FAQ on Prokaryotic Protein Expression

1. What preliminary preparations are needed to ensure successful prokaryotic protein expression?

Answer: We can consult online databases to determine the protein’s molecular weight, the presence or absence of a signal peptide, transmembrane domains, and hydrophilicity/hydrophobicity; predict the protein’s secondary and tertiary structures; review the literature to summarize precautions for protein expression; analyze whether to express a fusion protein or add a tag; and perform codon optimization.

2. What should be done if protein expression levels are low?

Answer: It is best to conduct preliminary experiments before proceeding with large-scale induced expression. Key factors include the concentration of the inducer IPTG, the induction time, and the induction temperature. Through preliminary experiments involving single-factor variations, we can determine the conditions that yield the highest protein expression levels.

3. What should I do if soluble expression fails?

A: To promote soluble protein expression, try adding a fusion tag to the N-terminus or C-terminus of the target protein; common examples include GST and SUMO tags. However, you must determine whether tag removal is necessary based on the protein’s downstream application.

4. What should I do if inclusions form during expression?

A: If the protein’s intended application does not require biological activity, the presence of inclusions is not a problem. However, the subsequent protein purification process must be carried out under denaturing conditions.

5. The sequence contains an affinity tag, but why isn’t the protein binding to the column? How should we address this?

A: There are two common reasons. First, the tag is short, while the target peptide chain is relatively large; the tag is not exposed because it is buried within the chain. We can improve this by inserting a linker between the tag and the target fragment. Second, the structure is incorrect, leading to high levels of aggregation, so the tag is not exposed. First, we can change the construction strategy; second, we can optimize the buffer to ensure the tag is fully exposed.

6. How can we address protein degradation during purification?

A: We need to address the issue based on when the degradation occurs. If it occurs during cultivation, we need to optimize the cultivation conditions—it may be due to an excess of nutrients, for example—and can be improved by optimizing the culture medium and cultivation temperature. If we have ruled out degradation occurring during cultivation, then it may be occurring during the purification process. The solution is to add protease inhibitors, use a low-temperature environment, and reduce physical shear stress.

7. What are the commonly used vectors for prokaryotic expression?

Answer: The pET series of vectors is the most commonly used in prokaryotic systems, with lactose or IPTG serving as inducers—both of which are common types of inducers. The advantage of the pET series is the variety of tag formats available, which facilitates purification; the disadvantage is that high expression levels can easily lead to the formation of inclusions. The pGEX-4T-1 vector is a commonly used prokaryotic expression vector. Its full sequence contains a tac promoter and a GST tag sequence, enabling high-level expression of GST-tagged proteins in expression strains such as BL21 and Rosstea. Furthermore, the GST tag can be cleaved from the target protein using thrombin, facilitating downstream protein purification.