| Comparing to cell-based in vivo protein expression, the cell-free protein synthesis system has great advantages in the aspect of high-throughput expression potential due to its open nature. By converging the studies of cell-free expression using linear template, the improvement of cell-free expression efficiency via the introduction of DNA gel, and the integration of robust inkjet printing technique with cell-free expression platform, this paper had comprehensively explored the high-throughput biosynthesis of proteins in the Escherichia coli derived cell-free protein synthesis system (E. coli-CFPS system).Bacillus subtilis xylanase A was selected as a model protein to fulfill the high efficiency of E. coli-CFPS system. To improve its functional expression level in such system, the second codon of the signal peptide sequence of xylA gene was mutated to AAA triplets. Furthermore, the over-expression of molecular chaperons GroES-GroEL in the E. coli cell extract and the addition of detergent Triton X-100were also adopted to enhance the solubility and activity of the in vitro synthesized xylanase A. With the rational intrinsic manipulation and external modification, a combined strategy was established to increase the functional expression level of xylanase A as much as6.1-fold in CFPS system. This strategy was further applied to produce four other representative enzymes (including alkaline phosphatase, glucose dehydrogenase, penicillin G acylase and lipase) in vitro. The overall expression improvements of these enzymes ranging from3.2-fold to5.3-fold suggested a great potential of our combined strategy in the production of other industrially important enzymes in vitro.For the high-throughput cell-free protein expression, linear PCR template was preferred than plasmid template due to the elimination of the time-consuming gene cloning steps. However, the presence of endonuclease in the cell-free extract could degrade the linear templates and lead to the extremely low expression efficiency. DNA gel technique was introduced into the CFPS system for the improvement of expression efficiency, by covalently immobilizing linear target fragment inside the gel. The oil/water emulsion-based fabrication method was developed to produce DNA gel particles. With this novel approach, the high-throughput fabrication of μm-scaled DNA gel particles was achieved. Using this DNA gel technique, the expression efficiency of xylanase A was improved nearly10-fold comparing to that of the conventional CFPS system directly using linear DNA templates. Further modifications of DNA gel fabrication through colliding and membrane filtering were also evaluated. With these efforts, the DNA gel particles with the scale down to3μM were obtained.With the progresses in CFPS system and DNA gel technique, as well as the established approach for liposome production, a high-throughput method was developed for the construction of μm-scaled cell-free micro-bioreactor, or "artificial cell", which possessed phospholipid bilayer membranes, DNA gel inner core with immobilized target templates and cell-free protein synthesis components. Such "artificial cell" mimics the structure and the function of a nature cell, which was biologically important. Moreover, the "artificial cell" model was extremely useful for the investigation of high efficient cell-free expression in a micro system, especially for membrane proteins.Finally, the basic characteristics of commercial inkjet printer used as a kind of high-throughput tool for biological material deposition was investigated. Combining the CFPS system with inkjet printing technique, the feasibility of the in-vitro high-throughput protein synthesis technique was further demonstrated. |
PDF Full Text Request