26 April 2010

One of the ambitions of synthetic biology is the design and construction of entirely novel, orthogonal life-forms. Their incompatibility with existing life-forms would not only make such orthogonal systems relatively safe to use, they could also be designed to produce new types of proteins that can be assessed for potential medical and industrial applications. From this perspective, the work of a research group from Cambridge University, in the UK is groundbreaking. They designed a novel system that can incorporate unnatural amino acids in biosynthesis of peptides and proteins far more efficiently than present technologies.

In natural living systems, the cell’s DNA is translated into proteins or peptides in three steps. First of all, the cell copies the genetic code from its DNA to  messenger RNA. The messenger RNA then takes this copy to the part of the cell that produces peptides, the ribosome. On the ribosome so-called triplets, codons of three nucleotides (i.e. the building blocks of the genetic code: A, C, U or G), are linked to an amino acid (see figure). Thus, the ribosome transcribes the genetic code step-by-step in a peptide chain or a protein. There is a total of 64 possible combinations of A, C, U and G in triplets (4³). One of the triplets is the so-called start codon, which makes the transcription process begin. Another three triplets are stop codons that make the process end. That leaves 60 remaining triplets encoding for linkage to amino acids. Most amino acids are encoded by several different triplets. Therefore, the natural system can link only 20 different amino acids instead of 60 (see the table at the bottom of the article).

Solid-phase peptide synthesis (SPPS) was developed in the 1960's as a chemical method for creating peptides and proteins in the lab. This technology can be used to synthesize natural peptides, which are difficult to express in bacteria and to incorporate unnatural amino acids. Although SPPS is relatively simple to apply, there remain some constraints concerning the yield, length of the peptides (a maximum of 70 – 100 amino acids) and the type of peptides and proteins that can be synthesized.

Jason Chin’s research group in Cambridge redesigned several pieces of the cell’s protein-building machinery to construct a so-called orthogonal ribosome. Based on transcription of quadruplets, the orthogonal ribosome contains codons of four bases, and uses the cell’s normal protein translation machinery. This raises the number of possible combinations of A, C, U and G to 256 (4⁴) allowing the system to produce peptides and proteins with unnatural amino acids without the constraints of SPPS.

Chin’s team took the gene that codes for the calcium binding protein calmodulin and synthesized pieces of DNA designed to enhance the capability of the ribosome system, to decode quadruplets. They put this synthesized DNA in the calmodulin gene and integrated unnatural amino acids. This resulted in a protein that is more condensed and stable, allowing the protein to survive in a much wider range of environments.

Chin’s research could lead to new drugs that can be swallowed without being destroyed by the acids in the digestive tract, and to polymers with entirely new characteristics for industrial uses.

Some scientists already warn that the synthesis of new proteins is not without risk. New polymers may interfere with existing cellular processes, and should therefore be carefully assessed before their use outside the lab.

Sources:

Guzman F, Barberis S, Illanes A. Peptide synthesis: chemical or enzymatic. Electronic Journal of Biotechnology [online] 2007; 10(2).

Chin, Jason W. Modular approaches to expanding the functions of living matter. Nature Chemical Biology Vol. 2, nr 6, p. 304-311

Heinz Neumann et al. Encoding multiple unnatural amino acids via evolution of a quadruplet-decoding ribosome. Nature Vol. 464, 441-444 (18 March 2010) | doi:10.1038

Life’s code rewritten in four-letter words, New Scientist 2748, 17 February 2010.