Cell-free protein synthesis (CFPS) 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.

Writing in Frontiers in Bioengineering and Biotechnology, Michael W. Black, PhD, and colleagues at California Polytechnic State University report that eliminating the lac operators (lacO) and N-terminal tags in the vector backbone enabled the pET vector to support high titers of protein expression for cell-free protein synthesis and, therefore, produce higher yields.

“Many researchers who commonly express/purify proteins using bacterial systems construct their expression plasmids using the pET vector system. As people transition to cell-free protein synthesis systems, they often use the same plasmids and find that expression is extremely low,” Black says. “This paper identifies regions within the expression cassette of the pET vectors that could be easily changed to improve synthesis in CFPS.”

They began by characterizing NEBExpress, PURExpress, and CFAI-based E. coli extracts. First, they ascertained that the pET30 vector they studied contained a complete T7 promotor because an incomplete promoter decreases sfGFP production. Then they looked at the individual and combined effects of removing the pET30 lacO and the N-terminal tags on superfolder green fluorescent protein (sfGFP) expression.

sfGFP expression improved notably

In each of the three systems, sfGFP expression improved notably when the lacO and N-terminal tags were removed. In the CFAI-based system and the NEBExpress system, removing lacO improved sfGFP expression more than removing the N-terminal tags alone. In contrast, they wrote, “The PURExpress system was less sensitive to the lacO element.” There, sfGFP expression increased significantly when the N-terminal tag was removed.

Analyzing fluorescence data showed maximum yields of sfGPF were more than 1,000 µg/ml in the CFAI system, compared to approximately 800 µg/ml in the NEB Express system and about 200 µg/ml in the PURExpress system.

“These changes improve the pET system so it expresses at a level comparable to other vectors that traditionally have been used for cell-free protein synthesis,” Black says. “DNA elements that are commonly found in bacterial expression vectors may negatively impact protein expression when converting to cell-free systems.”

Therefore, manufacturers wanting to optimize cell-free protein expression levels “could use these results to determine if their vectors include DNA elements or purification tags that may reduce protein yield,” he adds.

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