Optimizing cell free protein expression using PCR templates

Initially commercial DNA-based, coupled (e.g., transcription/translation) cell-free expression systems were optimized to use plasmid DNA containing the gene of interest. To meet the growing demands of using PCR-generated DNA as the template, we developed the TNT T7 Quick for PCR DNA system.

As referenced, for optimal translation the initiation ATG codon should be in the context of a Kozak consensus sequence. (1). The presence of poly (A) tail on the 3´ end of the mRNA template also appears to enhance translation (1).

To investigate how these elements contribute to the optimized expression of a protein in TNT system based on PCR templates, several different forward and reverse primers were used to generate template DNA.

The forward primers contain either:

  • the endogenous APC start codon, (which is not a perfect Kozak consensus sequence)
  • a APC start codon in a perfect Kozak sequence
  • or a minimal T7 promoter and ATG codon

The reverse primers contain either a stop codon or a stop codon followed by a poly(T) 30 tail, which introduces a poly (A) 30 tail into the RNA transcript. Results indicate that the context of the ATG start codon and a poly (A) 30 tail are critical for optimal expression.

To summarize, the presence of a Kozak consensus sequence produces optimal expression, but a nonperfect start codon context can be compensated by the addition of a poly(T)30 tail into the PCR product template. Expression levels were lowest when only using minimal T7 promoter plus ATG start codon were incorporated into the PCR product.

For additional details regarding these experiments visit http://www.promega.com/pnotes/77/9028_19/9028_19.html

References
1. Sachs, A.B. and Varani, G. (2000) Eukaryotic translation initiation: there are (at least) two sides to every story. Nat. Struct. Biol. 7, 356–61.

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Gary Kobs

Strategic Marketing Manager at Promega Corporation
Gary earned his B.S. in Bacteriology, UW-Madison in 1982. From 1982–1986 he served as Research Tech at UW-Madison. From 1986 to the present Gary has been with Promega Corporation serving in many capacities including as the very first editor of Promega Notes. He was also Manager Tech Services and Training, Product Manager Restriction/Modifying Enzymes, Product Manager Protein Analysis, and is now Marketing Manager Protein Analysis.

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