Cell-free synthesis of functional proteins using transcription/translation machinery entrapped in silica sol-gel matrix

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Cell-free synthesis of functional proteins using transcription/translation machinery entrapped in silica sol-gel matrix Kyeong-Ohn Kim, Seong Yoon Lim, Geun-Hee Hahn, Sahng Ha Lee, Chan Beum Park, and Dong-Myung Kim, Biotechnol Bioeng 102(1), 303-307

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Motivation and Methods

Sol-gel created by precipitating TEOS in a basic solution with ammonium hydroxide as a catalyst.

The translation of mRNA into proteins is a complex biological process. Many industrial products, such as pharmaceuticals, are synthesized in living cells, which is an expensive and difficult to optimize process. The development of better cell-free protein synthesis systems is therefore critically important. This paper builds one such system by showing that the cellular synthesis machinery can be immobilized on a sol-gel.

The sol-gels were made from combinations of tetramethoxysilane (TMOS), tetraethoxysilane (TEOS), and methyltrimethoxysilane (MTMOS). The silanes were mixed with an equal volume of 5 mM HCl and ultra-sonicated. The resulting precursors were then mixed first with a neutralizing HEPES-KOH (pH 7.2) buffer in order increase the pH to physiological levels.

The S30 ribosomal protein and its associated components was obtained by synthesis in vivo in E. coli and subsequent purification. A plasmid encoding a variant of a green fluorescent protein (pIVEX2.3d-EGFP) or its PCR products were used as the DNA template. The DNA and S30 extract were added to the sol-gel precurosor, after which the sol-gel was formed by curing for 12 h at 4 C. Synthesis was performed by adding a translation-initiating solution (57 mM pH 8.2 HEPES-KOH, 1.2 mM ATP, 0.85 mM dNTPs, 2 mM DTT, 0.17 mg/mL E. coli tRNA mixture, 90 mM potassium glutamate, 80 mM ammonium acetate, 12 mM magnesium acetate, 34 ug/mL folinic acid, 1.5 mM complete amino acids, 2% PEG, 67 mM creatine phosphate) and incubating the sol-gel at 30 C. The synthesis of a fluorescent protein was used as a read-out.

Kim2009 fig1.jpg

Results

SEM images of TEOS+MTOS sol-gel matrices prepared with different additives. Insets show fluorescent microscopy images after a 50 h protein synthesis reaction. (A) No additives. (B) polyvinyl alcohol. (C) Alginate. (D) Polyethylene glycol. Scale bars = 2 microns.

Cells are not bags filled with dilute enzymes. Rather, their contents are dense and vastly numerous. This limits diffusion and makes it difficult for all components of a reaction to efficiently find each other. The cells often solve this problems by co-localizing the molecules by immobilizing them on a scaffold. This experiment was motivated by the same logic.

The sol-gel immobilized synthesis system was able to synthesize a protein from both plasmid DNA and PCR-amplified linear DNA. However, the efficiency (in terms of both speed of synthesis and final product amount) of immobilized system was never directly compared to a solution-based system, which made it unclear whether a true improvement in the process was achieved.

The most interesting result was that certain sol-gel additives could affect the amount of protein synthesized. For instance polyethylene glycol (PEG) lead to a 250%-350% increase in protein synthesis. Since the addition of PEG also led to a significantly larger sol-gel pore size, the authors hypothesized that certain pore sizes were optimal for solid-phase in vitro translation. However, no experiments were performed to specifically test this hypothesis while controlling for reaction chemistry. The additives did not affect synthesis in a solution-phase reaction, suggesting that the affects were not the result of a direct interaction between the additives and the synthesis machinery.

Relevance to Soft Matter: Sol-Gels

This paper demonstrated the use of sol-gels in a biotechnology arena. Sol-gels consist of a gel-like network of solid particles in a liquid phase (solution, or sol). The geometries of the solid phase can vary widely: in general, base-catalyzed solutions cause the particles to self-assemble into colloids; while acid-catalyzed reactions produce 3D branched polymer networks. Commonly used precursors include metal alkoxides and metal chlorides, and the most common reactions for the sol-gel phase separation catalysis are polycondensation and hydrolysis. Sol-gels have been used to make ceramics, and are often relied on in biosensor, drug release, and optoelectronic applications.