Q1: What preliminary preparations do we need to make for smooth expression of prokaryotic protein?
A: You can use databases to find out information about proteins, such as molecular weight, signal peptide, transmembrane domains, hydrophilicity, etc. It is also possible to know the precautions for protein expression by referring to the literature, and analyze whether fusion protein expression, labeling and codon optimization are needed.
A: It is advisable to conduct preliminary experiments in advance before conducting large amounts of induced expression. The most suitable condition for protein expression can be obtained through the preliminary experiment of single factor change. The main factors include the concentration of inducer IPTG, induction time and induction temperature.
Q3：Protein soluble expression can not come out how to do?
A: In order to promote the soluble expression of proteins, fusion labels can be added to the N-terminal or C-terminal of the target proteins, such as GST and SUMO labels. According to the downstream application of the protein, the need for label removal can be determined.
Q4：What about inclusion of body expression in the experiment?
A: Inclusion body expression does not matter if the protein is not required to be active for practical use. However, the subsequent protein purification process needs to be carried out under denaturation conditions.
Q5：Destination sequence with affinity label, but why not hang column? How do we deal with this?
A: There are two common reasons for this. First, the label is short, the target peptide chain is relatively large, and the label is not exposed because it is wrapped. We can improve it by adding a linker between the label and the target fragment. Second, the structure is not correct, there is a high degree of aggregation, and the label is not exposed, we can change the construction strategy, or optimize the buffer, so that the label is fully exposed.
Q6：How can protein degradation during purification be improved?
A: We need to deal with it according to when degradation occurs. If it occurs in the culture process, we need to optimize the culture conditions, which may be due to the excessive abundance of nutrients, etc., which can be improved by optimizing the culture medium and culture temperature; If we rule out degradation during culture, then it may be degradation during purification. The solution is to add protease inhibitors and a low-temperature treatment environment to reduce the physical shear force.
Q7：What are the common vectors for prokaryotic expression?
A: The use of pET series vectors is most common in prokaryotes, and the inducers are lactose or IPTG, which are also common inducers. pET series carrier has the advantage of various label forms, and is easy to purify; The disadvantage is high expression, which will be easy to form an inclusion body. pGEX-4T-1 vector is also a commonly used prokaryotic expression vector. The whole sequence of the vector contains tac promoter and GST tag sequence, which can highly express the GST tag fusion protein in BL21, Rosstea and other expression strains, and the GST tag of the target protein can be cut off by thrombin, which is convenient for downstream protein purification.