1、For customized recombinant expression services, what preliminary information is needed?
Answer: Preliminary information includes the following points:
(1) Research species: Specific types, such as animals, plants, microorganisms.
(2) Protein sequence information: Whether there is sequence information, technical analysis based on the provided sequence.
(3) Antibody sequence information: Whether there is antibody sequence; if not, reference sequence needs to be searched, and then a plan is devised.
(4) Quotation: Based on the specific sequence information of the project, the difficulty of analysis is determined to provide a final quotation reference.
2、Briefly describe the advantages of your company's platforms?
Answer: Currently, we have two core platforms: the mammalian cell protein expression platform and the B-cell antibody development platform. Our technical team has over 10 years of experience in recombinant protein and antibody expression, with over 2,000 protein expression cases. The project is initially assessed and planned by technical colleagues for free, ensuring attentive service and timely responses.
3、How much quantity can be delivered for proteins and antibodies, and how much purification is performed?
Answer: For recombinant proteins, we generally calculate based on the expression volume. The expression levels vary for each protein, ranging from 50 μg to several milligrams in a fixed expression volume. We deliver all expressed and purified proteins with a purity generally above 85%. If higher purity is required, secondary purification can be performed at an additional cost.
4、How is the pricing for expressing 1 mg of protein?
Answer: For recombinant proteins, we generally calculate based on the expression volume. Since each protein has different characteristics, the expression levels vary in a fixed expression volume. If pricing is based on yield, technical colleagues evaluate the difficulty of protein expression, and the final quotation is determined accordingly.
5、What is the timeline for the protein expression process?
Answer: The timeline is as shown below. It generally starts with gene synthesis (1-2 weeks), vector construction for one week, expression testing (1 week for prokaryotes, 1-2 weeks for eukaryotes), and large-scale production (1 week for prokaryotes, 2-3 weeks for eukaryotes). The total timeline is 3-4 weeks for prokaryotes and 4-5 weeks for eukaryotes.
6、Is there a guarantee for the functional activity of proteins?
Answer: Recombinant expressed proteins cannot be guaranteed for activity before activity validation. We can only ensure that the expressed protein sequence is consistent with the molecular design. Based on our experience, proteins expressed in eukaryotic mammalian cells generally have functional activities similar to native proteins.
7、Can functional activity validation be performed for proteins?
Answer: Since the standards for functional activity testing vary for each protein, involving the development of testing methodologies, including the development of protein-related receptors or other protein detections in cellular pathways, we suggest that researchers perform testing based on their experimental needs or evaluate pricing based on specific testing methodologies.
8、How long can proteins generally be stored?
Answer: Proteins are typically stored at -80°C for 1-3 months. If stored at 4°C, they can be kept for 1 to 2 weeks. For proteins with poor stability in vitro, storage time is shorter.
9、Do proteins retain activity in inclusion bodies?
Answer: Proteins are typically soluble under denaturing conditions, but they have generally lost activity. Inclusion body refolding operations can lead to significant protein loss and reduced yield, and the stability of the refolded protein is also low. It is recommended to consider changing solubility tags or trying a different eukaryotic expression system.
10、Can antibody isotypes be changed?
Answer: Yes, but changing antibody isotypes generally involves altering the antibody framework structure, which may affect antibody expression levels and affinity.
11、If there is target information but you want to check if there are known antibody sequences publicly available, can the sequences be used for recombinant expression?
Answer: We can help customers search databases and assess whether there are suitable antibody sequences for the intended application.
12、How is failure charged for expression?
Answer: Failure is determined based on delivering bands with a clear purpose using SDS-PAGE. Regarding protein degradation issues, optimization will be attempted during the experiment, but complete prevention of protein degradation is not guaranteed. For inclusion body issues, since inclusion bodies are a form of protein present in vivo, they also meet the delivery standards. If the above conditions are not met, the project is considered a failure. We share half of the expression testing fee with the researcher, and the researcher only needs to pay for gene synthesis, vector construction and 50% of the expression testing fee. The remaining 50% of the expression testing fee is not charged to the researcher.
13、If the protein is a transmembrane protein and you want to express the full length, how is it done?
Answer: Full-length expression of transmembrane proteins is generally challenging due to their high overall hydrophobicity. Even if expressed, the expression level will not be too high because the limited cell membrane area is insufficient to support the attachment of too many membrane proteins. Additionally, the yield of purification in the later stages is also low. Our technical colleagues can analyze based on the researcher's protein application sequence and evaluate intracellular or extracellular truncation expression, as membrane protein function mainly depends on extracellular or intracellular structural domains. If full-length protein expression is still required, it is recommended to try a cell-free expression system.
14、Can expression be done in E. coli, yeast, insects, and mammalian cells?
Answer: Expression can be done in all these systems. Currently, our company has the technical capabilities for these platforms. However, considering the optimization of platform expression success rates and cost-effectiveness, we mainly focus on prokaryotic E. coli and eukaryotic mammalian cell expression platforms. Other platforms are gradually being phased out due to their higher costs and longer timelines.
15、Can you work directly with provided expression plasmids?
Answer: Yes, it can be done. Generally, in such cases, the researcher needs to provide plasmid construction maps and sequencing files. If there are no issues confirmed by us, expression testing can proceed, and if expression fails, the remaining payment for expression testing still needs to be collected. Therefore, it is recommended that we synthesize and construct the expression vector, as we usually unify and optimize codons together with the tag, increasing the success rate of expression.
16、Does a large fusion tag affect protein activity?
Answer: Generally, it does not affect activity. Currently used large fusion tags have been determined after a long period of trial and error. Tags like Fc are commonly used in the development of functionally active proteins by many pharmaceutical companies. We have conducted Fc-tagged protein-receptor interaction tests for over 200 different targets and found that activity data is not affected by the tag.