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 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.

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.

Development of a process for large scale production of PfRH5 in E. coli expression system

2021 ◽  
Author(s):  
Arjun Singh Raghuwanshi ◽  
Ankit Kumar ◽  
Navdeep Raghuwanshi ◽  
Shravan Kumar Singh ◽  
Avinash Kumar Singh ◽  
...  
Keyword(s):  
Large Scale ◽  
Expression System ◽  
Scale Production ◽  
E Coli ◽  
Large Scale Production

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.

scholarly journals High efficient extracellular production of recombinant Streptomyces PMF phospholipase D in Escherichia coli

2020 ◽  
Author(s):  
Jing Wang ◽  
Sheng Xu ◽  
Yang Pang ◽  
Xin Wang ◽  
Kequan Chen ◽  
...  
Keyword(s):  
Phospholipase D ◽  
Signal Peptide ◽  
Large Scale ◽  
Expression System ◽  
Cost Effective ◽  
Scale Production ◽  
Secretory Expression ◽  
E Coli ◽  
Cost Effective Method ◽  
High Efficient

Abstract Background Currently, Streptomyces is widely used in the preparation of phospholipase D (PLD) with high transphosphatidylation activity. However, the yield of PLD from Streptomyces was low and the culture period was long. Therefore, an efficient and cost-effective method is needed urgently.Results Firstly, PLDs from Streptomyces PMF and Streptomyces racemochromogenes were separately over-expressed in E. coli to compare their transphosphatidylation activity based on the synthesis of phosphatidylserine (PS), and PLDPMF was determined to have higher activity. To further improve PLDPMF synthesis, a secretory expression system suitable for PLDPMF was constructed and optimized with different signal peptides. The highest secretory efficiency was observed when the PLDPMF gene was expressed together with its native signal peptide (Nat) and the signal peptide PelB from E. coli. For the application of recombinant PLD to PS synthesis, the PLD properties were characterized and 30.2 g/L of PS was produced after 24 h of bioconversion when 50 g/L phosphatidylcholine (PC) was added.Conclusions We succeeded in over-expressing PLD from Streptomyces PMF in E. coli with high transphosphatidylation activity and enhanced the yield by secretory expression. The secreted PLD was successfully used in the production of PS. Our work makes the large-scale production of PLD and PS feasible.

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.

Implementation of a novel self-induced promoter for the expression of pharmaceutical peptides in Escherichia coli: YY(3-36) peptide

2019 ◽  
Vol 0 (0) ◽  
Author(s):  
Amir Hossein Momen ◽  
Naser Harzandi ◽  
Azam Haddadi ◽  
Bijan Bambai
Keyword(s):  
Escherichia Coli ◽  
Large Scale ◽  
Peptide Yy ◽  
Signal Sequence ◽  
Expression System ◽  
Promoter Sequence ◽  
Scale Production ◽  
Signal Sequences ◽  
T7 Promoter ◽  
E Coli

Abstract Background Increasing the expression rate of recombinant mammalian hormones in Escherichia coli by combining efficient promoters and signal sequences is a never ending process. A self-induced promoter will have some beneficial gains compared to the classical T7 promoter or its variants with isopropyl β-D-1-thiogalactopyranoside (IPTG) as the inducer. Obesity is the prime suspect in widespread frequency of diabetes type II and cardiovascular diseases worldwide. YY (tyrosine-tyrosine) peptide is a local acting hormone, controlling appetite. Excitingly, it was has been shown that a truncated version of the YY peptide, YY(3-36) peptide, has potential as a worthy biopharmaceutical agent in the fight against obesity. Materials and methods To develop an economical expression system for the large scale production of the peptide in Gram-negative bacteria, we introduced a promoter sequence upstream of a chimeric gene for the extracellular expression of this peptide with the assistance of a signal sequence of asparaginase II from E. coli. This system has the advantage of producing a complete sequence of a truncated YY peptide, YY(3-36), without any extra tags that would require further removal with the assistance of expensive specific proteases and reduced the downstream steps, significantly. Results Recombinant production of YY(3-36) peptide under a self-induced promoter proves the efficacy of the asparaginase II signal sequence as a communicator of foreign peptides and proteins into the extracellular space of E. coli. Conclusions The application of fusion protein expression of biopharmaceuticals, especially mammalian hormones, in prokaryotic systems with the help of native signal sequences makes some common tags with expensive proteases for the removal of the attached protein Tag redundant.

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.

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