Quantitative relationship between the mRNA secondary structure of translational initiation region and the expression level of heterologous protein in Escherichia coli
Abstract:Translational efficiency in Escherichia coli is strongly influenced by mRNA secondary structure of translational initiation region (TIR). We have previously reported that the expression of heterologous protein is directly related to the minimal folding free energy (ΔG) of the local secondary structure. However, identifying biologically relevant maximum and minimum levels of expression, or exploring the optimal level between them, is a key to successful optimization of heterologous protein expression. To system… Show more
“…The minimum free energy ( ΔG ) of the optimal secondary structure in dot-bracket notation was − 770.9 kcal·mol − 1 (1 cal = 4.2 J), while the ΔG of non-optimized codons was − 774.10 kcal·mol − 1 by ViennaRNA Web Services. Yin et al reported that ΔG is closely related to translation efficiency and increasing ΔG can enhance the expression level [27]. Amaral et al demonstrated that the folding free energy of the 5′ end of mRNA transcripts could have significant effects on translation efficiency and overall protein abundance [18].…”
BackgroundCyclodextrin glucanotransferase (CGTase) can transform L-ascorbic acid (L-AA, vitamin C) to 2-O-α-D-glucopyranosyl-L-ascorbic acid (AA-2G), which shows diverse applications in food, cosmetic and pharmaceutical industries.ResultsIn this study, the cgt gene encoding α-CGTase from Paenibacillus macerans was codon-optimized (opt-cgt) and cloned into pET-28a (+) for intracellular expression in E. coli BL21 (DE3). The Opt-CGT was purified by Ni2+-NTA resin with a 55% recovery, and specific activity was increased significantly from 1.17 to 190.75 U·mg− 1. In addition, the enzyme was adopted to transform L-AA into 9.1 g/L of AA-2G. Finally, more economic substrates, including β-cyclodextrin, soluble starch, corn starch and cassava starch could also be used as glycosyl donors, and 4.9, 3.5, 1.3 and 1.5 g/L of AA-2G were obtained, respectively.ConclusionsN-terminal amino acid is critical to the activity of CGTase suggested by its truncation study. Furthermore, when the Opt-CGT was flanked by His6-tags on the C- and N-terminal, the recovery of purification by Ni2+-NTA resin is appreciably enhanced. α-cyclodextrin was the ideal glycosyl donor for AA-2G production. In addition, the selection of low cost glycosyl donors would make the process of AA-2G production more economically competitive.
“…The minimum free energy ( ΔG ) of the optimal secondary structure in dot-bracket notation was − 770.9 kcal·mol − 1 (1 cal = 4.2 J), while the ΔG of non-optimized codons was − 774.10 kcal·mol − 1 by ViennaRNA Web Services. Yin et al reported that ΔG is closely related to translation efficiency and increasing ΔG can enhance the expression level [27]. Amaral et al demonstrated that the folding free energy of the 5′ end of mRNA transcripts could have significant effects on translation efficiency and overall protein abundance [18].…”
BackgroundCyclodextrin glucanotransferase (CGTase) can transform L-ascorbic acid (L-AA, vitamin C) to 2-O-α-D-glucopyranosyl-L-ascorbic acid (AA-2G), which shows diverse applications in food, cosmetic and pharmaceutical industries.ResultsIn this study, the cgt gene encoding α-CGTase from Paenibacillus macerans was codon-optimized (opt-cgt) and cloned into pET-28a (+) for intracellular expression in E. coli BL21 (DE3). The Opt-CGT was purified by Ni2+-NTA resin with a 55% recovery, and specific activity was increased significantly from 1.17 to 190.75 U·mg− 1. In addition, the enzyme was adopted to transform L-AA into 9.1 g/L of AA-2G. Finally, more economic substrates, including β-cyclodextrin, soluble starch, corn starch and cassava starch could also be used as glycosyl donors, and 4.9, 3.5, 1.3 and 1.5 g/L of AA-2G were obtained, respectively.ConclusionsN-terminal amino acid is critical to the activity of CGTase suggested by its truncation study. Furthermore, when the Opt-CGT was flanked by His6-tags on the C- and N-terminal, the recovery of purification by Ni2+-NTA resin is appreciably enhanced. α-cyclodextrin was the ideal glycosyl donor for AA-2G production. In addition, the selection of low cost glycosyl donors would make the process of AA-2G production more economically competitive.
“…S2 ), implying that use of the eSD2 did not change the transcription levels and mRNA stability. We suppose that the increased gene expression may be a result of the enhanced translational efficiency with the eSD2 sequence after transcription 32 – 35 . Figure 2 Verification of the isolated eSD2.…”
The lactic acid bacteria (LAB) Leuconostoc citreum are non-sporulating hetero-fermentative bacteria that play an important role in the fermented food industry. In this study, for the enhanced and reliable production of recombinant proteins in L. citreum, we developed a bicistronic design (BCD) expression system which includes a short leader peptide (1st cistron) followed by target genes (2nd cistron) under the control of a single promoter. Using superfolder green fluorescent protein (sfGFP) as a reporter, the functionality of BCD in L. citreum was verified. Further, to improve the expression in BCD, we tried to engineer a Shine-Dalgarno sequence (SD2) for the 2nd cistron and a promoter by FACS screening of random libraries, and both strong SD2 (eSD2) and promoter (P710V4) were successfully isolated. The usefulness of the engineered BCD with P710V4 and eSD2 was further validated using three model proteins—glutathione-s-transferase, human growth hormone, and α-amylase. All examined proteins were successfully produced with levels highly increased compared with those in the original BCD as well as the monocistronic design (MCD) expression system.
“…Previous studies demonstrated that increasing the ΔG of TIR increases protein expression. 36,37 In addition, the start AUG codon was demonstrated as a key limiting factor for protein expression in E. coli. 38 The AUG start codons in TIR-pelB and TIR-ompA were fully exposed, compared with that in TIR-Sp.…”
Metalloprotease PT121 and its mutant Y114S (Tyr114 was substituted to Ser) are effective catalysts for the synthesis of Z-aspartame (Z-APM). This study presents the selection of a suitable signal peptide for improving expression and extracellular secretion of proteases PT121 and Y114S by Escherichia coli. Co-inducers containing IPTG and arabinose were used to promote protease production and cell growth. Under optimal conditions, the expression levels of PT121 and Y114S reached >500 mg/L, and the extracellular activity of PT121/Y114S accounted for 87/82% of the total activity of proteases. Surprisingly, purer protein was obtained in the supernatant, because arabinose reduced cell membrane permeability, avoiding cell lysis. Comparison of Z-APM synthesis and caseinolysis between proteases PT121 and Y114S showed that mutant Y114S presented remarkably higher activity of Z-APM synthesis and considerably lower activity of caseinolysis. The significant difference in substrate specificity renders these enzymes promising biocatalysts.
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