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  • In recent years advances in the field of D protein

    2020-08-03

    In recent years, advances in the field of D-protein synthesis accelerated. The chaperone GroEL/ES was found to catalyze similarly the folding of both the D- and the natural L-form of the E. coli protein DapA (Weinstock et al., 2014). The mirror-image D-version of the ribonuclease barnase was used for screening an aptamer library of natural RNA molecules (Olea et al., 2015). The identified aptamers were prepared as L-RNA copies and in turn acted as inhibitors of the barnase in its natural protein conformation. A crucial step toward the establishment of a self-replicating in vitro system was the synthesis of a chirally mirrored version of the 174-residue African swine fever virus polymerase X, which acts as both an RNA and a DNA polymerase (Wang et al., 2016). This was complemented by a D-version of a heat-stable mutant of Sulfolobus solfataricus DNA polymerase IV (Pech et al., 2017), by which PCR could be performed. This development could have consequences in practical biotechnology applications, such as the selection of aptamers from libraries made of synthetic L-DNA molecules. The ribosomal synthesis of D-proteins is not currently feasible; the best effort undertaken so far was limited to the translational incorporation of two D-amino acids into the nascent protein chain by means of modified ribosomes (Dedkova et al., 2006). We therefore apply solid-phase peptide synthesis (SPPS) in concert with native chemical ligation for the synthesis of D-proteins. Conceptually developed in the 1960s, SPPS routinely allows the synthesis of peptides of about 30–50 INT-777 in length, depending on the actual sequence (Kent, 2009). Native chemical ligation (Dawson et al., 1994) permits the synthetic merger of unprotected peptide fragments in aqueous solution, enabling the synthesis of entire protein domains (Dawson and Kent, 2000, Bondalapati et al., 2016, Kulkarni et al., 2018) and has been used to synthesize proteins of up to some 300 amino acids in length (Wang et al., 2016, Xu et al., 2017). Here, we report the synthesis of a DNA-ligase activity in D-conformation for the production of long stretches of L-DNA from synthetic oligonucleotides. It represents another important piece to the molecular puzzle needed for the establishment of an orthogonal self-replicating system. We did not aim at the production of an enantiomeric molecule for comparative studies of the structures or biochemistry of the D- and L-form, the utility of which has been discussed (Siegel, 1992), but went for the activity mainly.
    Results
    Discussion Synthesis of a DNA-ligase in D-conformation adds another component to the portfolio of proteins required for the assembly of an orthogonal self-replicating system. Currently, chemical synthesis is the sole means available for producing such molecules. While chemical synthesis is rather complex in comparison with the production of recombinant enzyme, it could nevertheless be sufficient for many applications. Relatively large amounts of protein could be produced that could last for multiple reactions in molecular biology systems. Also, once produced, the enzyme is expected to be covalently robust, since it is not susceptible to enzymatic degradation. Any unfolding that occurs over time could be reversed by the process used for protein folding in the first place. However, the chemical synthesis of the large proteins required for ribosomal activity, for example, could be a challenge; enzymes such as aminoacyl-tRNA synthetases are more than 1,000 residues in size. Still, chemically, the task is overall more the number of proteins needed rather than their actual length. Another problem is appropriate protein folding to yield functional proteins. However, it may not be that much of an obstacle for many proteins. In vitro synthesis of very large numbers of proteins yielded a surprisingly high percentage of molecules that were functional or recognized their partners specifically in interaction studies (e.g., Syafrizayanti et al., 2017). Still, assembling a structure such as a ribosome and keeping it in its functional state is likely to be a major aspect of getting a self-replicating molecular system going. In vitro ribosomal assembly and function has been studied (Jewett et al., 2013, Sashital et al., 2014, Earnest et al., 2015), optimizing the yields to be comparable with components purified in vivo. Ultimately, it may be possible to produce mirror proteins through the synthesis of a fully functional mirror ribosome. In principle, an alternative for production could be non-ribosomal peptide synthetase enzymology, which permits incorporation of D-amino acids (Hur et al., 2012). However, this approach is currently still lagging well behind chemical synthesis in terms of yield and length of the products.