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.

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.

Production of Recombinant Melittin by Auto-Induction in Escherichia coli

2013 ◽  
Vol 798-799 ◽  
pp. 1007-1012
Author(s):  
Wen He Zhu ◽  
Wei Zhang ◽  
Yan Li ◽  
Jun Jie Xu ◽  
Shi Jie Lv
Keyword(s):  
Fusion Protein ◽  
Large Scale ◽  
Expression Vector ◽  
Human Cancer ◽  
Expression System ◽  
Scale Production ◽  
Recognition Sequence ◽  
Large Scale Production ◽  
N Terminus ◽  
Gst Fusion Protein

Melittin is a novel peptide of biological activity isolated from bee venom. It has potential application value in medicine and agriculture. Here we encoded melittin gene with the EK recognition sequence in the N-terminus into expression vector pGEX-2T.The expressed fusion protein, which is about 29KDa, identified by Western Blot. To facilitate large-scale production of recombinant GST-fusion protein, we optimized different expression conditions to increase the overall production of the fusion protein. The production of the protein had increased about 10-fold when we used an auto-inducing medium. The GST fusion protein showed an equivalent activity with the natural melittin after digested by EK and can inhibited the proliferations of several human cancer lines. The expression system described in this study provides a feasible way for producing melittin in further studies.

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 Expression of Hybrid Peptide EF-1 in Pichia pastoris, Its Purification, and Antimicrobial Characterization

Molecules ◽  
2020 ◽  
Vol 25 (23) ◽  
pp. 5538
Author(s):  
Zhongxuan Li ◽  
Qiang Cheng ◽  
Henan Guo ◽  
Rijun Zhang ◽  
Dayong Si
Keyword(s):  
Pichia Pastoris ◽  
High Performance ◽  
Large Scale ◽  
Expression System ◽  
Reversed Phase ◽  
E Coli ◽  
Cell Membrane Permeabilization ◽  
Bactericidal Activities ◽  
The Individual ◽  
Industrial Level

EF-1 is a novel peptide derived from two bacteriocins, plantaricin E and plantaricin F. It has a strong antibacterial activity against Escherichia coli and with negligible hemolytic effect on red blood cells. However, the chemical synthesis of EF-1 is limited by its high cost. In this study, we established a heterologous expression of EF-1 in Pichia pastoris. The transgenic strain successfully expressed hybrid EF-1 peptide, which had a molecular weight of ~5 kDa as expected. The recombinant EF-1 was purified by Ni2+ affinity chromatography and reversed-phase high performance liquid chromatography (RP-HPLC), which achieved a yield of 32.65 mg/L with a purity of 94.9%. The purified EF-1 exhibited strong antimicrobial and bactericidal activities against both Gram-positive and -negative bacteria. Furthermore, propidium iodide staining and scanning electron microscopy revealed that EF-1 can directly induce cell membrane permeabilization of E. coli. Therefore, the hybrid EF-1 not only preserves the individual properties of the parent peptides, but also acquires the ability to disrupt Gram-negative bacterial membrane. Meanwhile, such an expression system can reduce both the time and cost for large-scale peptide production, which ensures its potential application at the industrial level.

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 Efficient Production of l-Ribose with a Recombinant Escherichia coli Biocatalyst

2008 ◽  
Vol 74 (10) ◽  
pp. 2967-2975 ◽  
Author(s):  
Ryan D. Woodyer ◽  
Nathan J. Wymer ◽  
F. Michael Racine ◽  
Shama N. Khan ◽  
Badal C. Saha
Keyword(s):  
Escherichia Coli ◽  
Large Scale ◽  
Active Form ◽  
Apium Graveolens ◽  
Efficient Production ◽  
Scale Production ◽  
Gene Encoding ◽  
Large Scale Production ◽  
One Step ◽  
Rare Sugars

ABSTRACT A new synthetic platform with potential for the production of several rare sugars, with l-ribose as the model target, is described. The gene encoding the unique NAD-dependent mannitol-1-dehydrogenase (MDH) from Apium graveolens (garden celery) was synthetically constructed for optimal expression in Escherichia coli. This MDH enzyme catalyzes the interconversion of several polyols and their l-sugar counterparts, including the conversion of ribitol to l-ribose. Expression of recombinant MDH in the active form was successfully achieved, and one-step purification was demonstrated. Using the created recombinant E. coli strain as a whole-cell catalyst, the synthetic utility was demonstrated for production of l-ribose, and the system was improved using shaken flask experiments. It was determined that addition of 50 to 500 μM ZnCl2 and addition of 5 g/liter glycerol both improved production. The final levels of conversion achieved were >70% at a concentration of 40 g/liter and >50% at a concentration of 100 g/liter. The best conditions determined were then scaled up to a 1-liter fermentation that resulted in 55% conversion of 100 g/liter ribitol in 72 h, for a volumetric productivity of 17.4 g liter−1 day−1. This system represents a significantly improved method for the large-scale production of l-ribose.

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.

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