CFPS (cell-free protein synthesis) eliminates the need to maintain living cells and allows greater control of reaction conditions than in vivo expression systems, but is limited by its sensitivity to plasmid type. Consequently, the commonly used pET series of bacterial expression vectors are unable to produce the high levels of proteins that manufacturers desire.
Michael W. Black, PhD, and colleagues at California Polytechnic State University argue that eliminating lac operators (lacO) and N-terminal tags in the vector backbone enabled the pET vector to achieve high protein expression levels for cell-free protein synthesis and, therefore, higher yields.
Many scientists who use bacterial methods to express/purify proteins often construct their expression plasmids using the pET vector system. They often use the same plasmids and find that expression is extremely low, according to Black. This paper identifies regions within the pET vectors that may be easily modified to enhance synthesis in CFPS.
The researchers started by characterizing E. coli extracts from NEBExpress, PURExpress, and CFAI. First, they verified that the pET30 vector they studied contained a complete T7 promotor because an incomplete promoter reduces sfGFP production. Then they studied the individual and combined effects of removing the pET30 lacO and the N-terminal tags on sfGFP expression.
When the lacO and N-terminal tags were removed, sfGFP expression improved significantly in both the CFAI-based system and the NEBExpress system, according to the authors, the removal of the lacO element improved sfGFP expression more than the removal of the N-terminal tags alone.
Analyzing fluorescence data revealed that sfGPF maximum yields were greater than 1,000 mg/ml in the CFAI system, compared to around 800 mg/ml in the NEB Express system and about 200 mg/ml in the PURExpress system.
Black adds that these changes improve the pET system, which can express at a level comparable to other cell-free protein synthesis vectors. DNA elements that are commonly found in bacterial expression vectors might disrupt protein expression when converted to cell-free systems.
Therefore, manufacturers who want to improve cell-free protein expression levels may use these findings to investigate if their vectors include DNA elements or purification tags that might reduce protein yield, according to him.