Key Questions to Ask When Ordering recombinant proteins

Introduction

Recombinant proteins play a crucial role in cell therapy by providing essential growth factors, cytokines, and signaling molecules that regulate cell proliferation, differentiation, and survival. In cell therapy, recombinant proteins such as interleukins, growth factors, and stem cell factors support the maintenance and functional enhancement of therapeutic cells, ensuring their efficacy in clinical applications.

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This guide provides a step-by-step approach in choosing the best recombinant proteins for your next research project.

1. Define your research objectives

Before selecting a recombinant protein, clearly define your research goals. Questions to consider before getting started:

• What level of purity and bioactivity is required?
• Are you studying a disease pathway, developing a drug, or testing a diagnostic tool?
• What specific biological function must the protein fulfill?

2. Choose the appropriate expression system

Recombinant protein expression systems are biological systems used to produce proteins by introducing a foreign gene (recombinant DNA) into a host organism. These systems enable the large-scale production of proteins for research and is widely used in biotechnology, pharmaceuticals (e.g., insulin, monoclonal antibodies), and vaccine development.

In determining which system to use, your lab should also consider the cell type that produces the protein, promoter, gene of interest, and an induction system to control timing and level of protein expression (like His-tag or GST-tag). The choice of expression system will also impact protein yield, structure, and functionality. Expression system options include:

• Bacterial (E. coli): High yield, fast, and cost-effective, but may lack proper post-translational modifications (PTMs).
• Yeast (Pichia pastoris, Saccharomyces cerevisiae): Suitable for eukaryotic proteins, improved PTMs, and scalability, yet sometimes leads to glycosylation.
• Mammalian (HEK293, CHO cells): Best for human-like PTMs and bioactivity, but more expensive.
• Insect (Sf9, Sf21, baculovirus system): Good for complex proteins with moderate PTMs.
• Plant-based and Cell-free Systems: Scalable, but complex and may lead to emerging technologies with unique advantages.

3. Assess protein purity and bioactivity

A protein’s purity and activity are crucial for reproducible results. Consider the following quality aspects:

• Purity Level: Confirm the percentage of pure protein (e.g., >95% for therapeutic studies).
• Endotoxin Levels: Low endotoxin content (<0.1 EU/µg) is essential for in vivo applications.
• Biological Activity: Verify using functional assays such as enzymatic activity, ligand binding, or cell-based assays.

4. Verify structural and functional integrity

Proper folding and structural integrity influence protein activity, consider the following:

• Post-Translational Modifications: Ensure necessary glycosylation, phosphorylation, or disulfide bonds are present.
• Protein Folding and Aggregation: Check for monomeric forms using SDS-PAGE, SEC-HPLC, or DLS.
• Stability and Storage: Evaluate the protein’s stability at different temperatures and buffer conditions.

5. Choose a supplier and source reliability

Choosing a reliable supplier is critical to the success of your research. In addition to quality, ensuring you have a primary and secondary supplier for your proteins early on, helps avoid problems later down the road. Other metrics to consider:

• Proof of Batch-to-Batch Consistency: Reliable sources provide consistent purity and activity levels.
• Verifying the Certificate of Analysis (CoA): Confirm specifications, testing results, and compliance with regulatory standards.
• Validating a Sample: You are the champion of your research, regardless of the supplier, always validate against your control to get expected results.

6. Review scalability and regulatory compliance

For therapeutic applications, ensure the recombinant protein meets clinical and regulatory standards of not only your region but consider other global regulatory requirements as well. Consider the following:

• Scalability: The expression system should support large-scale production without loss of quality.
• Ancillary Material: If progressing to clinical trials, the protein should be available as Research-grade and/or cGMP-grade proteins.
• Regulatory Approval: Confirm compatibility with FDA, EMA, PDMA, or other relevant regulatory guidelines.

7. Consider key systems today used in research

Cytokines, growth factors, and interleukins play a crucial role in stem cell research by regulating cell proliferation, differentiation, and maintenance. A fast way to choose starting material for your project is to look at some of the most popular proteins being used in stem cell research today. These include:

• Interleukin-7 (IL-7) – Plays a crucial role in the proliferation and differentiation of stem cells, particularly in hematopoiesis and immune regulation.
• Interleukin-15 (IL-15) – Supports the immune system and stimulates T cell, B cell, and NK cell activities..
• Stem Cell Factor (SCF) – Essential for hematopoietic stem cell survival, proliferation, and differentiation.
• Fibroblast Growth Factor-2 (FGF-2 or bFGF) – Promotes self-renewal and maintenance of pluripotency in embryonic and induced pluripotent stem cells.
• Transforming Growth Factor-Beta (TGF-β) – Regulates stem cell differentiation and plays a role in tissue homeostasis and immune modulation.

Summary

Selecting the right recombinant protein for therapeutic research involves careful consideration of expression system, purity, bioactivity, structure, supplier reliability, and regulatory factors. By following this guide, researchers can ensure the best protein choice for their specific application, ultimately leading to more effective and reliable therapeutic discoveries.

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Depending upon the product, the instructions for reconstituting the lyophilized protein are provided in either the product manual, product data sheet, or Certificate of Analysis (COA), which can be found on the product page. We recommend that the container be first centrifuged to concentrate the powder at the bottom of the tube. Most proteins can be reconstituted with the addition of sterile, distilled water. However, the product data sheet or COA will indicate when a diluent other than water is required. Recommended solutions, carrier protein concentrations, and extended storage conditions can also be found within these documents.

Reconstitution to a concentration of 0.1 to 1.0 mg/mL is recommended. For example, for 100 µg of protein, the amount of water that should be added should be between 100 µL and 1 mL, resulting in a protein solution with a concentration between 1 mg/mL and 0.1 mg/mL.

In general, we recommend storing lyophilized recombinant proteins at -20 degrees C upon arrival, but short-term storage at 4 degrees C (up to 6 months) or room temperature (up to 30 days) is permissible.

For reconstituted protein solutions, we recommend storing working aliquots (containing at least 10 µL of protein solution with carrier protein) at -20 degrees C to -80 degrees C for extended storage. Do not allow multipe freeze-thaw cycles.

As these storage conditions are protein-dependent, we do recommend checking the product-specific storage recommendations that are provided in either the product manual, data sheet, or certificate of analysis (CoA).

Carrier proteins help improve the stability of proteins in dilute solutions, extending storage. Protein solutions are generally not very stable when frozen at low concentration. Upon freeze and thaw, some proteins in the solution may stick to the wall of the container, resulting in significant reduction of protein concentration if the starting concentration was low. Carrier proteins are used to reduce such loss. The most commonly used carrier proteins include bovine serum albumin (BSA), human serum albumin (HSA), or fetal bovine serum (FBS). These carrier proteins are generally used at 0.1% concentration. As a rule of thumb, if the concentration of the recombinant protein is less than 0.5 mg/mL, it is a good idea to add some carrier protein.

The recombinant proteins provided by Thermo Fisher Scientific™ are usually produced in different expression systems such as E. coli, insect cells, or mammalian cells. The major differences in recombinant proteins produced in different expression systems are in the post-translational modifications present, such as glycosylation. Recombinant proteins produced in E. coli are not glycosylated. Recombinant proteins produced in insect cells are partially glycosylated without galactose and sialic acid and not branched. Recombinant proteins produced in mammalian cells are fully glycosylated.

Note: Mimic™ Sf9 Insect Cells (a derivative of the Sf9 insect cell line that has been modified to stably express a variety of mammalian glycosyltransferases) can be used for production of complex N-glycans with terminal sialic acid and galactose.

In most cases, glycosylation of a growth factor or cytokine does not affect how it binds to a receptor directly, so its biological activity is not significantly affected by glycosylation in in vitro studies. However, the glycosylated protein is usually less sensitive to protease degradation and exhibits much longer half life in vivo than the same protein without glycosylation. Therefore, for in vivo studies, selecting a recombinant protein produced in a mammalian expression system or insect expression system might be a better choice than the same recombinant protein produced in E. coli.

Bioassays are intended to measure the biological activity of a given growth factor or cytokine. In most cases, the bioassays are cell-based tests using different indicator cells such as primary cells or cell lines. The most commonly used bioassays include cell proliferation assay, chemotaxis assay, cytokine production assay, and cytotoxicity assay. The biological activity of a given cytokine is expressed as ED50, which represents the concentration of the cytokine that induces 50% of the maximum response. This method of expressing potency should only be used for cytokines whose dose-response curves are sigmoidal in shape.

The specific activity of a bioactive protein can be determined using the following equation:

1 x 10E6/ED50 (ng/mL) = specific activity (units/mg)

The ED50 can be found on the Certificate of Analysis (COA) for the recombinant protein, but we advise determining the ED50 of a given recombinant protein in your particular functional assay system.

For additional information on ED50, and its relationship with specific activity, please refer to our Tech Tip:https://assets.thermofisher.com/TFS-Assets/BID/Technical-Notes/converting-ed50-ng-ml-specific-activity-units-mg-tech-note.pdf

Where possible, Thermo Fisher Scientific obtains International Unit (IU) values through multiple side-by-side comparisons of our product(s) against the analogous WHO Reference Standard within our biological activity assay. Performing multiple comparison tests allows us to account for any outliers due to possible variations with the assay (e.g., product, handling, assay protocol, etc.) Using the results of these comparisons, we can provide a reliable quantification of our product's activity in relation to the activity of the WHO Reference Standard.

Information pertaining to whether a specific product has been tested against the WHO Reference Standard can typically be located on the product page or Certificate of Analysis (COA).

Besides the species difference between LIF Recombinant Mouse Protein, Embyonic Stem Cell-Qualified (Cat. Nos. A, A, A) and LIF Recombinant Human Protein (Cat. No. PHC), the other main difference is that ESC-qualified mouse LIF is derived from plant tissue, whereas LIF Recombinant Human Protein is produced in E. coli. ESC-qualified mouse LIF is verified to contain less than 0.005 ng/µg of endotoxin, which is more than ten times lower than that in E. coli-derived recombinant human LIF protein. These two recombinant proteins also have different reconstitution instructions, different storage conditions, and shelf life.