The expression system used in the generation of recombinant proteins has a considerable effect on the resulting product. The use of E. coli by Genentech to produce recombinant human insulin was an important landmark in the development of active human proteins. As research has moved forwards in the years since this discovery the needs of active recombinant proteins have changed. In some instances, glycosylation and post translation modifications (PTMs) are needed to realise the desired activity for a protein; something that is available through use of eukaryotic expression systems exclusively. This led to the development of both insect, and mammalian (Chinese Hamster Ovary – CHO) systems. For human applications and research, a human expression system is ideal. Proteintech’s HumanKine recombinant proteins are human embryonic kidney cell (HEK293) derived.
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Recombinant proteins derived from a human cell (HEK293) expression system possess many advantages over proteins from bacterial systems such as E. coli, due to their ability to produce human recombinant proteins with native PTMs and glycosylation (like CHO). However, Proteintech’s HumanKine proteins have advantages over proteins derived from other eukaryotic systems such as insect or CHO – these are discussed below.
The final product does not contain any constituent that is either an animal tissue, body fluid or components derived from it. All materials from procurement to final products are stored and handled in dedicated animal free facility. Final product and the process does not involve the use of materials from non- human animal sources or recombinant materials made from non-human animal sources.
No tags are used for expression and purification of proteins. Inclusion of a tag can often result in changes to the structure of the protein of interest. Sometimes a tag interferes with the active site of the protein resulting in altered biological activity. Presence of a tag can increase immunogenicity of some protein, which makes a tag-free recombinant protein more desirable for in vivo applications.
Though CHO and insect cells are eukaryotic, their ability to process human proteins does not match human cells in many cases. For example, human cells can generate more mature Activin A dimers than CHO cells (figure 1).
The breadth of information provided by the company on their recombinant proteins should provide an insight into the depth of validation performed by the company, and therefore the product quality. This information should include bioactivity (with bioassay validation), purity, endotoxin concentration, and SDS-PAGE validation.
The bioactivity of a recombinant protein can be important when comparing products from different companies. They are often provided as an ED50 or EC50 (dose which provides 50% maximal activity – lower is better) in ug/mL or pg/mL. Alternatively, you will see units/mg (higher is better). These concentrations are assay-specific, so whilst they are useful for a comparison of the activity of proteins from different companies, it is always a failsafe to perform parallel benchmarking of competing products to know which will work best in your own applications.
The purity of a recombinant protein has an influence on the overall quality of the final product. It is important to use proteins with the highest purity possible (ideally >95%) to ensure the product you use is as free from other extraneous proteins, endotoxins, or anything else that could interfere with the physiological mechanism of action of your recombinant protein.
Endotoxins in commercially available recombinant proteins have been known to interfere with the biological activity of the protein, or the cultured cells. Endotoxins such as LPS can activate immune cells in culture; even at ‘commercially acceptable levels’ (1). It is important that the recombinant protein you buy is endotoxin-free or has extremely low levels of endotoxins.
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Bioassays are intended to measure the biological activity of a given growth factor or cytokine. In most of the cases, the bioassays are cell-based tests using different indicator cells such as primary cells or cell lines. 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.
The specific activity of a biologically active protein can be determined using the following equation:
1 x 106 / ED50 (ng/mL) = specific activity (units/mg)
The ED50 (AKA EC50) can be found on the CoA for the recombinant protein, but we advise determining the ED50 of a given recombinant protein in your functional assay system.
For additional information on ED50 and its relationship with specific activity, please refer to our Tech Tip.
First, centrifuge the container to concentrate the powder at the bottom of the tube. In general, we recommend reconstitution to a concentration of 0.1 to 1.0 mg/mL. 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 on these documents.
In general, we recommend that lyophilized recombinant proteins be stored at –20°C, but short-term storage at 4°C or room temperature is permissible. For reconstituted protein solutions, we recommend that you make working aliquots containing at least 10 μL of protein solution and store at –20°C to –80°C. Do not allow multiple freeze-thaw cycles. Product-specific instructions for storage of lyophilized and reconstituted recombinant proteins are provided on either the product manual, data sheet, or certificate of analysis (CoA).
Recombinant proteins are usually formulated without carrier proteins or additives (e.g., BSA, HSA, sucrose, etc.). As a result, during lyophilization the protein product may be deposited on the vial as a thin, and sometimes invisible, film instead of a pellet. The size of the pellet, if any, is not directly related to the quantity of the recombinant protein in the vial.
To ensure complete recovery of protein product, before opening a vial of lyophilized recombinant protein, we recommend centrifuging it for 20–30 seconds to drive any protein that may be lodged in the cap or on the side to the bottom of the vial. After reconstitution, you can confirm the presence of product protein by running a small amount on SDS-PAGE. In general, a protein band with expected size should be visible with as little as 10 ng of protein loaded on an acrylamide gel.
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