The pCS vector system is an efficient system for expressing recombinant proteins in E. coli at low temperatures, typically 15°C. It is based on the E. coli cold-shock gene cspA, and also includes part of the lac operon regulatory system. It is considered to be complementary to the pET and pBAD systems that are typically used to express recombinant proteins at 37°C.
Temperature can have a significant impact on the folding of recombinant proteins, which in turn affects protein solubility and stability. As such, many proteins that exhibit poor solubility or stability at 37°C show improved properties at lower temperatures. When a recombinant protein is difficult to produce in pET or pBAD at 37°C, the pCS system could be a useful alternative to try.
The gene of interest is placed into the pCS vector downstream of the cspA promoter, lac operator (LacO), and the 5’ UTR of cspA. A short translation enhancing element (TEE) from the N-terminus of cspA is also included.
Transcription from the cspA promoter is very inefficient at 37°C, because of instability of the 5’ UTR at this temperature, but when the host bacteria is shifted to 15°C, the 5’ UTR adopts a highly stable secondary structure. The TEE sequence enhances translation initiation of the gene of interest through a ribosome trapping process.
The plasmid also carries the natural promoter and coding sequence for LacI. The LacI protein acts on the LacO sequence, adjacent to the cspA promoter, to block transcription of the gene of interest. In the absence of IPTG, this inhibition prevents any leaky expression of the downstream gene. Addition of IPTG blocks the inhibitory action of LacI. This, coupled with shifting of the culture from 37°C to 15°C, can efficiently induce gene expression.
For further information about this vector system, please refer to the papers below.