期刊
JALA
卷 19, 期 4, 页码 366-374出版社
SAGE PUBLICATIONS INC
DOI: 10.1177/2211068213501497
关键词
microreactors; protein synthesis; cell free; vertical orientation; array
资金
- National Science Foundation [OISE-0968313]
- Defense Advanced Research Projects Agency (DARPA)
- University of Florida
- Office Of Internatl Science &Engineering
- Office Of The Director [968313] Funding Source: National Science Foundation
Cell-free protein synthesis (CFPS) has been used as an alternative to cell-based recombinant technology for protein production in academic and industrial labs. The continuous-exchange format generally has higher expression yield by constantly supplying a nutrient solution and removing inhibitory by-products through a porous membrane. Because of the concern of possible membrane clogging by large molecules in the CFPS solution, we investigated the effects of membrane orientation on protein synthesis. We fabricated a miniaturized array device called Vertical-I with its membrane oriented vertically in reference to the table surface and found that the protein synthesis yield in the Vertical-I device was 144% higher than the Horizontal Device reported previously. The reaction time was also faster; beta-glucuronidase reached the synthesis yield plateau after 2 h in the Vertical-I device versus 4 h in the Horizontal Device. Possible clogging of membrane pores was confirmed by fluorescein diffusion measurement. Using these results, we designed a device called Vertical-II that would fit into a 96 well plate holder for compatibility with commercial reagent dispensers and microplate readers. The experimentally optimized device increased protein expression 406% over the Horizontal Device and consumes 5 times fewer reagents than a commercial device, showing the potential for high-throughput protein synthesis.
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