T7-lac Promoter Vectors Derepression Caused by Plant-Derived Growth Media Can Lead to Serious Expression Problems: A Systematic Evaluation.

2021 ◽  
Author(s):  
Daria Krefft ◽  
Maciej Prusinowski ◽  
Paulina Maciszka ◽  
Aleksandra Skokowska ◽  
Joanna Zebrowska ◽  
...  
Keyword(s):  
Culture Media ◽  
Wide Spectrum ◽  
Expression System ◽  
Correct Selection ◽  
Systematic Evaluation ◽  
Growth Media ◽  
Pharmaceutical Drugs ◽  
Expression Systems ◽  
E Coli ◽  
Cloned Gene

Abstract BackgroundThe widespread usage of protein expression systems in Escherichia coli (E. coli) is a workhorse of molecular biology research and practical applications in biotechnology industry, including pharmaceutical drugs production. Various factors can highly affect successful clones construction and their stable maintenance as well as obtained biosynthesis levels. These include correct selection of recombinant hosts, expression systems, promoters regulation, repression level at uninduced state, growth temperature, codon usage, codon context, mRNA secondary structure, translation kinetics and chaperons presence/absence, among others. However, the optimization of the growth media compositions is often overlooked. We systematically evaluate this factor, which can have dramatic effect on biosynthesis of recombinant proteins, especially those, which are toxic to a recombinant host. Results Commonly used animal tissue- and plant-based media were evaluated using a series of clones in pET vector, containing expressed ORFs with wide spectrum of toxicity to the recombinant E. coli: (i) gfpuv (nontoxic); (ii) tp84_28 – coding thermophilic endolysin (moderately toxic) and (iii) tthHB27IRM – coding for thermophilic restriction endonuclease-methyltransferase (REase-MTase) (very toxic). The use of plant-derived peptones (soy peptone and wheat extract) in culture media causes leakage of the T7-lac expression system. We show, that the presence of raffinose and stachyose (galactoside derivatives) in those peptones causes premature and uncontrolled induction of gene expression, which affects the course of cultures, clones stability and biosynthesis levels.Conclusions The use of plant-derived peptones in culture media when using the T7-lac hybrid promoter expression systems, such as Tabor-Studier, can lead to uncontrolled production of a recombinant protein. These conclusions also extend to other, lac operator-controlled promoters. In the case of proteins toxic to a recombinant host, this can result in the expression vector and/or cloned gene mutations, deletions, host’s death or highly decreased expression levels. This phenomenon is caused by certain saccharides content in plant peptones, some of which (galactosides) may act as T7-lac promoter inducers by theirinteraction with Lac repressors. Thus, when attempting to overexpress toxic proteins, it is recommended to either not use plant-derived media or use them with caution and to pilot-scale evaluate the derepression effect on the case-by-case basis.

scholarly journals Preliminary Investigation of Naringenin Hydroxylation with Recombinant E. coli Expressing Plant Flavonoid Hydroxylation Gene

2010 ◽  
Vol 5 (5) ◽  
pp. 1934578X1000500
Author(s):  
Ilef Limem-Ben Amor ◽  
Nidhal Salem ◽  
Emmanuel Guedon ◽  
Jean-Marc Engasser ◽  
Leila Chekir-Ghedrira ◽  
...  
Keyword(s):  
Biomass Yield ◽  
Culture Media ◽  
Plasmid Stability ◽  
Biological Activities ◽  
Preliminary Investigation ◽  
Petroselinum Crispum ◽  
Growth Media ◽  
Hydroxyl Groups ◽  
E Coli ◽  
Cloned Gene

Flavonoid hydroxylation is one way to increase the biological activities of these molecules and the number of hydroxyl groups needed for polymerization, esterification, alkylation, glycosylation and acylation reactions. These reactions have been suggested as a promising route to enhance flavonoid solubility and stability. In our preliminary study we hydroxylated naringenin (the first flavonoid core synthesized in plants) with recombinant E. coli harboring flavanone 3 hydroxylase (F3H). We demonstrated that recombinant E. coli harboring the F3H from Petroselinum crispum, can convert naringenin to dihydrokaempferol. The whole cell hydroxylase activity was often influenced by the stability of the plasmid harboring the cloned gene and the biomass yield. When the composition of the growth media became richer the amount of formed product decreased about twofold; the naringenin bioconversion yield in LB media was 70% and decreased to 33% in TB. However, the enrichment of culture media increased the biomass yield nearly threefold in LB media, only 0.5 g/L of bacteria was formed, but in TB there was 1.6 g/L. Thus, LB constitutes the best medium for naringenin bioconversion using the recombinant E. coli harboring the F3H; this allows for maximum bioconversion yield and plasmid stability when compared with the fourth tested culture medium. Consequently, E. coli harboring F3H from Petroselinum crispum can be used to produce flavonoids hydroxylated in position 3 that can serve in additional reactions like polymerization, glycosylation, and acylation,

Characterization of Codon Usage Bias in UL13 Gene of the Duck Plague Virus

2011 ◽  
Vol 393-395 ◽  
pp. 641-650
Author(s):  
Xi Xia Hu ◽  
An Chun Cheng ◽  
Ming Shu Wang
Keyword(s):  
Codon Usage ◽  
Codon Usage Bias ◽  
Codon Position ◽  
Expression System ◽  
Expression Systems ◽  
Yeast Expression System ◽  
E Coli ◽  
Synonymous Codons ◽  
Suitable Expression

A comprehensive analysis of codon usage bias of DPV UL13 gene (GenBank Accession No. EU195098) was performed to provide a basis for understanding the relevant mechanism for its biased usage of synonymous codons and for selecting suitable expression systems to improve the expression of UL13 genes. Our study showed that codon usage bias of DPV UL13 gene strongly prefered to the synonymous with A and T at the third codon position. And ENC value and GC3s contents of the codon usage bias of UL13 gene in DPV were significantly different compared with those in other 21 reference herpesviruses. The phylogentic analysis about the putative protein of DPV UL13 and the 21 reference herpesviruses revealed that DPV was evolutionarily closer to the AnHV-1. In addition, the codon usage bias of DPV UL13 gene was compared with those of E. coli, yeast and human. There are 23 codons showing distinct usage differences between DPV and E. coli, 12 codons between DPV and yeast, 21 codons between DPV and human. Therefore, the yeast expression system is more appropriate for heterologous expression of the DPV UL13 gene.

scholarly journals Escherichia coli σ70 promoters allow expression rate control at the cellular level in genome-integrated expression systems

2020 ◽  
Author(s):  
Artur Schuller ◽  
Monika Cserjan-Puschmann ◽  
Christopher Tauer ◽  
Johanna Jarmer ◽  
Martin Wagenknecht ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Recombinant Protein ◽  
Recombinant Protein Production ◽  
Protein Production ◽  
Expression System ◽  
Cellular Level ◽  
Lac Repressor ◽  
Expression Systems ◽  
E Coli ◽  
Basal Expression

Abstract Background The genome-integrated T7 expression system offers significant advantages, in terms of productivity and product quality, even when expressing the gene of interest (GOI) from a single copy of. Compared to plasmid-based expression systems, this system does not incur a plasmid-mediated metabolic load, and it does not vary the dosage of the GOI during the production process. However, long-term production with T7 expression system leads to a rapidly growing non-producing population, because the T7 RNA polymerase (RNAP) is prone to mutations. The present study aimed to investigate whether two σ 70 promoters, which were recognized by the Escherichia coli host RNAP, might be suitable in genome-integrated expression systems. We applied a promoter engineering strategy that allowed control of expressing the model protein, GFP, by introducing lac operators ( lacO ) into the constitutive T5 and A1 promoter sequences. Results We showed that, in genome-integrated E. coli expression systems that used σ 70 promoters, the number of lacO sites must be well balanced. Promoters containing three and two lacO sites exhibited low basal expression, but resulted in a complete stop in recombinant protein production in partially induced cultures. In contrast, expression systems regulated by a single lacO site and the lac repressor element, lacI Q , on the same chromosome caused very low basal expression, were highly efficient in recombinant protein production, and enables fine-tuning of gene expression levels on a cellular level. Conclusions Based on our results, we hypothesized that this phenomenon was associated with the autoregulation of the lac repressor protein, LacI. We reasoned that the affinity of LacI for the lacO sites of the GOI must be lower than the affinity of LacI to the lacO sites of the endogenous lac operon; otherwise, LacI autoregulation could not take place, and the lack of LacI autoregulation would lead to a disturbance in lac repressor-mediated regulation of transcription. By exploiting the mechanism of LacI autoregulation, we created a novel E. coli expression system for use in recombinant protein production, synthetic biology, and metabolic engineering applications.

scholarly journals Escherichia coli σ70 promoters allow expression rate control at the cellular level in genome-integrated expression systems

2019 ◽  
Author(s):  
Artur Schuller ◽  
Monika Cserjan-Puschmann ◽  
Christopher Tauer ◽  
Johanna Jarmer ◽  
Martin Wagenknecht ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Recombinant Protein ◽  
Recombinant Protein Production ◽  
Protein Production ◽  
Expression System ◽  
Lac Repressor ◽  
Expression Systems ◽  
E Coli ◽  
Basal Expression ◽  
T7 Expression System

Abstract Background The genome-integrated T7 expression system offers significant advantages, in terms of productivity and product quality, even when expressing the gene of interest (GOI) from a single copy of. Compared to plasmid-based expression systems, this system does not incur a plasmid-mediated metabolic load, and it does not vary the dosage of the GOI during the production process. However, long-term production with T7 expression system leads to a rapidly growing non-producing population, because the T7 RNA polymerase (RNAP) is prone to mutations. The present study aimed to investigate whether two σ 70 promoters, which were recognized by the Escherichia coli host RNAP, might be suitable in genome-integrated expression systems. We applied a promoter engineering strategy that allowed control of expressing the model protein, GFP, by introducing lac operators ( lacO ) into the constitutive T5 and A1 promoter sequences.Results We showed that, in genome-integrated E. coli expression systems that used σ 70 promoters, the number of lacO sites must be well balanced. Promoters containing three and two lacO sites exhibited low basal expression, but resulted in a complete stop in recombinant protein production in partially induced cultures. In contrast, expression systems regulated by a single lacO site and the lac repressor element, lacI Q , on the same chromosome caused very low basal expression, were highly efficient in recombinant protein production, and enables fine-tuning of gene expression levels on a cellular level.Conclusions Based on our results, we hypothesized that this phenomenon was associated with the autoregulation of the lac repressor protein, LacI. We reasoned that the affinity of LacI for the lacO sites of the GOI must be lower than the affinity of LacI to the lacO sites of the endogenous lac operon; otherwise, LacI autoregulation could not take place, and the lack of LacI autoregulation would lead to a disturbance in lac repressor-mediated regulation of transcription. By exploiting the mechanism of LacI autoregulation, we created a novel E. coli expression system for use in recombinant protein production, synthetic biology, and metabolic engineering applications.

scholarly journals Incompatibility of DFHBI based fluorescent RNA aptamers with particular commercial cell-free expression systems

2021 ◽  
Author(s):  
Alexander J Speakman ◽  
Katherine E Dunn
Keyword(s):  
Expression System ◽  
In Vitro Transcription ◽  
Rna Aptamers ◽  
Free Expression ◽  
Reaction Products ◽  
Expression Systems ◽  
E Coli ◽  
Cell Free Expression System

Fluorescent RNA aptamers are an increasingly used tool for quantifying transcription and for visualising RNA interactions, both in vitro and in vivo. However when tested in the commercially available, E. coli extract based Expressway™ cell-free expression system, no fluorescence is detected. The same experimental setup is shown to successfully produce fluorescent RNA aptamers when tested in another buffer designed for in vitro transcription, and RNA purification of the Expressway™ reaction products show that transcription does occur, but does not result in a fluorescent product. In this paper we demonstrate the incompatibility of a narrow selection of RNA aptamers in one particular cell-free expression system, and consider that similar issues may arise with other cell-free expression systems, RNA aptamers, and their corresponding fluorophores.

scholarly journals Recent Developments of Recombinant Protein Expression for Therapeutic Purposes

2021 ◽  
Vol 3 (4) ◽  
Author(s):  
Anam Amir
Keyword(s):  
Recombinant Protein ◽  
Recombinant Proteins ◽  
Expression System ◽  
Cell Penetrating Peptides ◽  
Ovary Cell ◽  
Expression Systems ◽  
E Coli ◽  
Mammalian Expression ◽  
Mammalian Expression System ◽  
High Level

In the most recent seven to eight years, the therapeutic recombinant proteins have rapidly expanded in the biotechnology domain due to its wide variety of needs. There has been significant development in the mammalian expression system for fine purification and increased level of expressed recombinant proteins [1,2]. Many drugs like tetracycline have been demonstrated on the Chinese Hamster Ovary cell line for promising multi control strategies and effective cytotoxicity. Mammalian expression system improves the proper glycosylation of recombinant proteins which are very helpful to increase solubility of product [3-6].             Meanwhile on the prokaryotic expression system, E. coli has proven to be an easier to handle, friendly and economical strain [2]. Recently these expression systems are using to work on antibody fragment productions and their proper folding with co-expression of chaperones [7]. Moreover E. coli has been used for the production of cancer cell penetrating peptides which promises the targeted delivery of drugs to specific effector cells only.  Yeast systems are also being used for the antibody fragments production and the high level production of insulin. Interestingly cell free expression systems are also participating in this game and that would be very fascinating to see in the coming years about cell extract medium for production of high level recombinant protein [8, 9]. Purification and optimization of recombinant protein has always been a challenging situation for scientists and they paid more attention to increase the overall yield of the product. Many affinity chromatography techniques has been introduced for efficient purification of protein of interest [10]. Despite these research and developments in methodologies to produce and purify the recombinant therapeutic protein, scientists still face the hurdles and challenges with all expression systems. Rationally E. coli produces inclusion bodies and many mammalian cell types do not show the same results with the same recombinant protein. [11]. So there is a requirement for adding the appropriate features to the expression systems focused to better improvising recovery, production and purification of recombinant protein. Copyright(c) The Author

The Influence of Glycosylation on the Catalytic and Fibrinolytic Properties of Pro-Urokinase

1992 ◽  
Vol 68 (05) ◽  
pp. 539-544 ◽  
Author(s):  
Catherine Lenich ◽  
Ralph Pannell ◽  
Jack Henkin ◽  
Victor Gurewich
Keyword(s):  
Escherichia Coli ◽  
Mammalian Cell ◽  
Human Gene ◽  
Culture Media ◽  
Mammalian Cell Culture ◽  
Glycosylation Site ◽  
Clot Lysis ◽  
Cell Culture Media ◽  
E Coli ◽  
The Uk

SummaryWe previously found that human pro-UK expressed in Escherichia coli is more active in fibrinolysis than recombinant human pro-UK obtained from mammalian cell culture media. To determine whether this difference is related to the lack of glycosylation of the E. coli product, we compared the activity of E. coli-derived pro-UK [(-)pro-UK] with that of a glycosylated pro-UK [(+)pro-UK] and of a mutant of pro-UK missing the glycosylation site at Asn-302 [(-) (302) pro-UK]. The latter two pro-UKs were obtained by expression of the human gene in a mammalian cell. The nonglycosylated pro-UKs were activated by plasmin more efficiently (≈2-fold) and were more active in clot lysis (1.5-fold) than the (+)pro-UK. Similarly, the nonglycosylated two-chain derivatives (UKs) were more active against plasminogen and were more rapidly inactivated by plasma inhibitors than the (+)UK.These findings indicate that glycosylation at Asn-302 influences the activity of pro-UK/UK and could be the major factor responsible for the enhanced activity of E. coli-derived pro-UK.

Purification of Urokinase from Complex Mixtures Using Immobilized Monoclonal Antibody Against Urokinase Light Chain

1983 ◽  
Vol 49 (01) ◽  
pp. 024-027 ◽  
Author(s):  
David Vetterlein ◽  
Gary J Calton
Keyword(s):  
Monoclonal Antibody ◽  
Light Chain ◽  
Culture Media ◽  
Biological Fluids ◽  
Single Step ◽  
High Yield ◽  
Step Method ◽  
Commercial Preparation ◽  
E Coli ◽  
Tissue Culture Media

SummaryThe preparation of a monoclonal antibody (MAB) against high molecular weight (HMW) urokinase light chain (20,000 Mr) is described. This MAB was immobilized and the resulting immunosorbent was used to isolate urokinase starting with an impure commercial preparation, fresh urine, spent tissue culture media, or E. coli broth without preliminary dialysis or concentration steps. Monospecific antibodies appear to provide a rapid single step method of purifying urokinase, in high yield, from a variety of biological fluids.

scholarly journals Functional expression of the eukaryotic proton pump rhodopsin OmR2 in Escherichia coli and its photochemical characterization

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Masuzu Kikuchi ◽  
Keiichi Kojima ◽  
Shin Nakao ◽  
Susumu Yoshizawa ◽  
Shiho Kawanishi ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Proton Pump ◽  
Expression System ◽  
Spectroscopic Characterization ◽  
Functional Expression ◽  
Proton Pumping ◽  
E Coli ◽  
Oxyrrhis Marina ◽  
Domains Of Life

AbstractMicrobial rhodopsins are photoswitchable seven-transmembrane proteins that are widely distributed in three domains of life, archaea, bacteria and eukarya. Rhodopsins allow the transport of protons outwardly across the membrane and are indispensable for light-energy conversion in microorganisms. Archaeal and bacterial proton pump rhodopsins have been characterized using an Escherichia coli expression system because that enables the rapid production of large amounts of recombinant proteins, whereas no success has been reported for eukaryotic rhodopsins. Here, we report a phylogenetically distinct eukaryotic rhodopsin from the dinoflagellate Oxyrrhis marina (O. marina rhodopsin-2, OmR2) that can be expressed in E. coli cells. E. coli cells harboring the OmR2 gene showed an outward proton-pumping activity, indicating its functional expression. Spectroscopic characterization of the purified OmR2 protein revealed several features as follows: (1) an absorption maximum at 533 nm with all-trans retinal chromophore, (2) the possession of the deprotonated counterion (pKa = 3.0) of the protonated Schiff base and (3) a rapid photocycle through several distinct photointermediates. Those features are similar to those of known eukaryotic proton pump rhodopsins. Our successful characterization of OmR2 expressed in E. coli cells could build a basis for understanding and utilizing eukaryotic rhodopsins.

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