Overcoming challenges for amplified expression of recombinant proteins using Escherichia coli

Ahmad, Irshad; Nawaz, Nighat; Darwesh, Nizam M.; Rahman, Sadeeq ur; Mustafa, Mohammad Z.; Khan, Sher Bahadar; Patching, Simon G.

doi:10.1016/j.pep.2017.11.005

Cited by 61 publications

(36 citation statements)

References 188 publications

(57 reference statements)

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“…What are molecular properties that determine protein expression and genetic compatibility? Many factors have been proposed such as GC content, codon usage, and mRNA folding energy, however, the evidence from independent studies is often contradictory (11,37,(41)(42)(43)(44)(45). Indeed, a recent study that assessed 200 diverse antibiotic resistance genes in E. coli demonstrated that many proposed factors mentioned above cannot sufficiently explain the compatibility of the heterologous genes.…”

Section: Discussionmentioning

confidence: 99%

The Molecular Mechanisms Underlying Hidden Phenotypic Variation among Metallo-β-Lactamases

Socha

Chen

Tokuriki

2019

Journal of Molecular Biology

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Genetic variation among orthologous genes has been largely formed through neutral genetic drift to maintain the same functional role. In some circumstances, however, this genetic variation can create critical phenotypic variation, particularly when genes are transferred to a new host by horizontal gene transfer (HGT). Unveiling "hidden phenotypic variation" through HGT is especially important for genes that confer resistance to antibiotics, which continue to disseminate to new organisms through HGT.Despite this biomedical importance, our understanding of the molecular mechanisms that underlie hidden phenotypic variation remains limited. Here we sought to determine the extent of hidden phenotypic variation in the B1 metallo-β-lactamase (MBL) family, as well as to determine its molecular basis by systematically characterizing eight MBL orthologs when they are expressed in three different organisms (E. coli, P. aeruginosa, and K. pneumoniae). We found that these MBLs confer diverse levels of resistance in each organism, which cannot be explained by variation in catalytic efficiency alone; rather, it is the combination of the catalytic efficiency and abundance of functional periplasmic enzyme that best predicts the observed variation in resistance. The level of functional periplasmic expression varied dramatically between MBL orthologs and between hosts. This was the result changes at multiple levels of each enzyme's functional: 1) the quantity of mRNA; 2) the amount of MBL expressed; and 3) the efficacy of functional enzyme translocation to the periplasm. Overall, we see that it is the interaction between each gene and the host's underlying cellular processes (transcription, translation, and translocation) that determines MBL genetic incompatibility thorough HGT. These hostspecific processes may constrain the effective spread and deployment of MBLs to certain host species, and could explain the current observed distribution bias..

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Section: Discussionmentioning

confidence: 99%

The Molecular Mechanisms Underlying Hidden Phenotypic Variation among Metallo-β-Lactamases

Socha

Chen

Tokuriki

2019

Journal of Molecular Biology

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“…Bacterial growth phase at the time of induction as well as inducer concentration also affect the production of recombinant proteins (Ahmad et al 2018 ). Accordingly, the effects of these parameters on our target protein yield were next examined individually.…”

Section: Discussionmentioning

confidence: 99%

Expression optimization of recombinant cholesterol oxidase in Escherichia coli and its purification and characterization

et al. 2018

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Cholesterol oxidase is a bacterial flavoenzyme which catalyzes oxidation and isomerization of cholesterol. This enzyme has a great commercial value because of its wide applications in cholesterol analysis of clinical samples, synthesis of steroid-derived drugs, food industries, and potentially insecticidal activity. Accordingly, development of an efficient protocol for overexpression of cholesterol oxidase can be very valuable and beneficial. In this study, expression optimization of cholesterol oxidase from Streptomyces sp. SA-COO was investigated in Escherichia coli host strains. Various parameters that may influence the yield of a recombinant enzyme were evaluated individually. The optimal host strain, culture media, induction time, Isopropyl ß-d-1-thiogalactopyranoside concentration, as well as post-induction incubation time and temperature were determined in a shaking flask mode. Applying the optimized protocol, the production of recombinant cholesterol oxidase was significantly enhanced from 3.2 to 158 U/L. Under the optimized condition, the enzyme was produced on a large-scale, and highly expressed cholesterol oxidase was purified from cell lysate by column nickel affinity chromatography. Km and Vmax values of the purified enzyme for cholesterol were estimated using Lineweaver–Burk plot. Further, the optimum pH and optimum temperature for the enzyme activity were also determined. We report a straightforward and easy protocol for cholesterol oxidase production which can be performed in any laboratory.

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“…A variety of expression vectors are available, but the most commonly used ones are plasmid vectors that contain a number of sequence elements, including an origin of replication, a promoter, a multi-cloning site, affinity tags, a transcription terminator and selection markers [7]. Choosing a suitable vector requires detailed understanding of these features and their usefulness can be carefully evaluated according to the characteristics of the target protein [8]. For selection of anticipated cells carrying the desirable plasmid, and to prevent the growth of plasmid-free cells, a resistance marker is generally included in the plasmid.…”

Section: Introductionmentioning

confidence: 99%

Novel Expression Vectors Based on the pIGDM1 Plasmid

et al. 2019

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Escherichia coli is one of the most widely used hosts for the production of heterologous proteins. Within this host, the choice of cloning vector constitutes a key factor for a satisfactory amplified expression of a target gene. We aimed to develop novel, unpatented expression vectors that enable the stable maintenance and efficient overproduction of proteins in E. coli. A series of expression vectors based on the ColE1-like pIGDM1 plasmid were constructed. The vectors named pIGDMCT7RS, pIG-DM4RS and pIGDMKAN carry various antibiotic resistance genes: chloramphenicol, ampicillin or kanamycin, respectively. Two derivatives contain the inducible T7 promoter while the third one bears the constitutive pms promoter from a clinical strain of Klebsiella pneumoniae. The pIGDM1-derivatives are compatible with other ColE1-like plasmids commonly used in molecular cloning. The pIGDMCT7RS and pIGDM4RS vectors contain genes encoding AGA and AGG tRNAs, which supplement the shortage of these tRNAs, increasing the efficiency of synthesis of heterologous proteins. In conclusion, pIG-DMCT7RS, pIGDM4RS and pIGDMKAN vectors, with significantly improved features, including compatibility with vast majority of other plasmids, were designed and constructed. They enable a high-level expression of a desired recombinant gene and therefore constitute a potential, valuable tool for pharmaceutical companies and research laboratories for their own research or for the production of recombinant biopharmaceuticals. Keywords Expression vectors • Recombinant protein production • Bacterial expression system • E. coli Abbreviations bla gene AMPICILLIN RESISTANCE GENE cat gene Chloramphenicol resistance gene kan gene Kanamycin resistance gene MCS Multiple cloning site SD Shine Dalgarno sequence IPTG Isopropyl ß-D-1-thiogalactopyranoside CPH The yeast cyclophilin gene IFNA13 The interferon alpha 13 gene Ubi HGH: ubiquitin-human growth hormone fusion gene

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Overcoming challenges for amplified expression of recombinant proteins using Escherichia coli

Cited by 61 publications

References 188 publications

The Molecular Mechanisms Underlying Hidden Phenotypic Variation among Metallo-β-Lactamases

The Molecular Mechanisms Underlying Hidden Phenotypic Variation among Metallo-β-Lactamases

Expression optimization of recombinant cholesterol oxidase in Escherichia coli and its purification and characterization

Novel Expression Vectors Based on the pIGDM1 Plasmid

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