Protein Expression in Bacterial System

Escherichia coli is one of the most commonly used bacterial hosts in the production of heterologous proteins. It has been proven as the preferred system for recombinant protein production as it is advantageous in several important ways, including fast rate of reproduction, ease of culture, and ample knowledge about its genetics.

Backed by years of development and research experience in the field of recombinant protein expression, Biologics International Corp (BIC) offers comprehensive, high-quality recombinant custom protein services. All you need to do is simply provide protein sequences, and our experienced team of scientists will help you achieve your research goals quickly and efficiently. Contact us for project quotations and more detailed information. Learn more about our bacterial expression system.

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Service Highlights

• Guaranteed: no-success no-fee guarantee.
• Customization: quantity, purity, tags, and endotoxin levels are customizable and RNase removal is optional.
• Modifications: isotope labeling, protein biotinylation and other labeling services.

Bacterial Protein Expression Service

	Service Process	Deliverables
Design strategy	The optimal plan will be designed according to the customer’s requirements and protein characteristics.	Cloning vector QC data Final report >90% purity Quantity as per customer requirements (≥3 mg soluble proteins)
Codon optimization & gene synthesis (2 weeks)	With our improved codon optimization technology, the expression levels of target proteins can be significantly improved.
Protein expression evaluation & optimization (1 week)	We will conduct a series of pilot expression tests using diverse expression conditions and take SDS-PAGE and western blot to confirm the expression levels. At this step, we will optimize your protein expression conditions.
Scale up & protein purification (2-3 weeks)	We will scale up your protein expression using optimized conditions. After multistep protein purification, the protein purity will be >90%, and endotoxin level will be <1 EU/μg.
Notes: According to your specific requirements, His, GST, or other affinity tags are available. A tag-free protein service is also offered. We perform QC detection using SDS-PAGE and western blot methods without charge. Other detection methods are optional (additional charges) , such as mass spectrometry (MS). In a standard package, we deliver 3-5 mg soluble proteins with more than 90% purity. Furthermore, BIC has the ability to provide recombinant proteins with specific quantity and purity that can meet your requirements. Based on our customized solutions and extensive experience, we provide the following four optional services: Endotoxin removal As endotoxin interferes with biological responses, endotoxin removal is critical for in vivo and cell-based applications. BIC understands that a reliable and accurate endotoxin test and removal of endotoxin from your biological sample are extremely important for downstream applications. We offer an endotoxin removal service (detection levels below 1.0 EU/μg). Protein labeling Protein labeling technique refers to the covalent attachment of molecules such as enzymes to specific sites on the target protein. BIC has developed a series of protein labeling techniques, including HRP conjugates, isotope labeling and biotinylation. RNase removal Most lysis methods cause the release of RNA which may cause viscosity problems or interfere with subsequent steps, such as chromatography. Our RNase removal service will solve this problem. Tag-free protein Tags may have an impact on protein structure and biological functions. A recombinant protein with exogenous amino acids has a limited opportunity to pass the FDA approval. Efficient tag-free protein services offered by BIC generate high yields of tag-free protein with the desired amino acid sequence.

If you have any questions, please contact us, and let us see what we can do for you.

Bacterial Expression Systems

Bacterial protein expression systems are advantageous in several important ways, including their fast rate of reproduction, ease of culture, and production of recombinant protein with high yields. As they can grow to high densities with inexpensive media, bacteria are also highly suitable for large-scale fermentations.

Comparison of Other Expression Systems

Characteristics	Mammalian Systems	Bacterial Systems	Yeast Systems
Cost	High	Low	Low
Timeline	Slow	Fast	Slow
Glycosylation	Complex, authentic	No	Simple
Phosphorylation	Yes	No	Yes
Acetylation	Yes	No	Yes

Multi-domain eukaryotic proteins expressed in E.coli often fail to undergo the required post-translational modifications or molecular folding, resulting in loss of functions. E.coli has limited eukaryotic post-translational machinery function, which is considered as a key disadvantage when producing the eukaryotic phosphoproteins. Glycosylation is another complex process of post-translational modification. It is responsible for the formation of cellular glycans which are often attached to proteins and lipids. Glycoproteins, which are commonly distributed in eukaryotic cells, are rarely presented in prokaryotic organisms because cellular organelles essential for glycosylation are missing in these organisms. In addition, if you plan to use your recombinant proteins in animal cells, endotoxin will be a threat. In these cases, you may need endotoxin removal or a mammalian expression system.

Codon Optimization

Most of amino acids are encoded by more than one codon, heterologous genes with the codons which are rarely used in E.coli, may not be efficiently expressed, leading to translation errors. Translation errors arise from rare codon bias and include mistranslational amino acid substitutions, frameshifting events, or premature translational termination. Codon usage optimization and gene synthesis may improve the situation without any side effects.

Inclusion Bodies and Refolding

Inclusion bodies are the main problems when proteins are expressed in the cytoplasm. Cytoplasmic folding is often enhanced at low temperatures; thus, the use of cold temperature is often accompanied by misfolding and segregation into insoluble aggregates known as inclusion bodies. Aggregation can be minimized through the control of parameters such as temperature, expression rate and host metabolism. Though formation of inclusion bodies facilitates protein purification, there is no guarantee that the in vitro refolding will generate large amounts of biologically active products. If possible, protein refolding should be avoided.

Case Study

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