Issues related to protein expression

2026-07-10

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What types of expression systems and protein services does KMD Bioscience offer?

Currently, we provide E. coli, mammalian, yeast, and baculovirus (insect) expression systems. We offer a wide range of production and purification services, including tag removal, lyophilization, endotoxin removal, fermentation, N-terminal sequencing, and QC testing. Additionally, we provide gene synthesis and subcloning services to prepare for protein production.

When should the E. coli expression system be used?

The E. coli expression system remains the most cost-effective and consistent production method available. We are capable of scaling up production from milligrams to grams.Although E. coli does not incorporate post-translational modifications (PTMs) into recombinant proteins, many proteins expressed in E. coli still exhibit native-like activity—as demonstrated by our extensive catalog of active recombinant proteins.However, bacterial cells produce endotoxins, so if you plan to use the protein in animal cell studies or assays, endotoxin removal is highly recommended.

When should the baculovirus (insect) expression system be used?

The baculovirus (insect) expression system is ideal for producing mature, properly folded and processed recombinant proteins. Compared to E. coli, insect cells are capable of incorporating post-translational modifications and are particularly well-suited for expressing secreted proteins.

When should a mammalian expression system be used?

Proteins produced in mammalian cells offer the advantage of containing complete post-translational modifications (such as proper folding, glycosylation, phosphorylation, etc.) of the expressed protein.

Do you use affinity tags to purify my protein? What if I need the tag removed?

Yes, we will use affinity tags (such as His or GST) to purify your protein. For challenging-to-express proteins, we can even try alternative tags (including SUMO or thioredoxin) to improve solubility and expression. If required, we can absolutely cleave the tag and provide tag-free protein as the final product.

Do you use adherent cells or suspension cells?

We exclusively use suspension-adapted cell lines.

Can you express membrane proteins?

Currently, our focus is on intracellular and secreted proteins (e.g., antibodies, cytokines) as well as secretory proteins containing signal peptides.

Can you guarantee the amount of expressed protein?

Unfortunately, this service does not guarantee the expression level or yield of the target protein. We recommend first confirming expression through small-scale testing to determine the optimal cultivation time, followed by expression and purification at a 1 L scale. Based on the results, larger-scale cultivation can then be pursued. We do not guarantee the enzymatic activity or the preservation of the higher-order structure of the target protein.

Can I remove the affinity tag from the protein after purification?

Yes, we use tags that can be cleaved and removed from the purified protein after purification (e.g., thrombin or TEV protease cleavage sequences introduced during gene synthesis—note that some residual amino acids may remain on the protein after cleavage).

My target protein seems to have "disappeared" during purification. What should I do?

Reason	Remedy
The protein may have been degraded by proteases.	Add protease inhibitors to the sample and buffer to prevent proteolytic digestion.Pass the sample through media such as Benzamidine Sepharose 4 Fast Flow to remove trypsin-like serine proteases.
Protein adsorption occurred during sample preparation filtration.	Use filters with different membrane types. Regenerated cellulose, PVDF, and PES typically exhibit low protein binding and serve as a good starting point for determining the most suitable membrane type for your application.
Sample precipitation.	This issue may be caused by salt removal or inappropriate buffer conditions.
Hydrophobic proteins. The proteins remain attached to the ligand.	Use chaotropic agents, polarity-reducing agents, or detergents.

His-tagged protein

Reason	Remedy
The starting material does not contain His-tagged protein.	Verify the presence of His-tagged protein in the starting material, for example, by Western blot analysis.
The elution conditions are too mild (the His-tagged protein remains bound).	Elute by increasing imidazole concentration or decreasing pH.
Protein precipitation occurred in the column.	Reduce sample load or protein concentration by using a linear imidazole gradient instead of step elution. Consider adding detergents or adjusting NaCl concentration.
Nonspecific hydrophobic or other interactions are taking place.	Include a nonionic detergent (e.g., 0.2% Triton X-100) in the elution buffer or modify NaCl concentration.
The His-tagged protein was not fully eluted.	Elute with a larger volume of elution buffer or increase imidazole concentration.
Nickel ions are leaching from the resin.	If sample causes resin stripping (e.g., when purifying His-tagged proteins secreted into eukaryotic cell culture supernatant), switch to Ni Sepharose Excel instead.

Strep-tag® II protein

Reason	Remedy
Protein was detected in the flow-through.	The buffer/sample composition is not optimal. Check the pH and composition of the sample and binding buffer. The pH should typically be pH 7 or higher.
Strep-tag™ II is absent.	Use a protease-deficient E. coli expression strain. Add protease inhibitors during cell lysis.
Strep -tag™ II is inaccessible.	Fuse the Strep-tag™ II with the other protein terminus. Use a different linker.
The ligand is blocked by biotinylated proteins present in the extract.	If purifying extracts containing biotin, add avidin. The biotin content in the soluble fraction of total E. coli cell lysate is approximately 1 nmol per liter of culture (A550 = 1.0). Add 2 to 3 nmol of avidin monomer per nmol of biotin.

GST-tagged protein

Reason	Remedy
Insufficient elution volume.	Increase the volume of elution buffer used. In some cases, particularly after on-column cleavage of tagged proteins, a larger volume of buffer may be required to elute the tagged protein.
Insufficient elution time.	Increase the time available for elution by reducing the flow rate during elution. For GSTrap columns, optimal results are achieved using flow rates of 0.2 to 1 mL/min (for 1 mL HiTrap columns) and 0.5 to 5 mL/min (for 5 mL HiTrap columns) during sample application. For centrifugation methods, reduce the centrifugation speed during elution.
Insufficient glutathione concentration.	Increase the concentration of glutathione in the elution buffer: For most applications, the recommended 10 mM in this protocol should suffice, but exceptions exist. Try using 50 mM Tris-HCl, 20 to 40 mM reduced glutathione, pH 8.0, as the elution buffer.
The pH of the elution buffer is too low.	Increase the pH of the elution buffer. Raising the pH of the elution buffer to 8–9 can improve elution without increasing the glutathione concentration used for elution.
The ionic strength of the elution buffer is too low.	Increase the ionic strength of the elution buffer. Adding 0.1 to 0.2 M NaCl to the elution buffer can also improve results.
The glutathione in the elution buffer is oxidized.	Use fresh elution buffer. Add DTT.
Non-specific hydrophobic interactions lead to non-specific binding with the medium or protein aggregation, thereby preventing the solubilization and elution of the labeled protein.	Include a nonionic detergent in the elution buffer. Adding 0.1% Triton X-100 or 2% n-octyl glucoside can significantly improve the elution of some GST-tagged proteins.

MBP-tagged protein

Reason	Remedy
Factors in the crude extract interfere with binding.	Add glucose to the growth medium to suppress amylase expression.
The MBP tag is absent.	Use a protease-deficient E. coli expression strain. Add protease inhibitors during cell lysis.
The MBP tag is inaccessible.	Fuse an MBP tag to the C-terminus of the target protein. Use a different linker.
Due to high protein concentration, the protein precipitates in the column.	Clean the column according to the instructions. In subsequent runs, reduce the sample load or lower the protein concentration by using a linear gradient elution instead of step elution. Try using a cleaning agent or adjusting the NaCl concentration.If using an MBPTrap HP 1 ml column, switch to a larger MBPTrap HP 5 ml column. This will reduce the final concentration if the same sample amount is applied.For rapid scale-up, connect two or more columns in series by screwing the end of one column into the top of the next. However, note that connecting columns in series will increase backpressure.

KMD Biotech is a leader in protein production with advanced technologies and extensive expertise, offering a wide range of protein expression services across multiple systems, including yeast, prokaryotic (e.g., E. coli), insect, and mammalian cells. Our services are designed to meet diverse needs in scientific research and the biopharmaceutical industry, supporting projects from basic research to drug development. By selecting the optimal expression system tailored to the characteristics of the target protein, we maximize expression levels, stability, and bioactivity to deliver high-quality protein products.Our comprehensive services include gene synthesis, vector construction, host cell transfection, expression optimization, protein purification, and quality control. We provide customized solutions for various protein types and project requirements, including but not limited to recombinant protein expression, antibody production, and functional enzyme preparation.With our high-efficiency protein expression platform, KMD Biotech empowers clients to accelerate scientific discovery and therapeutic development, providing robust support for the translation of research breakthroughs and the advancement of novel drug candidates.

Protein Expression Services

E. coli System

Eukaryotic Protein Expression

Yeast Protein Expression

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