Heterologous Expression, Characterization and Evolution Prediction of a Diaphorase From Geobacillus sp. Y4.1MC1

2021 ◽  
Author(s):  
Jinzhao Shang ◽  
Shuohao Yue ◽  
Fang Zeng ◽  
Yun Chen ◽  
Longgang Jia
Keyword(s):  
Large Scale ◽  
Ketone Bodies ◽  
Scale Production ◽  
Hydroxybutyric Acid ◽  
E Coli ◽  
Ph Values ◽  
Structure Changes ◽  
Large Scale Production ◽  
Expression Characterization ◽  
The Stability

Abstract β-hydroxybutyric acid is the most sensitive indicator in ketoacidosis detection, and accounts for nearly 78% of the ketone bodies. Diaphorase is commonly used to detect the β-hydroxybutyric acid in clinical diagnosis. However, the extraction of diaphorase from animal myocardium is complex and low-yield, which is not convenient for large-scale production. In this study, a diaphorase from Geobacillus sp. Y4.1MC1 was efficiently heterologous expressed and purified in E. coli a yield of 110 mg/L culture. The optimal temperature and pH of this recombinant diaphorase (rDIA) were 55 °C and 6.5, respectively. It was proved that rDIA was a dual acid- and thermo-stable enzyme, and which showed much more accurate detection of β-hydroxybutyric acid than the commercial enzyme. Additionally, we also investigated the molecular interaction of rDIA with the substrate, and the conformation transition in different pH values by using homology modeling and molecular dynamics (MD) simulation. The results showed that 141-161 domain of rDIA played important role in the structure changes and conformations transmission at different pH values. Moreover, it was predicted that F105W, F105R and M186R mutants were able to improve the binding affinity of rDIA, and A2Y, P35F, Q36D, N210L, F211Y mutants were benefit for the stability of rDIA.

scholarly journals Cloning, expression, and analysis of the group 2 allergen from Dermatophagoides farinae from China

2010 ◽  
Vol 82 (4) ◽  
pp. 941-951 ◽  
Author(s):  
Cui Yu-bao ◽  
Ying Zhou ◽  
Shi Weihong ◽  
Ma Guifang ◽  
Li Yang ◽  
...  
Keyword(s):  
Large Scale ◽  
Alpha Helix ◽  
Random Coil ◽  
Reference Sequence ◽  
Scale Production ◽  
Dermatophagoides Farinae ◽  
E Coli ◽  
Large Scale Production ◽  
Solid Foundation ◽  
Group 2

To obtain the recombinant group 2 allergen product of Dermatophagoides farinae (Der f 2), the Der f 2 gene was synthesized by RT-PCR. The full-length cDNA comprised 441 nucleotides and was 99.3% identical to the reference sequence (GenBank AB195580). The cDNA was bound to vector pET28a to construct plasmid pET28a(+)-Der f 2, which was transformed into E. coli BL21 and induced by IPTG. SDS-PAGE showed a specific band of about 14kDa in the hole cell lysate. s estiated by chroatography, about 3.86 g of the recobinant product as obtained, which conjugated with serum IgE from asthmatic children. The protein had a signal peptide of 17 amino acids. Its secondary structure comprised an alpha helix (19.86%), an extended strand (30.82%), and a random coil (49.32%). The subcellular localization of this allergen was predicted to be at mitochondria. Furthermore, its function was shown to be associated with an MD-2-related lipid-recognition (ML) domain. The results of this study provide a solid foundation for large-scale production of the allergen for clinical diagnosis and treatent of allergic disorders.

scholarly journals Advances in the Metabolic Engineering of Escherichia coli for the Manufacture of Monoterpenes

Catalysts ◽  
2019 ◽  
Vol 9 (5) ◽  
pp. 433 ◽  
Author(s):  
Si-si Xie ◽  
Lingyun Zhu ◽  
Xin-yuan Qiu ◽  
Chu-shu Zhu ◽  
Lv-yun Zhu
Keyword(s):  
Large Scale ◽  
Metabolic Flux ◽  
Process Development ◽  
Pathway Engineering ◽  
Scale Production ◽  
Future Studies ◽  
E Coli ◽  
Large Scale Production ◽  
Rate Limiting ◽  
Dependent Pathway

Monoterpenes are commonly applied as pharmaceuticals and valuable chemicals in various areas. The bioproduction of valuable monoterpenes in prokaryotic microbial hosts, such as E. coli, has progressed considerably thanks to the development of different outstanding approaches. However, the large-scale production of monoterpenes still presents considerable limitations. Thus, process development warrants further investigations. This review discusses the endogenous methylerythritol-4-phosphate-dependent pathway engineering and the exogenous mevalonate-dependent isoprenoid pathway introduction, as well as the accompanied optimization of rate-limiting enzymes, metabolic flux, and product toxicity tolerance. We suggest further studies to focus on the development of systematical, integrational, and synthetic biological strategies in light of the inter disciplines at the cutting edge. Our review provides insights into the current advances of monoterpene bioengineering and serves as a reference for future studies to promote the industrial production of valuable monoterpenes.

cDNA cloning and heterologous expression of a wheat proteinase inhibitor of subtilisin and chymotrypsin (WSCI) that interferes with digestive enzymes of insect pests

2005 ◽  
Vol 386 (4) ◽  
pp. 383-389 ◽  
Author(s):  
Simone Di Gennaro ◽  
Anna G. Ficca ◽  
Daniela Panichi ◽  
Elia Poerio
Keyword(s):  
Proteinase Inhibitor ◽  
Large Scale ◽  
Insect Pests ◽  
Expression System ◽  
Physiological Role ◽  
Scale Production ◽  
Insect Larvae ◽  
E Coli ◽  
Large Scale Production ◽  
Degenerate Oligonucleotide

Abstract A cDNA encoding the proteinase inhibitor WSCI (wheat subtilisin/chymotrypsin inhibitor) was isolated by RT-PCR. Degenerate oligonucleotide primers were designed based on the amino acid sequence of WSCI and on the nucleotide sequence of the two homologous inhibitors (CI-2A and CI-2B) isolated from barley. For large-scale production, wsci cDNA was cloned into the E. coli vector pGEX-2T. The fusion protein GST-WSCI was efficiently produced in the bacterial expression system and, as the native inhibitor, was capable of inhibiting bacterial subtilisin, mammalian chymotrypsins and chymotrypsin-like activities present in crude extracts of a number of insect larvae (Helicoverpa armigera, Plodia interpunctella and Tenebrio molitor). The recombinant protein produced was also able to interfere with chymotrypsin-like activity isolated from immature wheat caryopses. These findings support a physiological role for this inhibitor during grain maturation.

scholarly journals Call for papers on special issue “Perovskite photovoltaics and optoelectronic devices”

2020 ◽  
Vol 2 (1) ◽  
pp. 049-049
Keyword(s):  
Solar Cells ◽  
Large Scale ◽  
Perovskite Solar Cells ◽  
Optoelectronic Devices ◽  
Scale Production ◽  
Special Issue ◽  
Device Structure ◽  
Stability Performance ◽  
Large Scale Production ◽  
The Stability

Aim & Scope: Metal halide perovskitehave been regarded as promising classes of materials for photovoltaics and optoelectronic devices, owing to the unique characteristics, such as long charge carrier diffusion lengths, precise tunable bandgaps, high light absorption coefficients, and high defect tolerance. Research on perovskite in the fields including photovoltaics, light-emitting diodes, lasers, X-ray imaging, and photodetectors has been gaining increasingly interest over the past years. Up to now, the efficiency of perovskite solar cells has grown from 3.8% in single-junction solar cells in 2009 to more than 25%, catching up the efficiency level of commercial silicon cells. Up to now, the key issues of perovskite photovoltaics and optoelectronic devices have become the stability, performance and large-scale production. This requires optimization of the film morphology, interface, device structure and the fabrication process. A lot work has been done on this issue and has made remarkable progress. We kindly invite you to submit a manuscript(s) for this Special Issue. Full papers, communications, and reviews are all welcome.

scholarly journals Secretory Expression of YY(3-36) Peptide in E. coli

2019 ◽  
Author(s):  
Sorush Niknamian
Keyword(s):  
Large Scale ◽  
Peptide Yy ◽  
Signal Sequence ◽  
Expression System ◽  
Complete Sequence ◽  
Scale Production ◽  
E Coli ◽  
Large Scale Production ◽  
Human Experiments ◽  
Clinical Experiments

Obesity is the prime suspect in a wide frequency of diabetes type II and cardiovascular diseases worldwide. Recombinant YY (tyrosine-tyrosine) peptide is a locally acting hormone, controlling secretion in the digestive tract. Interestingly, it was later shown that a truncated version of YY peptide, YY(3-36) peptide, has the potential as an important biopharmaceutical in a fight against obesity. This peptide has shown promising results in human clinical experiments in appetite reduction in human experiments. To develop an economical expression system for large-scale production of the peptide in gram-negative bacteria, we have developed a chimeric gene for extracellular expression of this peptide with the assistance of signal sequence of asparaginase II from Escherichia coli. This system has the advantage of producing the complete sequence of YY(3-36) without any extra tags that require further removal with the assistance of expensive specific proteases and reduce the downstream steps significantly. Our results pave the way for the recombinant production of YY(3-36) peptide and further proves the efficacy of asparaginase II signal sequence as a communicator of foreign peptides and proteins into extracellular space of E. coli.

Construction and Expression of Mouse-human Chimeric Antibody SZ-51 Specific for Activated Platelet P-selectin

1997 ◽  
Vol 77 (04) ◽  
pp. 755-759 ◽  
Author(s):  
Jianming Gu ◽  
Yue Liu ◽  
Lijun Xia ◽  
Haiying Wan ◽  
Peixia Li ◽  
...  
Keyword(s):  
Large Scale ◽  
Variable Region ◽  
Expression Vectors ◽  
Chimeric Antibody ◽  
Scale Production ◽  
Fab Fragment ◽  
Variable Regions ◽  
E Coli ◽  
Large Scale Production

SummaryA murine monoclonal (mAb) SZ-51 specific for human P-selectin may be used for in vivo thrombus imaging and for the targeting of fibrinolytic agents to thrombi. In order to reduce the immunogenicity of the murine mAb SZ-51 in humans, we cloned and sequenced the cDNAs encoding the variable region of mAb SZ-51 in order to develop mouse/human chimeric reagents. The E. coli expression vector. pHENl-SZ51 Fab/Hu was constructed by fusing the variable regions of mAb SZ-51 with human IgG γICHI and Cκ genes. The constructs were introduced into E. coli HB2151 for expression of soluble chimeric Fab fragment. We also constructed two fusion products by joining the variable regions of mouse antibody to the appropriate constant regions of human Igγl and κ. These chimeras were cloned into two eukaryotic selectable expression vectors separately, which were then cotransfected into a non-Ig secreting murine myeloma line SP2/0 with lipofectin reagent. Six cell lines remained positive for Ig secretion. The highest producing cell line, which showed stable integration and expression at 5 mg/1 of culture, was selected for the large scale production of chimeric antibody. Immunoblotting analysis demonstrated that both of the chimeric antibodies (SZ51Fab/Hu, SZ51/Hu) in the culture supernatants, like the native mAb SZ-51, bind P-selectin. In addition, the whole chimeric antibody can compete for binding to activated platelets with murine SZ-51. Therefore, the SZ-51 chimeric antibody may be a potential agent for diagnosis and treatment of thrombotic diseases in the future.

Computer controlled large scale production of ?-interferon by E. coli

1994 ◽  
Vol 10 (4) ◽  
pp. 145-153 ◽  
Author(s):  
B. Wipf ◽  
E. K. Weibel ◽  
S. Vogel
Keyword(s):  
Large Scale ◽  
Scale Production ◽  
E Coli ◽  
Large Scale Production ◽  
Computer Controlled

scholarly journals Novel Expression System for Large-Scale Production and Purification of Recombinant Class IIa Bacteriocins and Its Application to Piscicolin 126

2004 ◽  
Vol 70 (6) ◽  
pp. 3292-3297 ◽  
Author(s):  
Gerard M. Gibbs ◽  
Barrie E. Davidson ◽  
Alan J. Hillier
Keyword(s):  
Fusion Protein ◽  
Large Scale ◽  
Expression System ◽  
Reversed Phase ◽  
Scale Production ◽  
Gene Encoding ◽  
Strong Activity ◽  
E Coli ◽  
Large Scale Production ◽  
Bacterial Cell Membrane

ABSTRACT Piscicolin 126 is a class IIa bacteriocin isolated from Carnobacterium piscicola JG126 that exhibits strong activity against Listeria monocytogenes. The gene encoding mature piscicolin 126 (m-pisA) was cloned into an Escherichia coli expression system and expressed as a thioredoxin-piscicolin 126 fusion protein that was purified by affinity chromatography. Purified recombinant piscicolin 126 was obtained after CNBr cleavage of the fusion protein followed by reversed-phase chromatography. Recombinant piscicolin 126 contained a single disulfide bond and had a mass identical to that of native piscicolin 126. This novel bacteriocin expression system generated approximately 26 mg of purified bacteriocin from 1 liter of E. coli culture. The purified recombinant piscicolin 126 acted by disruption of the bacterial cell membrane.

scholarly journals Production of Human Cu,Zn SOD with Higher Activity and Lower Toxicity inE. colivia Mutation of Free Cysteine Residues

2017 ◽  
Vol 2017 ◽  
pp. 1-12
Author(s):  
Kun Zhang ◽  
Yuejuan Zhang ◽  
Jing Zi ◽  
Xiaochang Xue ◽  
Yi Wan
Keyword(s):  
Large Scale ◽  
Soluble Form ◽  
Scale Production ◽  
Superoxide Dismutase 1 ◽  
Wild Type ◽  
Lower Toxicity ◽  
E Coli ◽  
Promising Strategy ◽  
Large Scale Production ◽  
Cell Components

Although, as an antioxidant enzyme, human Cu,Zn superoxide dismutase 1 (hSOD1) can mitigate damage to cell components caused by free radicals generated by aerobic metabolism, large-scale manufacturing and clinical use of hSOD1 are still limited by the challenge of rapid and inexpensive production of high-quality eukaryotic hSOD1 in recombinant forms. We have demonstrated previously that it is a promising strategy to increase the expression levels of soluble hSOD1 so as to increase hSOD1 yields inE. coli. In this study, a wild-type hSOD1 (wtSOD1) and three mutant SOD1s (mhSOD1s), in which free cysteines were substituted with serine, were constructed and their expression in soluble form was measured. Results show that the substitution of Cys111 (mhSOD1/C111S) increased the expression of soluble hSOD1 inE. coliwhereas substitution of the internal Cys6 (mhSOD1/C6S) decreased it.Besides, raised levels of soluble expression led to an increase in hSOD1 yields. In addition, mhSOD1/C111S expressed at a higher soluble level showed lower toxicity and stronger whitening and antiradiation activities than those of wtSOD1. Taken together, our data demonstrate that C111S mutation in hSOD1 is an effective strategy to develop new SOD1-associated reagents and that mhSOD1/C111S is a satisfactory candidate for large-scale production.

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