Site-Specific Chemoselective Pyrrolysine Analogues Incorporation Using the Cell-Free Protein Synthesis System

Cell-free protein synthesis (CFPS) is a fast and convenient way to synthesize proteins for analytical studies and applications. CFPS, when equipped with a suitable orthogonal pair, allows for protein-site-directed labeling with desired functionalities such as fluorescent dyes or therapeutic groups that are needed to tailor proteins for analytical applications. In this context, chemo­selective reactive pyrrolysine analogues (CR-OAs) are of particular value, as this class of unnatural amino acids, among other useful properties, covers a wide range of different chemo­selective reactions. In this study, we present a flexible approach that facilitates incorporation of CR-OAs in CFPS systems. In particular, a fairly simple addition of two expression plasmids in our cell-free system, one encoding pyrrolysyl-tRNA synthetase and the other one the target protein, enabled ribosomal synthesis of proteins in the half-milligram range with the pre-installed orthogonal reactivity, easily modifiable by using mild, copper-free bioorthogonal chemistry. Our CFPS system allows rapid and highly customizable expression, as shown by several examples of successful site-directed fluorescence labeling. The feasibility of our CFPS system for protein analytics is further proved by demonstrating the functional integrity of a labeled protein by interaction measurements using microscale thermophoresis

ESI-MS analyses of hSOD1 variants detected on the immunoblot in <b>Figure 2</b>.

<p>The o-aaRS which was used for amino acid incorporation is given in brackets. hSOD1(W33Y) (TyrRS), replicate 1 (A); hSOD1(W33Y) (TyrRS), replicate 2 (B); hSOD1(W33Y) (AzRS3) (C); hSOD1(W33AmF) (AzRS3), replicate 1 (D); hSOD1(W33AmF) (AzRS3), replicate 2 (E); hSOD1(W33AmF) (AzRS3), replicate 3 (F); hSOD1(W33PxF) (PxRS1) (G); hSOD1(W33Bpa) (BpaRS1) (H). For the interpretation of the main peaks (bold mass labels) refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031992#pone-0031992-t002" target="_blank">Table 2</a>; none of the minor peaks (standard mass labels) corresponds to the calculated mass of hSOD1 variants containing either a canonical amino acid at position 33 or an ncAA shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031992#pone-0031992-g001" target="_blank"><b>Figure 1</b></a> (analysis details not shown).</p

In vivo incorporation of Tyr and its analogs by amber suppression.

<p>hSOD1(W33TAG) was expressed in the presence of an o-pair and one of the analogs shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031992#pone-0031992-g001" target="_blank">Figure 1</a> as indicated below. The variant proteins were immunodetected using a monoclonal anti-<i>Strep</i>-tag II antibody (refer to the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031992#s4" target="_blank">Materials and Methods</a> section for experimental details). The average calculated molecular weight of hSOD1(W33X) is 17 kDa. (A) 1, TyrRS/tRNA_CUA + Tyr (replicate 1); 2, AzRS1/tRNA_CUA + AzF; 3, AzRS1/tRNA_CUA + AmF; 4, AzRS6/tRNA_CUA + AzF; 5, AzRS6/tRNA_CUA + PxF; 6, AzRS6/tRNA_CUA + AmF; 7, PxRS1/3SUP-tRNA_CUA + PxF; M; molecular weight marker; +, wild type hSOD1 with a C-terminal <i>Strep</i>-tag II. (B) 1, TyrRS/tRNA_CUA + Tyr (replicate 2); 2, AzRS3/3SUP-tRNA_CUA + AzF; 3, AzRS3/3SUP-tRNA_CUA + Tyr; 4, AzRS3/3SUP-tRNA_CUA + AmF (replicate 1); 5, AzRS3/3SUP-tRNA_CUA + AmF (replicate 2); 6, AzRS3/3SUP-tRNA_CUA + AmF (replicate 3); M and + as indicated in (A); -, empty lane. (C) 1, BpaRS/tRNA_CUA + Bpa; M and + as indicated in (A).</p

Structures of tyrosine and its analogs used in this study.

<p>Structures, names and abbreviations are shown.</p

Details of the orthogonal pairs used for the in vivo incorporation of Tyr analogs into hSOD1(W33TAG).

<p>The pairs consist of <i>E. coli</i> TyrRS, or a mutant descendant, and <i>E. coli</i> amber suppressor tRNA_CUA. The aaRSs are expressed under the strong, constitutive <i>ADH1</i> promoter (refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031992#pone.0031992.s001" target="_blank">Figure S1</a> for plasmid map details).</p><p>*The improved promoter consists of a yeast <i>PGK</i>1 promoter followed by three copies of the <i>E. coli</i> tRNA_CUA gene (with an internal B-box), each flanked by 55 bp upstream and 30 bp downstream sequences of the yeast <i>SUP4</i> gene <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031992#pone.0031992-Chen2" target="_blank">[29]</a>.</p

Activation of Tyr, AzF, and AmF by TyrRS and the AzRSs.

<p>Tyr, AzF and AmF were used at a concentration of 5 mM. No amino acid was added to the negative control (w/o aa). TyrRS (A) was added at 1 µM and AzRS1 (B), AzRS3 (C), and AzRS6 (D) at a concentration of 5 µM. The data for each o-aaRS were collected in one series of experiments (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031992#s4" target="_blank">Materials and Methods</a> for details). The average of duplicate determinations is shown; the bars indicate the discrete values.</p

Activation of Tyr, Bpa, AzF, and AmF by BpaRS.

<p>Tyr, Bpa, AzF and AmF were used at a concentration of 5 mM. In the negative control, the amino acid was omitted (w/o aa). BpaRS was added at a concentration of 3 µM. The data were all recorded in one row of experiments and each value was determined in duplicate. The bars denote the discrete values.</p

ESI-MS analyses of selected hSOD1 variants.

<p>Only variant proteins for which defined mass spectra were obtained are shown. The same hSOD1 variants were detected on the immunoblot in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031992#pone-0031992-g002" target="_blank">Figure 2</a>. The corresponding ESI-MS spectra are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031992#pone-0031992-g003" target="_blank">Figure 3</a>. All hSOD1 variants were found with the N-terminal methionine cleaved off and acetylated alanine at position 2, as reported in the literature <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031992#pone.0031992-Hallewell1" target="_blank">[41]</a>. The occasionally attached sodium ions (+22.99 Da) most probably originated from the <i>Strep</i>-Tactin elution buffer which contained 150 mM NaCl. The buffer was not exchanged during sample concentration in order to avoid protein loss. In some of the protein preparations we found a known disulfide bond (S-S, −2 Da; between C57 and C146 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031992#pone.0031992-Lindberg1" target="_blank">[70]</a>).</p>1<p>All hSOD1 masses were calculated without N-terminal methionine, acetylated alanine at position 2 and with completely reduced cysteines.</p

DataSheet1_Efficient Unnatural Protein Production by Pyrrolysyl-tRNA Synthetase With Genetically Fused Solubility Tags.pdf

Introducing non-canonical amino acids (ncAAs) by engineered orthogonal pairs of aminoacyl-tRNA synthetases and tRNAs has proven to be a highly useful tool for the expansion of the genetic code. Pyrrolysyl-tRNA synthetase (PylRS) from methanogenic archaeal and bacterial species is particularly attractive due to its natural orthogonal reactivity in bacterial and eukaryotic cells. However, the scope of such a reprogrammed translation is often limited, due to low yields of chemically modified target protein. This can be the result of substrate specificity engineering, which decreases the aminoacyl-tRNA synthetase stability and reduces the abundance of active enzyme. We show that the solubility and folding of these engineered enzymes can become a bottleneck for the production of ncAA-containing proteins in vivo. Solubility tags derived from various species provide a strategy to remedy this issue. We find the N-terminal fusion of the small metal binding protein from Nitrosomonas europaea to the PylRS sequence to improve enzyme solubility and to boost orthogonal translation efficiency. Our strategy enhances the production of site-specifically labelled proteins with a variety of engineered PylRS variants by 200–540%, and further allows triple labeling. Even the wild-type enzyme gains up to 245% efficiency for established ncAA substrates.</p

DataSheet1_Characterization of Polymer Degrading Lipases, LIP1 and LIP2 From Pseudomonas chlororaphis PA23.PDF

The outstanding metabolic and bioprotective properties of the bacterial genus Pseudomonas make these species a potentially interesting source for the search of hydrolytic activities that could be useful for the degradation of plastics. We identified two genes encoding the intracellular lipases LIP1 and LIP2 of the biocontrol bacterium Pseudomonas chlororaphis PA23 and subsequently performed cloning and expression in Escherichia coli. The lip1 gene has an open reading frame of 828 bp and encodes a protein of 29.7 kDa whereas the lip2 consists of 834 bp and has a protein of 30.2 kDa. Although secondary structure analyses of LIP1 and LIP2 indicate a dominant α/β-hydrolase-fold, the two proteins differ widely in their amino acid sequences (15.39% identity), substrate specificities, and hydrolysis rates. Homology modeling indicates the catalytic serine in both enzymes located in a GXSXG sequence motif (lipase box). However, LIP1 has a catalytic triad of Ser152-His253-Glu221 with a GGX-type oxyanion pocket, whereas LIP2 has Ser138-His249-Asp221 in its active site and a GX-type of oxyanion hole residues. However, LIP1 has a catalytic triad of Ser152-His253-Glu221 with an oxyanion pocket of GGX-type, whereas LIP2 has Ser138-His249-Asp221 in its active site and a GX-type of oxyanion hole residues. Our three-dimensional models of LIP1 and LIP2 complexed with a 3-hydroxyoctanoate dimer revealed the core α/β hydrolase-type domain with an exposed substrate binding pocket in LIP1 and an active-site capped with a closing lid domain in LIP2. The recombinant LIP1 was optimally active at 45°C and pH 9.0, and the activity improved in the presence of Ca2+. LIP2 exhibited maximum activity at 40°C and pH 8.0, and was unaffected by Ca2+. Despite different properties, the enzymes exhibited broadsubstrate specificity and were able to hydrolyze short chain length and medium chain length polyhydroxyalkanoates (PHAs), polylactic acid (PLA), and para-nitrophenyl (pNP) alkanoates. Gel Permeation Chromatography (GPC) analysis showed a decrease in the molecular weight of the polymers after incubation with LIP1 and LIP2. The enzymes also manifested some polymer-degrading activity on petroleum-based polymers such as poly(ε-caprolactone) (PCL) and polyethylene succinate (PES), suggesting that these enzymes could be useful for biodegradation of synthetic polyester plastics. The study will be the first report of the complete characterization of intracellular lipases from bacterial and/or Pseudomonas species. The lipases, LIP1 and LIP2 are different from other bacterial lipases/esterases in having broad substrate specificity for polyesters.</p