, Tirrell DA. Biosynthesis of proteins incorporating a versatile set of phenylalanine analogues. Chembiochem. 2002;3:235–7.
Datta D, Wang P, Carrico IS, Mayo SL, Tirrell DA. A designed phenylalanyl-tRNA synthetase variant allows efficient in vivo incorporation of aryl ketone functionality into proteins. J Am Chem Soc. 2002;124:5652–3.
Carrico IS, Maskarinec SA, Heilshorn SC, Mock ML, Liu JC, Nowatzki PJ, et al. Lithographic patterning of photoreactive cell-adhesive proteins. J Am Chem Soc. 2007;129:4874–5.
Takasu A, Kondo S, Ito A, Furukawa Y, Higuchi M, Kinoshita T, et al. Artificial extracellular matrix proteins containing phenylalanine analogues biosynthesized in bacteria using T7 expression system and the PEGylation. Biomacromolecules. 2011;12:3444–52.
Studier FW, Moffatt BA. Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J Mol Biol. 1986;189:113–30.
Rosenberg AH, Lade BN, Chui DS, Lin SW, Dunn JJ, Studier FW. Vectors for selective expression of cloned DNAs by T7 RNA polymerase. Gene. 1987;56:125–35.
Studier FW, Rosenberg AH, Dunn JJ, Dubendorff JW. Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 1990;185:60–89.
Studier FW. Protein production by auto-induction in high-density shaking cultures. Protein Expres Purif. 2005;41:207–34.
Grabski A, Kehler M, Drott D. The overnight express autoinduction system: high-density cell growth and protein expression while you sleep. Nat Methods. 2005;2:233–5.
Ruxton GD. The unequal variance t test is an underused alternative to Student’s t test and the Mann–Whitney U test. Behav Ecol. 2006;17:688–90.
Malekian R, Jahanian-Najafabadi A, Fatemeh M, Ghavimi R, Mohammadi E, Akbari V. High-yield production of granulocyte-macrophage colony-stimulating factor in E.coli BL21(DE3) by an auto-induction strategy. Iran J Pharm Res. 2019;18:496–78.
Acknowledgements
AT is grateful to Prof. David A. Tirrell for the opportunity to work in his laboratory at the California Institute of Technology. The authors acknowledge support from the Nagoya Institute of Technology’s Research Promotion Program, from NIH grants EB1971 and GM 62523 and from the NSF Center for the Science and Engineering of Materials at the California Institute of Technology.
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Kataoka, K., Takasu, A. IPTG-independent autoinduction of extracellular matrix proteins using recombinant E. coli as the expression host. Polym J 53, 385–391 (2021). https://doi.org/10.1038/s41428-020-00411-9
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DOI: https://doi.org/10.1038/s41428-020-00411-9