Secretion of thermostable DNA polymerase using a novel baculovirus vector.

Mroczkowski, Barbara; Huvar, Arne; Lernhardt, Waldemar; Misono, Kunio S.; Nielson, K B; Scott, B K

doi:10.1016/s0021-9258(17)36862-x

Cited by 31 publications

(5 citation statements)

References 38 publications

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“…Therefore, how to produce a large quantity of this heat‐resistant and proofreading DNA polymerase becomes a major issue. Previously Pfu DNA polymerase had used the Baculovirus expression system and expressed in insect cells 5 . However, the Baculovirus expression system was less user‐friendly and its equipment were more expensive.…”

Section: Introductionmentioning

confidence: 99%

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High‐level expression of functional Pfu DNA polymerase recombinant protein by mimicking the enhanced green fluorescence protein gene codon usage

Chen¹,

Lin

et al. 2022

Biotech and App Biochem

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Pfu DNA polymerase is a vital enzyme in PCR-related experiments. However, it is not easy to achieve high-level expression and high purity through one-step purification. This paper illustrates the method to acquire the full-length open reading frame of Pfu DNA polymerase. Without altering its amino acids, we have modified the codon usage, based on that of the enhanced green fluorescence protein (eGFP), and named it rPfu. The synthesized rPfu gene has been subcloned into the pET28a plasmid and expressed in four Escherichia coli strains without the pLysS plasmid. Three strains have expressed a high level of soluble Pfu DNA polymerase. With the aid of Ni-NTA His•Bind R resin, we could obtain high purity (>95%) soluble recombinant protein. Compared with the commercial, proofreading DNA polymerase, rPfu's bioactivity was 12,987 U/mg; that is, 88,311 U of rPfu could be obtained from 50 mL cultured E. coli. The purified rPfu was able to amplify the length of DNA fragments at least 5.5 kb. The method of increasing soluble protein's yield using the eGFP codon usage may introduce a new possibility to the expression of other soluble recombinant proteins.

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Section: Introductionmentioning

confidence: 99%

“…Previously Pfu DNA polymerase had used the Baculovirus expression system and expressed in insect cells. 5 However, the Baculovirus expression system was less userfriendly and its equipment were more expensive. These factors made the mass production of commercial Pfu DNA polymerase infeasible.…”

Section: Introductionmentioning

confidence: 99%

High‐level expression of functional Pfu DNA polymerase recombinant protein by mimicking the enhanced green fluorescence protein gene codon usage

Chen¹,

Lin

et al. 2022

Biotech and App Biochem

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“…Pfu , Pwo and Taq were produced in E. coli at expression levels of approximately 24 mg/L, 26.6 mg/L and 95 mg/L, respectively (Dabrowski et al, 1998 ; Engelke et al 1990 ). Pfu polymerase was also produced into growth medium using Spodoptera frugiperda cells (sf-9) and Trichoplusia ni cells (hi-5) with final yields of 100 mg/L and 134 mg/L, respectively (Mroczkowski et al 1994 ). However, despite their ability to achieve higher production quantities, baculovirus systems are more expensive and more challenging to manipulate compared to bacterial systems.…”

Section: Discussionmentioning

confidence: 99%

Novel expression system based on enhanced permeability of Vibrio natriegens cells induced by D,D- carboxypeptidase overexpression

Kormanová,

Levarski,

Minich

et al. 2023

World J Microbiol Biotechnol

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Vibrio natriegens is a fast-growing, non-pathogenic marine bacterium with promising features for biotechnological applications such as high-level recombinant protein production or fast DNA propagation. A remarkable short generation time (< 10 min), robust proteosynthetic activity and versatile metabolism with abilities to utilise wide range of substrates contribute to its establishment as a future industrial platform for fermentation processes operating with high productivity.D,D-carboxypeptidases are membrane-associated enzymes involved in peptidoglycan biosynthesis and cell wall formation. This study investigates the impact of overexpressed D,D-carboxypeptidases on membrane integrity and the increased leakage of intracellular proteins into the growth medium in V. natriegens. Our findings confirm that co-expression of these enzymes can enhance membrane permeability, thereby facilitating the transport of target proteins into the extracellular environment, without the need for secretion signals, tags, or additional permeabilization methods. Using only a single step IMAC chromatography, we were able to purify AfKatG, MDBP or Taq polymerase in total yields of 117.9 ± 56.0 mg/L, 36.5 ± 12.9 mg/L and 26.5 ± 6.0 mg/L directly from growth medium, respectively. These results demonstrate the feasibility of our V. natriegens based system as a broadly applicable extracellular tag-less recombinant protein producer.

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“…As a eukaryotic cell system, insect cells perform post-translational modifications, such as phosphorylation, fatty acid acylation, and glycosylation, and eukaryotic recombinant proteins produced using insect cells exhibit characteristics similar to those of their native forms. , For the production of GPCRs, insect cells offer correct folding in cell membranes with certain Gα subunits. ,, Along with these advantages, insect cells used as host cell systems have shown high efficiency for the expression of GPCRs. Several reports suggested that the expression levels of receptors were 25–600 times higher than those in mammalian cells, and the receptors exhibited the correct function for ligand binding. − In addition, the fusion of some signal sequences of baculoviral, prokaryotic, or eukaryotic origin provides the ability to secrete soluble recombinant proteins and enhance the productivity. , ORs produced from insect cells were successfully applied as recognition elements for a bioelectronic nose by integration with a CNT-based sensor platform . Three mouse OR (mOR) proteins were overexpressed in Sf9 cells and were solubilized with a mild nonionic detergent, digitonin.…”

Section: Production Of Receptors For Combination With Conducting Nano...mentioning

confidence: 99%

“…482−485 In addition, the fusion of some signal sequences of baculoviral, prokaryotic, or eukaryotic origin provides the ability to secrete soluble recombinant proteins and enhance the productivity. 486,487 ORs produced from insect cells were successfully applied as recognition elements for a bioelectronic nose by integration with a CNT-based sensor platform. 488 Three mouse OR (mOR) proteins were overexpressed in Sf9 cells and were solubilized with a mild nonionic detergent, digitonin.…”

Section: Receptor Proteins Produced From Heterologous Cell Systemsmentioning

confidence: 99%

Conducting Nanomaterial Sensor Using Natural Receptors

et al. 2018

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One of the recently emerging topics in biotechnology is natural receptors including G protein-coupled receptors, ligand-gated ion channels, enzyme-linked receptors, and intracellular receptors, due to their molecular specificity. These receptors, other than intracellular receptors, which are membrane proteins expressed on the cell membrane, can detect extracellular stimuli. Many researchers have utilized cells with natural receptors embedded in the cellular membrane for human sense-mimicking platforms based on electrochemical impedance spectroscopy, quartz crystal microbalances, surface plasmon resonance, and surface acoustic waves. In addition, integration of conducting nanomaterials and natural receptors allows highly sensitive and selective responses toward target molecules, enabling, for example, nanobioelectronic noses for odorants, nanobioelectronic tongues for tastants, and G-protein-coupled receptor sensors for hormones, dopamine, cadaverine, geosmin, trimethylamine, etc. Moreover, as a part of nanobioelectronic sensors, natural receptors can be produced in various forms, such as peptides, proteins, nanovesicles, and nanodiscs, and each sensor can provide an ultralow limit of detection. In this Review, we discuss biosensors with natural receptors and then especially focus on natural receptor-conjugated conducting nanomaterial sensors. To provide a fundamental understanding, the sections encompass (1) the fabrication of conducting nanomaterials, (2) the production of natural receptors, (3) the characteristics of natural receptors, (4) the technology for immobilizing both components, and (5) their sensing applications. Finally, perspective is given on a new development in the use of natural receptors in a wide range of industries, such as food, cosmetics, and healthcare. In addition, artificial olfactory codes will be characterized by signal processing in the near future, leading to human olfactory standardization.

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Secretion of thermostable DNA polymerase using a novel baculovirus vector.

Cited by 31 publications

References 38 publications

High‐level expression of functional Pfu DNA polymerase recombinant protein by mimicking the enhanced green fluorescence protein gene codon usage

High‐level expression of functional Pfu DNA polymerase recombinant protein by mimicking the enhanced green fluorescence protein gene codon usage

Novel expression system based on enhanced permeability of Vibrio natriegens cells induced by D,D- carboxypeptidase overexpression

Conducting Nanomaterial Sensor Using Natural Receptors

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