There are two commonly used prokaryotic expression hosts: Escherichia coli and Bacillus subtilis. A comparison of the two is as follows:

Currently, commonly used recombinant protein expression plasmids integrate various components, including replicons, promoters, selection markers, multiple cloning sites (MCS), and fusion protein removal strategies, as shown in Figure 1:

Figure 1: Prokaryotic Expression Vector Map

Scientists at KMD Bioscience have modified the pET vector to obtain a dual-expression plasmid (BiPlasmid vector), which contains dual MCS sites, dual T7 promoters, dual lac operons, and dual ribosome binding sites. Using this plasmid, we can perform a single transfection operation to achieve independent expression of two proteins. Additionally, we have developed the pCold series vectors, which contain low-temperature induction elements for protein expression. These vectors can express high molecular weight proteins, such as a 150kDa active protein in supernatant, under IPTG induction at 16°C.

Affinity Tags: The use of affinity tag fusion in recombinant protein expression serves two main purposes. First, it makes the protein purification process easier. Second, it helps to solubilize certain insoluble proteins. Protein tags can be divided into two categories. One is short peptide tags, and the other is long peptide tags that co-express as fusion proteins (fused partners). The choice of protein tag should be analyzed based on specific cases. KMDhas summarized the properties of some commonly used protein tags, as shown in Table 1.

Recombinant protein expression is often not as smooth as the theory suggests. Various issues may arise during the actual expression process. When faced with difficulties in selecting an expression strategy, seemingly clumsy methods might turn out to be unexpected“successful shortcuts.”For example, when the project requires the expression of two recombinant proteins, constructing six different expression plasmids means there are 36 different expression condition combinations. Through high-throughput small-scale protein expression experiments, it is possible to obtain protein expression condition combinations all at once. The subsequent trial-and-error approach and cost control will become relatively easier.