Expression systems for biobetters
Several groups have been working on ways to develop protein therapeutics in the same way that small-molecule therapeutics
are developed. That is, they introduce systematic changes to the molecule, and assay how the changes affect properties, such
as metabolic stability, affinity, and efficacy. However, protein biochemistry is not as flexible as small-molecule chemistry.
The biological activity of a protein depends on correct three-dimensional structure, and small changes in protein stereochemistry
may dramatically affect protein stability, affinity for its receptor, or efficacy. Moreover, the amino acids that make up
mammalian proteins have a limited capacity for chemical modification. Reactive side groups are found on lysine and on cysteine,
but there is no way to target a single amino acid for modification. Any modification targeted, for instance, to lysine, would
potentially affect all the lysines in the protein, but would be more likely to produce proteins with a heterogeneous mixture
of modified and unmodified sites.
The ability of protein chemists to modify the naturally occurring form of a protein has been advanced by Ambrx of San Diego.
They have developed manufacturing-scale expression systems that allow insertion of nonnatural amino acids into defined positions
in the protein, says Dr. Ho Cho, chief technology officer at Ambrx. The purpose of doing so is two-fold. First it allows
one to introduce targeted changes to the protein, and to determine how these changes affect protein structure and function.
Second, it allows manufacturers to use the nonnatural amino acid as a site that can be selectively chemically modified. The
technology was originally developed in E. coli, and marketed as the ReCode expression system, which stands for Reconstituting Chemically Orthogonal Directed Engineering.
It has since been expanded to eukaryotic cells, (e.g., yeast and CHO cells), and is marketed as the EuCode expression system.
The EuCode expression systems are capable of producing fully glycosylated, multi-subunit proteins, such as antibodies and
blood factors, that incorporate nonnatural amino acids, says Cho.
In the EuCode system, cells contain an orthogonal transfer RNA (O-tRNA) that will read through (suppress) a stop codon called
amber. Ambrx has engineered tRNA synthtases that will amino acylate the O-tRNA with an Ambrx nonnatural amino acid. When the ribosomal
complex encounters the amber stop codon, the amino acylated O-tRNA will insert the nonnatural amino into the elongating protein. The O-tRNA and tRNA sythetases
are orthogonal, because they do not interfere with the cells' endogenous tRNAs, tRNA synthetases, or the incorporation of
naturally occurring amino acids into the protein. In the absence of an Ambryx amino acid, protein synthesis is terminated
at the amber codon.
The expression system is currently used to introduce a single amino acid substitution at a specific site to minimize disturbance
to the rest of the protein, to preserve the protein's normal structure and function. Orthogonal tRNA synthetases have been
developed for >50 nonnatural amino acids that possess unique functional properties. The nonnatural-amino-acid substitution
can be used to explore structure-function relationships in fully glycosylated proteins, or as sites of attachment for effector
molecules to enhance protein function, as in the case of drug-antibody conjugates or improve metabolic stability. The ReCODE
system has been successfully scaled to >50,000 L fermentation with consistant product titers of >5 g/L.
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