Synthesis of Proteins by Automated Flow Chemistry

Abstract

<p>Ribosomes produce most proteins of living cells in seconds. Here we report highly efficient chemistry matched with an automated fast-flow instrument for the direct manufacturing of peptide chains up to 164 amino acids over 328 consecutive reactions. The machine is rapid - the peptide chain elongation is complete in hours. We demonstrate the utility of this approach by the chemical synthesis of nine different protein chains that represent enzymes, structural units, and regulatory factors. After purification and folding, the synthetic materials display biophysical and enzymatic properties comparable to the biologically expressed proteins. High-fidelity automated flow chemistry is an alternative for producing single-domain proteins without the ribosome.<i></i></p>

Stirring Peptide Synthesis to a New Level of Efficiency

“…Since Route-2 applies much shorter reaction times compared to Route-1, it demonstrated that time is a limiting factor in reaching high conversion in each reaction step. Based on Fick's second law, 7,8,16,17 the temperature was elevated to 90 ºC while maintaining short reaction periods, fast stirring, and low reagent concentrations. Peptide-a was synthesized via Route-3 (30 sec reactions, 90 °C), which is almost identical to Route-2, only that the entire process was performed at 90 °C instead of 30 °C (Fig.…”

“…2,4,5 Increased reaction durations, high reagent concentrations, and elevation of reaction temperature are commonly applied to accelerate reactions on a solid support. 12,17 However, the effect of mixing efficiency on SPPS processes is not commonly discussed. 4,5,16 To perform an efficient SPPS process, one needs to consider all conditions that can affect the parameters related to Fick's second law of diffusion (Equation 1), including mixing, which is setup-dependent.…”

Stirring Peptide Synthesis to a New Level of Efficiency

“…The above results confirm that peptides containing His and Cys can be synthesized by HTFSPS without using special building blocks or deviating from the standard cycle protocol maintaining the high temperature. 17…”

“…2,4,5 Increased reaction durations, high reagent concentrations, and elevation of reaction temperature are commonly applied to accelerate reactions on a solid support. 12,17 However, the effect of mixing efficiency on SPPS processes is not commonly discussed. 4,5,16 To perform an efficient SPPS process, one needs to consider all conditions that can affect the parameters related to Fick's second law of diffusion (Equation 1), including mixing, which is setup-dependent.…”

“…In AFPS, a full cycle for the introduction of amino acids to the peptide is performed in a clave of 40 seconds, which is extremely fast compared to other setups (Fig 1A). 16,17,26 In AFPS the solid support is stationary and a large excess of reagents (6-60 equivalents) is used for coupling, proving that elevated temperature and flow-based mixing only partially suffice when the short reactions are employed. The above strategies show that for accelerating SPPS processes in reactors with poor mixing (decreasing t, Ficks' law), the use of high concentrations is inevitable (increasing C, Ficks' law).…”