Artificial Protein Synthesis

Why would we need more amino acids?

Several good functional groups are missing in the 20 natural amino acids: redox, double/triple bonds, ketone, ferrosyn

Why do we need artificial/post-translationally modified proteins?

• Therapeutic Proteins: Often modified for enhanced stability and pharmacokinetics, i.e. through pegylation.

• Protein-drug conjugates. Conjugates of proteins (commonly antibodies) and small molecule warheads.

• cells directly express therapeutic proteins

• artificial organism?

• More chemistry

How do we make artificial proteins?

chemoselective ligation or bio-orthogonal chemistry

Ribosomal Protein Synthesis

Translation of an mRNA message into a protein by the ribosome

Comparison of Amino Acid Activation in Cells to Chemical Carboxyl Activation

esters from acyl chloride (equivalent)

post-synthetase modification

alter amino acid AFTER charging tRNA - no further proofreading

manipulate traditional translational machinery

amber codon hijacks RF1, which usually inserts water, insert an orthogonal charged tRNA

“orthogonal” translation system

unlimited number of noncanonical; No cross charging!

codon reassignment

new number of terms in codon, such as 4; unlimited noncanonical possibilities

frameshift suppression

occassional 4-nucleotide codons, limited nonnatural mulceo tidw

breaking codon degeneracy

64 codons, >20 amino acids

nonsense suppression

use stop codon; Amber suppression

substrate promiscuity

alter the AA binding pocket to accommodate a slightly different AA

phenylalanyl tRNA-synthetase

Steps to Evolving Orthogonal tRNA/aaRS Pairs for Unnatural AA’s (UAA)

negative selection: endogenous aaRS charging tRNA kills cell (toxic barnase)

positive selection; orthogonal aaRS charging tRNA keeps cell alive (beta-lactamase amber)

positive selection; selects for mutant aaRS amino acylated tRNA (chloramphenicol acetyl transferase amber)

new nucleic bases

uses pi-stacking

The unnatural nucleoside triphosphates must be available inside the cell

Endogenous polymerases must be able to use the unnatural triphosphates to faithfully replicate DNA containing the unnatural base pair (UBP) within the complex cellular milieu

The UBP must be stable in the presence of pathways that maintain the integrity of DNA.

Unnatural amino acid chemistry

click chemistry