TABLE OF CONTENTS

Escherichia coli Expression System: A Powerful Tool for Recombinant Protein Production

Tags: Protein expression

Introduction

Recombinant proteins are essential components of current biotechnology with applications across all fields of biotech – drug discovery, vaccine development, enzyme engineering, basic biology. But many natural proteins are folding wrong, forming inclusion bodies or unstable when they're expressed in host cells, and therefore have limited activity. So it is essential to choose the right expression system in order to optimise the expression efficiency and performance of recombinant proteins.If you're looking for comprehensive and efficient protein expression solutions, CD Biosynsis offers a top-tier Recombinant Protein Expression service.

Recombinant protein productio(Hussain, et al, 2021)

General Overview of Standard Expression Systems

Most current recombinant protein expression systems consist of Escherichia coli, yeast, mammalian cells (China hamster ovary cells) and insect cells (baculovirus expression systems). For efficient and reliable protein production in bacterial systems, CD Biosynsis has the Bacterial Protein Expression Service.

Every system is better than none. For instance, Escherichia coli is cheap, fast-growing and simple to operate as an expression platform, but the proteins it can be expressed from might need additional post-translational modifications to be both structured and functional.

Advantages of Escherichia coli as an expression system

It is in the following areas that the advantages of Escherichia coli as an expression system are mainly apparent:

Fast and efficient: Escherichia coli spreads quickly and is easily raised at high density in a short period of time to make large quantities of protein.
Cost effective: Unlike yeast and mammals, Escherichia coli is less expensive to cultivate and it is easily scaled up.
Easy to control and genetic manipulation: Escherichia coli has the perfect genetic history and the mature genetic manipulation technology, so it is easy to perform genetic engineering.
Super expression: Super-expression of protein can be made possible by maximising promoter, signal peptide, mRNA stability, etc.

Even if the E.coli expression system has its drawbacks, like no eukaryotic post-translational modifications (glycosylation and disulfide bonding) and proteins can create inclusion bodies, those drawbacks can be overcome through genetic engineering and optimization of expression conditions. For instance, protein solubility and solubility can be improved through use of special promoters, signal peptides, chaperones, etc.CD Biosynsis specializes in cost-effective and scalable protein production with Protein Expression in E. coli.

Escherichia coli is also an ideal high-efficiency, high-speed and low-cost protein expression system because it's a recombinant protein. Now it's an expression engine widely employed in biopharmaceutical and fundamental science.

Overview of E.coli expression system

Escherichia coli (E. coli) is one of the most studied bacteria and has many biological features and biotechnical uses. What follows is for E. Basic biochemical features of coli, history of its use in biotechnology, basic chemistry of its expression system.

Scanning Electron Micrographs of E. coli(Blount, et al, 2015)

E. coli basic biological characteristics

Structure of the genome: E. The genome of coli has around 4639 protein-coding genes, half of which we know what they do and the rest are unknown. Its genome is mosaic: there is a primary genome and a second genome. The main genome carries out rudimentary activities in life; the second genome is involved in adaptive evolution.CD Biosynsis offers a powerful tool for precise gene knockout studies with Gene Knockout in E. coli.
Variation and diversity: E coli is very genetically diverse across hosts and environments. These varieties get this variety from genetic recombination and horizontal gene transfer, so the strain can quickly adapt to new ecological niches.
Metabolic capacity: E coli is a multicarbon, facultative anaerobic bacterium that differs across metabolic pathways in its range of metabolic capabilities. It has a specialised metabolic machinery: it produces and breakdowns carbohydrates, lipids, nucleic acids and other metabolites.
Virus pathogenicity and immunity: There are species of E. coli (eg, enteropathogenic Escherichia coli and enterohemorrhagic Escherichia coli) that may cause disease in humans and animals, and virulence factors can be studied for host immune response and disease mechanisms.

History of E.coli application in biotechnology

Drug and biotechnical: The first "gold molecule" in mass-production, E coli, is the maker of human insulin. Also used to create antibiotics, enzymes, vaccines and other living things.
Genetic engineering and genetic research: With its fast proliferation, low cost medium and low effort of production, E. coli is a perfect candidate for genetic engineering and genetic research. It has made important discoveries about how DNA replicates, how genes work and are controlled, and about mutations.To revolutionize your genetic engineering, CD Biosynsis provides E. coli CRISPR-Cas9 genome editing services.
Evolutionary studies: E. A good experimental model for studying genomic evolution, adaptive evolution and random mutations is coli.

Foundations of the E. coli expression system

Expression vector: Expression vectors like pET series and pGEX series can be easily expressed by E.coli expression systems. These vectors are programmed to release eukaryotic genes into prokaryotic cells (with some post-translational restrictions).
Protein distribution and secretion: Recombinant protein can be located in cytoplasm, membrane or outer membrane in E. In coli. A few proteins can be released into the culture medium.
Optimise expression strategies: To optimize expression, scientists have come up with many different optimization techniques such as changing the culture conditions, inducing expression using inducers and genetically engineering host strains. Maximize protein expression in E. coli by utilizing CD Biosynsis's Codon Optimization for E. coli Expression service.
Application area: E coli expression system is commonly used for the generation of antibody fragments, enzymes, vaccines and other biological molecules, especially for antibody drugs, and it offers many advantages as it does not require any post-processing.
E coli isn' t just a critical element in basic biological science: it's a fundamental building block of biotech and pharmaceuticals. Its expression system makes for a powerful and economical infrastructure for generating other biomolecules.

Molecular mechanism of recombinant protein expression in E. coli

Gene cloning and vector selection

There are many steps involved in recombinant protein expression in Escherichia coli, from gene cloning, vector selection and plasmid nature and selection. Gene cloning: inserting a target gene into a vector for replication and expression on a host cell. It is usually done by snipping the vector and target gene with restriction enzymes and then assembling them with a DNA ligase. -Type of vector to select, replication origin, copy number, resistance markers etc.

The vector can be the one used for the experiment, cloning expansion or expression. Most popular vectors are plasmids, bacteriophages and synthetic chromosome vectors. Plasmid vectors are the most commonly used vector because they are so straightforward to manipulate, and can replicate on their own in host cells.

Commonly used plasmid vectors

pET series: Commonly used to express recombinant proteins, since it contains tRNA genes of the Escherichia coli, which optimise the translation of proteins.
pGEX series: for expression of fusion proteins, especially glutathione S-transferase (GST) fusion proteins to be purified.
pUC-type series: Often used for cloning and expression experiments, contain ampicillin resistance marks.
pBluescript series: High-fidelity clone compatible and contains anti-kanamycin tags.

Species and selection algorithms of plasmids.

1. Characteristics of the plasmid:

Origin of replication: The origin of replication on a plasmid is a prime area for self replication and usually occurs on the 5' end of the plasmid.
Resistance markers: resistance genes, for example, antibiotic resistance, for the identification of transformed host cells that have already been transformed.
Multiple cloning site (MCS): Enables multiple restriction enzyme cleavage sites to introduce foreign genes.
Copy number: Changes the amount of plasmids in the host cell which influences the yield of the target protein.

2. Selection criteria:

Gene size: Genes smaller than 10kb can be used in little plasmid vectors.
Type of host cell: Choose the vector type according to the type of the host cell.
Promoters and terminators: Use the right promoters and terminators to enhance the expression of the target gene.
Antibiotic resistance markers: Use antibiotic resistance markers that will work for experiments.

E. coli promoter and regulatory elements

Promoters and regulatory elements are also involved in gene expression regulation in Escherichia coli (E. coli).

Mechanisms of transcription regulation by enhancers in eukaryotes(Bylino, et al, 2020)

E. coli promoter classification

Strong promoters: Strong promoters can be left active during growth and they will be efficient at transcriptional driving downstream genes. They tend to be found upstream of genes and their sequences enable RNA polymerase to recognise them easily and begin transcription.
Inducible promoters: This kind of promoter activity is modulated by a chemical or physical cue. Once these signals are available, inducible promoters can be switched on, and downstream genes can become much more transcribed. Promors like heat-inducible, light-inducible and wound-inducible, for instance, all fall into this category.

E.coli regulatory elements

Regulatory elements are involved in gene expression – the main ones are enhancers, boundary elements, insulators, and silencers. They regulate transcriptional activity by tying to trans-acting factors. Specifically:

Enhancers: Enhancers make the gene more efficient in transcription and are usually inside or near the gene.
Border elements: Border elements can deactivate enhancers on non-target genes, which narrows the target gene's activity.
Insulators: Insulators shut off enhancers and other cis-acting components and guard particular genes against attack.
Silencers: Silencers silence genes and halt the unneeded transcription.

Other exogenous agents can alter regulatory elements, for example chemicals, heat or light, which control gene expression by modifying the regulation elements' behaviour.

Promoters and regulators in E. coli ensure that genes are expressed at the right time in the right environment, and that their regulatory responses to environmental variations and cellular requirements are refined.

Commonly used E. coli expression strain

Escherichia coli is a model organism that is very common for protein expression and its different strains are of course capable of a variety of applications in genetic and metabolic engineering.

Most used E. coli strains (Kesidis, et al, 2020)

Commonly used strain BL21DE3

Characteristics:

BL21DE3 modified using Escherichia coli type B strain. It is anchored to the T7 RNA polymerase gene and under the austenimal control of the promoter lacUV5. : It can be efficiently transduced with foreign proteins through IPTG.

This strain doesn't have DNA or the endogenous protease OmpT and is therefore a great host for expressing non-toxic proteins.

BL21DE3 also has high acetic acid synthesis potential which allows for greater cell numbers and recombinant protein production.

Advantages:

Efficiency in protein expression: BL21DE3 is an efficient protein expression strain in many labs and especially with the T7 promoter system you can have the ability to express the foreign genes efficiently.
Cost Effective: BL21DE3 is cheap to grow and can be mass produced.
Easy to work with: BL21DE3 strain is simple to use and suitable for the beginner or big production projects.

Success stories of using BL21DE3

Production of allergen proteins: We commonly use BL21DE3 to make allergen proteins, including those expressed by the T7 expression system.
Produce industrial enzymes: BL21DE3 generates industrial enzymes like the lactose metabolic enzymes, better than other strains upon lactose stimulation.
BL21DE3 for protein folding and stability experiments: we used BL21DE3 to study the proper folding and stability of proteins, especially membrane proteins.

Other commonly used strains

DH5α

Details: DH5 is a K-12 cloneable, kanamycin resistant strain.
Application use-cases: Mainly employed in gene cloning, plasmid generation and DNA repair experiments.

JM109

Features: JM109 is a transformative efficient ampicillin resistant variant of K-12.
Applications: Ideal for gene cloning, plasmid making and experiments that require fast transformation rate.

HMS174

Details: HMS174 is a K-12 mutant strain. It is lactose metabolising and it is capable of pumping out protein under lactose stimulation.
Application context: Often used instead of BL21DE3 in the manufacturing of industrial enzymes requiring lactose induction.

Application examples of E. coli expression system

Expression systems for Escherichia coli (E. coli) are common in medicine, industrial enzyme production and research labs.

Examples of FDA-approved recombinant proteins produced in E. coli (Niazi, et al, 2023)

Applications in the pharmaceutical field

Production of recombinant drug proteins:

It's usually a host, such as Escherichia coli, which makes recombinant protein drugs such as monoclonal antibodies (mAbs), cheap, efficient and easy to work with.

Vaccine designers employ E.coli as well to create virus-like particles (VLPs) for vaccine antigens (eg, HPV vaccines). Ensure high-quality protein purification with CD Biosynsis's Custom Protein Purification from E. coli service.

Vaccine development:

e. coli expression system for production of vaccines like the SARS-CoV-2 vaccine that makes the vaccine more safe and effective by enhancing the protein structure.

Also, E.coli causes rabies and PCV2 vaccines.

Production of industrial enzymes:

We make industrial enzymes from Escherichia coli (peroxidase, glycosyltransferase, etc. Such enzymes are for dye elimination and biocatalysis.

Industrial enzymes : Types and applications.

Types of industrial enzymes:

E coli can also easily release a lot of industrial enzymes, such as peroxidase, glycosyltransferase etc. They're industrial enzymes for dyeing, chemicals etc.

Production process and case analysis:

If you have the right medium and conditions of fermentation, E coli can also express and purify industrial enzymes. Expression of glycosyltransferase for example can be pumped up with plasmid via pRARE. For rapid and controlled protein synthesis, CD Biosynsis provides advanced Cell-Free Protein Expression services.

Applications in scientific research

Structural biology research:

E. coli expresses and purifies proteins for X-ray crystallography or nuclear magnetic resonance in structural biology.

Applications in functional research:
coli expression system, is for protein action (e.g. to try the proteins' biology (say, by producing specific enzymes or receptor proteins). Discover the versatility of CD Biosynsis's Cell-Free Expression Toolkit for various biotechnological applications.

Because it's so efficient, cheap and handy, Escherichia coli expression systems have been used all over medicine, industrial enzyme manufacturing and science. That it is now being employed for vaccine research, recombinant protein production and functional studies are testaments to its stability and flexibility. Escherichia coli is very good, in glycosylation and post-translational modifications, but the creature must be better so that the expression system works better.

References

Hussain, Hirra, et al. "Predictive approaches to guide the expression of recombinant vaccine targets in Escherichia coli: a case study presentation utilising Absynth Biologics Ltd. proprietary Clostridium difficile vaccine antigens." Applied Microbiology and Biotechnology 105 (2021): 5657-5674.
Blount, Zachary D. "The unexhausted potential of E. coli." elife 4 (2015): e05826.
Bylino, Oleg V., Airat N. Ibragimov, and Yulii V. Shidlovskii. "Evolution of regulated transcription." Cells 9.7 (2020): 1675.
Kesidis, Athanasios, et al. "Expression of eukaryotic membrane proteins in eukaryotic and prokaryotic hosts." Methods 180 (2020): 3-18.
Niazi, Sarfaraz K., and Matthias Magoola. "Advances in Escherichia coli-based therapeutic protein expression: mammalian conversion, continuous manufacturing, and cell-free production." Biologics 3.4 (2023): 380-401.

Please note that all services are for research use only. Not intended for any clinical use.

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