Frequently Asked Questions About Recombinant Proteins

1. Why are proteins freeze-dried? What effects does freeze-drying have on proteins?

    Proteins are heat-sensitive. Freeze-drying preserves the activity of the vast majority of proteins, improves their stability, extends their shelf life, and reduces shipping costs.

2. Why are protectants added to protein solutions before freeze-drying? What are the common types of freeze-drying protectants?

     Protectants are used to protect proteins during freeze-drying and storage. Commonly used protectants or stabilizers include sugars, polyols, polymers, surfactants, certain proteins, and amino acids. We typically add 8% (by mass) trehalose and mannitol as freeze-drying protectants. Trehalose effectively prevents changes in the secondary structure of proteins as well as protein unfolding and aggregation during freeze-drying; mannitol is also a widely used freeze-drying protectant and bulking agent that can reduce post-freeze-drying aggregation in certain proteins.

Note: For most proteins, the resuspended solution can only be stored at 4°C for a short period (approximately 1 week). For long-term storage, please first prepare a diluent (which must contain a carrier protein, such as 0.1% BSA, 5% HSA, or 10% FBS), then aliquot and freeze at -20°C or -80°C. Be sure to avoid repeated freeze-thaw cycles, as each cycle causes partial inactivation of the protein.

3. How do I reconstitute the lyophilized powder?

    Please refer to the Certificate of Analysis included with your shipment for specific reconstitution instructions, as not all products are reconstituted under the same conditions. In general, we recommend using sterile water for reconstitution. Add the recommended volume of sterile water to the vial and gently shake to completely dissolve the protein. Do not vortex.

4. Why is the protein product barely visible in my tube?

     Protein products that do not contain carrier proteins or other additives (such as bovine serum albumin (BSA), human serum albumin (HSA), and sucrose) and are lyophilized in a solution with minimal salt content often fail to form a white mesh-like structure. Instead, trace amounts of protein deposit inside the vial during the lyophilization process, forming a very thin or transparent layer of protein that is invisible to the naked eye.

5. How should the species-cross-reactivity of cytokines be determined?

1) With a few exceptions, most human cytokines are active against mouse cells. 

2) Many mouse cytokines can also act on human cells, but their potency may be lower than that of the corresponding human cytokines. 

3) A small number of human cytokines, such as IL-7, exhibit greater potency against mouse cells than their corresponding mouse cytokines. 

4) Cytokines such as interferon, GM-CSF, IL-3, and IL-4 are species-specific and exhibit almost no activity on non-homologous cells. 

5) In contrast, fibroblast growth factors (FGFs) and neurotrophins are highly conserved and exhibit good activity on cells from different animal species.

6. What is a carrier protein?

    Carrier proteins such as HSA or BSA are used to enhance the stability of recombinant proteins and help prevent the product from adhering to the walls of the vial.

7. How should I store recombinant proteins?

     For long-term storage, the protein solution should be aliquoted with a carrier protein (e.g., 0.1% BSA or 0.1% HSA) and stored frozen at -20°C. Please keep in mind that each freeze-thaw cycle may cause protein denaturation. Unless otherwise specified on the certificate of analysis, most recombinant proteins have a shelf life of one year. This guarantee applies provided they are stored under the optimal storage conditions described on the certificate of analysis.

8. How is the amount of recombinant protein determined? Why do the protein quantities obtained from my assays differ from your results?

     We determine the quantity of recombinant proteins using methods such as BCA, SDS-PAGE, and HPLC. Different assays yield different quantification results. Sometimes, if you perform different assays, the differences can be significant. It is also possible for proteins to form aggregates during storage, leading to loss after recombination and centrifugation. We perform quality control testing on every batch, but some vials within the same batch may differ from others (this occurs rarely).

9. How to Distinguish Between Recombinant Proteins, Fusion Proteins, and Natural Proteins

    Recombinant proteins are produced using genetic engineering techniques, typically secreted by the mammary glands of transgenic animals, and play a significant role in the medical field as biopharmaceuticals. Through genetic engineering, mammals themselves can be transformed into “factories for mass-producing drugs.” Method: The gene encoding the therapeutic protein is recombined with regulatory elements—such as the promoter—of a mammary gland protein gene. This construct is then introduced into the fertilized eggs of mammals (since only mammals produce milk) via methods such as microinjection. The fertilized eggs are subsequently implanted into the mother’s uterus, where they develop into transgenic animals. Once the transgenic animals enter the lactation phase, they can produce the required protein drugs through their secreted milk; hence, this system is referred to as an animal mammary bioreactor or breast bioreactor.

    Fusion proteins are also known as “tagged proteins”; commonly used tags include His, GST, and Strep tags. Fusion proteins are produced by using DNA recombination technology to link the gene for the target protein to the fusion protein gene on an expression vector. The protein expressed through this method is a recombinant protein that contains both the target protein and the fusion protein. Fusion protein expression is a strategy for recombinant protein expression, and fusion expression is a specific method.

     Natural proteins exist in nature without any artificial modification or processing, such as the proteins found in soybeans and those on the surface of viruses.