scholarly journals Use of Amino Acids as Inducers for High-Level Protein Expression in the Single-Protein Production System

2010 ◽  
Vol 76 (18) ◽  
pp. 6063-6068 ◽  
Author(s):  
S. Thangminlal Vaiphei ◽  
Lili Mao ◽  
Tsutomu Shimazu ◽  
Jung-Ho Park ◽  
Masayori Inouye
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Protein Production ◽  
Target Protein ◽  
Complete Suppression ◽  
Tryptophan Residues ◽  
Single Protein ◽  
High Level ◽  
Isotopic Incorporation ◽  
Specific Mrna

ABSTRACT By taking advantage of MazF, an ACA codon-specific mRNA interferase, Escherichia coli cells can be converted into a bioreactor producing only a single protein of interest by using an ACA-less mRNA for the protein. In this single-protein production (SPP) system, we engineered MazF by replacing two tryptophan residues in positions 14 and 83 with Phe (W14F) and Leu (W83L), respectively. Upon the addition of an inducer (IPTG [isopropyl-β-d-thiogalactopyranoside]), the mutated MazF [MazF(ΔW)] can still be produced even in the absence of tryptophan in the medium by using a Trp auxotroph, while a target protein having Trp residues cannot be produced. However, at 3 h after the addition of IPTG, the addition of tryptophan to the medium exclusively induces production of the target protein at a high level. A similar SPP system was also constructed with the use of a His-less protein [MazF(ΔH)] and a His auxotroph. Using these dual-induction systems, isotopic enrichments of 13C, 15N, and 2H were highly improved by almost complete suppression of the production of the unlabeled target protein. In both systems, isotopic incorporation reached more than 98% labeling efficiency, significantly reducing the background attributable to the unlabeled target protein.

scholarly journals Use of Amino Acids as Inducers for High-Level Protein Expression in the Single-Protein Production System

2010 ◽  
Vol 76 (21) ◽  
pp. 7371-7371
Author(s):  
S. Thangminlal Vaiphei ◽  
Lili Mao ◽  
Tsutomu Shimazu ◽  
Jung-Ho Park ◽  
Masayori Inouye
Keyword(s):  
Amino Acids ◽  
Protein Expression ◽  
Production System ◽  
Protein Production ◽  
Single Protein ◽  
High Level

scholarly journals Random and combinatorial mutagenesis for improved total production of secretory target protein in Escherichia coli

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
David Gonzalez-Perez ◽  
James Ratcliffe ◽  
Shu Khan Tan ◽  
Mary Chen May Wong ◽  
Yi Pei Yee ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Protein Secretion ◽  
Protein Production ◽  
Secretory Protein ◽  
Target Protein ◽  
Carrier Protein ◽  
Signal Peptides ◽  
Carrier Proteins ◽  
E Coli ◽  
Combinatorial Mutagenesis

AbstractSignal peptides and secretory carrier proteins are commonly used to secrete heterologous recombinant protein in Gram-negative bacteria. The Escherichia coli osmotically-inducible protein Y (OsmY) is a carrier protein that secretes a target protein extracellularly, and we have previously applied it in the Bacterial Extracellular Protein Secretion System (BENNY) to accelerate directed evolution. In this study, we reported the first application of random and combinatorial mutagenesis on a carrier protein to enhance total secretory target protein production. After one round of random mutagenesis followed by combining the mutations found, OsmY(M3) (L6P, V43A, S154R, V191E) was identified as the best carrier protein. OsmY(M3) produced 3.1 ± 0.3 fold and 2.9 ± 0.8 fold more secretory Tfu0937 β-glucosidase than its wildtype counterpart in E. coli strains BL21(DE3) and C41(DE3), respectively. OsmY(M3) also produced more secretory Tfu0937 at different cultivation temperatures (37 °C, 30 °C and 25 °C) compared to the wildtype. Subcellular fractionation of the expressed protein confirmed the essential role of OsmY in protein secretion. Up to 80.8 ± 12.2% of total soluble protein was secreted after 15 h of cultivation. When fused to a red fluorescent protein or a lipase from Bacillus subtillis, OsmY(M3) also produced more secretory protein compared to the wildtype. In this study, OsmY(M3) variant improved the extracellular production of three proteins originating from diverse organisms and with diverse properties, clearly demonstrating its wide-ranging applications. The use of random and combinatorial mutagenesis on the carrier protein demonstrated in this work can also be further extended to evolve other signal peptides or carrier proteins for secretory protein production in E. coli.

Complementation of a polyamine-deficient Escherichia coli mutant by expression of mouse ornithine decarboxylase

1987 ◽  
Vol 7 (1) ◽  
pp. 564-567
Author(s):  
M Macrae ◽  
P Coffino
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Enzymatic Activity ◽  
Ornithine Decarboxylase ◽  
Polyamine Biosynthesis ◽  
Coli Mutant ◽  
Terminal Amino ◽  
High Level ◽  
Carboxy Terminal ◽  
Exogenous Source

Mouse ornithine decarboxylase (ODCase) cDNA was expressed at a high level in an Escherichia coli mutant deficient in polyamine biosynthesis. The expression of mouse ornithine decarboxylase relieved the dependence of the mutant on an exogenous source of polyamines, presumably by providing putrescine, the product of the enzyme. The effect on the enzymatic activity of deletions that removed carboxy-terminal amino acids of the protein was determined.

Development of Escherichia coli Strains That Withstand Membrane Protein-Induced Toxicity and Achieve High-Level Recombinant Membrane Protein Production

2016 ◽  
Vol 6 (2) ◽  
pp. 284-300 ◽  
Author(s):  
Dimitra Gialama ◽  
Kalliopi Kostelidou ◽  
Myrsini Michou ◽  
Dafni Chrysanthi Delivoria ◽  
Fragiskos N. Kolisis ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Membrane Protein ◽  
Protein Production ◽  
High Level

scholarly journals Single protein production (SPP) system in Escherichia coli

2007 ◽  
Vol 2 (7) ◽  
pp. 1802-1810 ◽  
Author(s):  
Motoo Suzuki ◽  
Lili Mao ◽  
Masayori Inouye
Keyword(s):  
Escherichia Coli ◽  
Protein Production ◽  
Single Protein

scholarly journals A Combination of sbmA and tolC Mutations in Escherichia coli K-12 Tn10-Carrying Strains Results in Hypersusceptibility to Tetracycline

2007 ◽  
Vol 190 (4) ◽  
pp. 1491-1494 ◽  
Author(s):  
Ricardo E. de Cristóbal ◽  
Paula A. Vincent ◽  
Raúl A. Salomón
Keyword(s):  
Escherichia Coli ◽  
Double Mutant ◽  
Phenotypic Expression ◽  
Tetracycline Resistance ◽  
Additive Effect ◽  
Complete Suppression ◽  
High Level ◽  
K 12 ◽  
Level Resistance

ABSTRACT Previously, we demonstrated that Escherichia coli tolC mutations reduce the high-level resistance to tetracycline afforded by the transposon Tn10-encoded TetA pump from resistance at 200 μg/ml to resistance at 40 μg/ml. In this study, we found that the addition of an sbmA mutation to a tolC::Tn10 mutant exacerbates this phenotype: the double mutant did not form colonies, even in the presence of tetracycline at a concentration as low as 5 μg/ml. Inactivation of sbmA alone partially inhibited high-level tetracycline resistance, from resistance at 200 μg/ml to resistance at 120 μg/ml. There thus appears to be an additive effect of the mutations, resulting in almost complete suppression of the phenotypic expression of Tn10 tetracycline resistance.

scholarly journals Alleviation of Proteolytic Sensitivity To Enhance Recombinant Lipase Production in Escherichia coli

2009 ◽  
Vol 75 (16) ◽  
pp. 5424-5427 ◽  
Author(s):  
Niju Narayanan ◽  
C. Perry Chou
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Recombinant Protein ◽  
Recombinant Protein Production ◽  
Double Mutant ◽  
Protein Production ◽  
Functional Expression ◽  
Lipase Production ◽  
Recombinant Lipase ◽  
Pseudozyma Antarctica

ABSTRACT Two amino acids, Leu149 and Val223, were identified as proteolytically sensitive when Pseudozyma antarctica lipase (PalB) was heterologously expressed in Escherichia coli. The functional expression was enhanced using the double mutant for cultivation. However, the recombinant protein production was still limited by PalB misfolding, which was resolved by DsbA coexpression.

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