E.G. Worst and A. Ott acknowledge financial support by the Deutsche Forschungsgemeinschaft (DFG) within the collaborative research center SFB 1027 as well as Saarland University. E.G. Worst, A. Ott and V. Noireaux further acknowledge financial aid by the Human Frontiers Science Program Organization (HFSPO). The authors thank Tobias Baumann and Stefan Oehm (Institute of Chemistry, Technische Universit&#228;t Berlin) for critical reading.

The authors have nothing to disclose.

An-easy-to use cell-free expression system as a viable strategy to residue-specifically incorporate ncAAs into proteins, is presented. To this end, the crude extract is supplemented with vector DNA coding for the protein of interest, the energy buffer and the corresponding amino acids. Note that the crude extract aliquot volume depends on the crude extract protein concentration34. The cell-free expression efficiency is optimized depending on the vector DNA construction concentration. The volumes of the energy buffer components vary as function of optimized Mg- and K-glutamate concentrations in order to enable high yields of the cell-free expressed model protein.
A preliminary evaluation of the incorporation experiment can be obtained by performing SDS-PAGE of the unpurified cell-free reaction medium. For a more detailed analysis, HPLC-ESI mass spectroscopy is proposed as a means to check for complete, residue-specific incorporation of the ncAA. As a preparation for the latter, spin column systems are used to enable His-tag purification and buffer exchange with the small volumes that we use in this protocol.
Including HPLC-ESI mass spectroscopy, the entire protocol can be performed within 2 days. It does not include any particularly critical steps. However, concentration optimizations of Mg- and K-glutamate as well as of vector DNA are crucial in order to express high yields of the model protein. The use of the highly efficient expression vector pBEST-OR2-Or1-Pr-UTR1-gene_of_model_protein-T500 is strongly recommended. Elution of His-tagged proteins is usually due to high concentration of imidazole (&#62; 150 mM) and other salts such as NaH2PO4 (&#62; 300 mM) or NaCl (&#62; 50 mM) that generate high background noise in mass spectroscopic analysis49. Exchange of such elution buffers with a suitable protein storage buffer stabilizes the model protein and drastically reduces background noise during mass spectroscopic analysis.
As a result, Can replaces Arg at all six positions within the model protein. In the expression system, no Arg residues can be detected. This simplifies the residue-specific incorporation of Arg analogs compared to other expression systems that require further depletion strategies29,30. The presented cell-free approach circumvents the inherent limitations of in vivo approaches that are due to Can toxicity, or the strong dependency on mRNA sequence in single protein production strategies24,31. Contrary to the employed in vitro system, in vivo cleavage of Can to homoserine and hydroxyguanidine does occur31.
However, the cell-free system retains a sufficient amount of Lys to compete with analogs such as Hyl. The HPLC-ESI mass spectroscopic analysis shows that the model protein contains both, the canonical as well as the noncanonical analog in different proportions. The residue-specific incorporation of Lys is possible in general, but for complete substitution, further depletion strategies, or specially engineered aaRS and tRNA optimized for the recognition of ncAAs need to be developed.
We achieved excellent yields of cell-free expressed, modified model proteins by adding the ncAA at the same concentration as the canonical ones. The incorporation efficiency depends on the nature of the ncAA to be incorporated. Even higher yields might still be realizable by optimizing the concentration of the ncAA.
The presented results demonstrate the applicability of the employed system for the residue-specific incorporation of ncAAs as long as they are accepted by the canonical endogenous translational system. For the residue-specific incorporation of specific ncAAs, one further needs to check if the residues of the analogous cAA disturb the expression system.
Cell-free transcription-translation systems can be engineered from different organisms to respond to different demands54. The all E. coli transcription-translation machineries of the here presented cell-free system enable the usage of bacteriophage and E. coli promoters, and they can act in parallel or consecutively in cascades55. The general applicability and usability make the method a potent tool for further research in amino acid toxicity and therapeutic application.

The genetic code is universal to the biosphere. It codes for a set of 20 canonical amino acids, which is sometimes extended by selenocysteine1 or pyrrolysine2. It is the ribosome that translates the genetic code with the help of tRNAs into chains of amino acids that fold into proteins. The functional groups of the canonical amino acids, in combination with posttranslational modifications, contribute to an exceptionally wide range of protein function3,4. In principle, functional limitations due to the limited set of canonical amino acids can be overcome by incorporating further, noncanonical amino acids (ncAAs) that enable new chemistries and new functionalities3,4.
There are two complementary approaches for the incorporation of ncAAs: the site- or the residue-specific incorporation. The former method entails considerable technical difficulties, since the canonical set of aminoacyl-tRNA-synthetases (aaRS) and tRNAs must be expanded by an orthogonal aaRS-tRNA pair that must not interact with the endogenous translation machinery. Based on careful engineering, this approach incorporates the ncAAs as single point mutations at the desired protein sites. Site-specific incorporation of ncAAs is genetically encoded by a codon that is not assigned to a canonical amino acid (cAA), usually a stop codon5-9. This method entails changes in function at a given site rather than across the entire protein10-13.
In contrast, residue-specific incorporation relies on erroneous recognition of the noncanonical amino acid by the canonical translation machinery. The incorporation occurs due to the lack of substrate specificity of the aaRS. The residue-specific incorporation of ncAAs, built on the work of Cohen and coworkers14, has led to important applications3,10, among them bio-orthogonal labeling15-17 of proteins or structure elucidation of proteins in X-ray crystallography18.
As natural aaRS generally prefer their cognate amino acid over an isostructural ncAA, efficient in vivo residue-specific incorporation usually requires an auxotrophic expression host not capable of synthesizing the canonical analog of the ncAA. The host cells are cultivated in growth medium that delivers only a low concentration of the analogous cAA. Its exhaustion in combination with the consecutive supplementation with the ncAA forces the expression host to incorporate the ncAA into the model protein at multiple, canonically prescribed sites. In contrast to the site-specific approach, this generally has a deep impact on the entire protein structure, leading to considerably modified physical and chemical properties of proteins19,20. However, most of the ncAAs are growth inhibitors for the expression host3, as they are incorporated into many other proteins besides those of interest during recombinant gene expression. This clearly limits the in vivo approach. The in vivo incorporation of amino acids that are toxic or have strong influence on the protein structure remains particularly challenging. However, these molecules are among the most promising to engineer proteins with extraordinary functions.
One example is the toxic, noncanonical, naturally occurring L-canavanine (Can), an analog of L-arginine (Arg). It affects and blocks Arg associated regulatory and catalytic reaction pathways, and its presence in the living cell can lead to immediate death3,21-23. Its incorporation into proteins at arginine positions can reduce protein stability21-23. Due to the resulting toxicity, expression of canavanine containing proteins in Escherichia coli (E. coli) and other common expression hosts remains a challenge. For these reasons, complete in vivo incorporation of Can at all Arg positions has appropriately been confirmed only once24, using an elaborated single-protein production system. However, Can has been proposed as an anti-cancer agent25-27, and as a stimulator for autoimmune diseases in humans28. Additionally, it is subject of various studies on its anti-metabolic, antibacterial, antifungal and antiviral properties25. These properties raise a demand for efficient and easy-to-perform methods to express Can containing proteins for pharmaceutic, medical and functional studies.
Although many problems that are connected to in vivo production can be circumvented using cell-free expression systems, in vitro residue-specific approaches have only been poorly explored. The cell-free residue-specific incorporation of an L-tryptophan analog29 and multiple ncAAs30 have been reported. These methods are based on the highly efficient T7 RNA polymerase. The T7 RNA polymerase entails bacteriophage-like transcription, thereby reducing genetic functionality in comparison to endogenous transcription.
The complete residue-specific incorporation of Can into a model protein at all Arg positions was recently reported31, using a cell-free expression system32. A slight modification of the same system enabled site-specific incorporation of different pyrrolysine analogs into a model protein via stop codon suppression33. The employed cell-free system31-33 is based on an all E. coli transcription-translation system. Nevertheless, it enables protein expression as efficiently as in current bacteriophage systems (0.5 - 1 mg/ml of recombinant protein)32, while retaining much of the original transcription-translation modularity.
In this work, a detailed protocol is provided on how the residue-specific incorporation of ncAAs can be realized, using this all E. coli cell-free system32. Additionally, further steps to prepare the expressed proteins for appropriate evaluation via HPLC-ESI mass spectroscopy are proposed. To expand the properties of this cell-free system, this work does not only refer to the published incorporation of Can31 but also presents new data related to the noncanonical L-lysine analog L-hydroxy-lysine.
The following protocol for the residue-specific incorporation of ncAAs is an adaptation of a protocol recently published in JoVE34. The latter protocol describes how to perform highly efficient cell-free expression with standard amino acids. Furthermore, it presents the preparation of the crude cell free extract, the amino acid solution, the energy stock solution and the energy buffer used in this approach. The following protocol focuses on modified steps in comparison to the previous protocol in order to enable the residue-specific incorporation of ncAAs. Calibrated pipets, low-binding pipette tips and micro-centrifuge tubes are recommended for the preparation. In the following, IUPAC abbreviations for the amino acids are used.

<table><tbody><tr><td>Protective eyewear</td><td>Sigma-Aldrich, St. Louis, USA</td><td>Z758841</td><td></td></tr><tr><td>Nitrile gloves (size S)</td><td>Sigma-Aldrich, St. Louis, USA</td><td>Z768960</td><td>Catalog numbers other sizes: Z768979 for M, Z768987 for L and Z768995 for XL</td></tr><tr><td>Eppendorf Safe-Lock Tube 1.5 ml (PCR clean)</td><td>Eppendorf, Hamburg, Germany</td><td>30123.328</td><td></td></tr><tr><td>Microbalance Discovery DV114CM</td><td>Ohaus, Greifensee, Switzerland</td><td>80104140</td><td></td></tr><tr><td>Microspatula (L 6 5/8 in., stainless steel, rod diam. 0.09 in.)</td><td>Sigma-Aldrich, St. Louis, USA</td><td>Z243213</td><td></td></tr><tr><td>L-Canavanine</td><td>Sigma-Aldrich, St. Louis, USA</td><td>C9758</td><td>Acute toxicity: wear eyeshields, dust mask, protective gloves</td></tr><tr><td>Hydroxylysine (racemic mixture)</td><td>Sigma-Aldrich, St. Louis, USA</td><td>H0377</td><td></td></tr><tr><td>Cryo-gloves (size S, water resistent)</td><td>Sigma-Aldrich, St. Louis, USA</td><td>Z183490</td><td>Catalog numbers other sizes: Z183512 for M, Z183520 for L and Z183539 for XL</td></tr><tr><td>RTS Amino Acid Sampler</td><td>Biotechrabbit, Hennigsdorf, Germany</td><td>BR1401801</td><td>For homemade preparation of amino acid stock solutions, follow this protocol&lt;sup&gt;35&lt;/sup&gt; and use the solid amino acid kit LAA21-1KT, L-proline (81709-25G), L-cysteine (30089-25G), L-histidine (53319-25G) and L-lysine (L5501-5G)&amp;nbsp; (all from Sigma-Aldrich, St. Louis, USA)</td></tr><tr><td>HEPES</td><td>Sigma-Aldrich, St. Louis, USA</td><td>H6147</td><td></td></tr><tr><td>ATP</td><td>Sigma-Aldrich, St. Louis, USA</td><td>A8937</td><td></td></tr><tr><td>CTP</td><td>Affymetrix, Santa Clara, USA</td><td>14121</td><td></td></tr><tr><td>GTP</td><td>Affymetrix, Santa Clara, USA</td><td>16800</td><td></td></tr><tr><td>UTP</td><td>Affymetrix, Santa Clara, USA</td><td>23160</td><td></td></tr><tr><td>tRNA (from &lt;em&gt;E. coli&lt;/em&gt;, pack size 100 mg)</td><td>Sigma-Aldrich, St. Louis, USA</td><td>10109541001</td><td>Catalog number for pack size of 500 mg is 10109550001</td></tr><tr><td>CoA</td><td>Sigma-Aldrich, St. Louis, USA</td><td>C4282</td><td></td></tr><tr><td>NAD (from yeast )</td><td>Sigma-Aldrich, St. Louis, USA</td><td>N6522</td><td></td></tr><tr><td>cAMP</td><td>Sigma-Aldrich, St. Louis, USA</td><td>A9501&amp;nbsp;</td><td></td></tr><tr><td>Folinic acid</td><td>Sigma-Aldrich, St. Louis, USA</td><td>F7878</td><td></td></tr><tr><td>3-PGA</td><td>Sigma-Aldrich, St. Louis, USA</td><td>P8877</td><td></td></tr><tr><td>Mg-glutamate</td><td>Sigma-Aldrich, St. Louis, USA</td><td>49605</td><td></td></tr><tr><td>K-glutamate</td><td>Sigma-Aldrich, St. Louis, USA</td><td>G1149</td><td></td></tr><tr><td>pBEST-OR2-OR1-Pr-UTR1-deGFP-T500</td><td>Addgene, Cambridge, USA</td><td>Plasmid #40019</td><td></td></tr><tr><td>4-20% precast Tris-Glycine Gels (10 cm x 10 cm x 1 mm, 10 courses)</td><td>Anamed Elektrophorese, Gro&amp;szlig;-Bieberau / Rodau, Germany</td><td>TG 81610</td><td></td></tr><tr><td>SDS running buffer (10 x concentrate, 5,000 ml)</td><td>Anamed Elektrophorese, Gro&amp;szlig;-Bieberau / Rodau, Germany</td><td>TG 50001</td><td></td></tr><tr><td>SDS loading buffer (2 x concentrate, 50 ml)</td><td>Anamed Elektrophorese, Gro&amp;szlig;-Bieberau / Rodau, Germany</td><td>TG 05002</td><td></td></tr><tr><td>Unstained protein marker, broad range (2-212 kDa)</td><td>New England Biolabs, Ipswich, USA</td><td>P7702S</td><td></td></tr><tr><td>Methanol</td><td>Merck, Darmstadt, Germany</td><td>1060091011</td><td>Toxic by inhalation, in contact with skin and if swallowed: wear protective gloves and work under fume hood</td></tr><tr><td>Acetic acid (99.8%)</td><td>VWR International, Darmstadt, Germany</td><td>20104.447</td><td></td></tr><tr><td>Coomassie Blue G-250 (10 g)</td><td>Biozym Scientific, Hessisch Oldendorf, Germany</td><td>902120</td><td></td></tr><tr><td>His-Spin Protein Miniprep kit</td><td>Zymo Research Europe, Freiburg, Germany</td><td>P2002</td><td>Product also distributed by Zymo Research Corporation, Irvine, USA&amp;nbsp;</td></tr><tr><td>Trizma Base</td><td>Sigma-Aldrich, St. Louis, USA</td><td>T1503</td><td></td></tr><tr><td>Hydrochloric acid</td><td>Sigma-Aldrich, St. Louis, USA</td><td>H1758</td><td></td></tr><tr><td>Glycerol, 99%</td><td>VWR International, Darmstadt, Germany</td><td>24397.296DB</td><td></td></tr><tr><td>CentriPure Z25 mini spin columns</td><td>Genaxxon bioscience, Ulm, Germany</td><td>CP-0205-Z100</td><td></td></tr><tr><td>Sodium chloride</td><td>Sigma-Aldrich, St. Louis, USA</td><td>S9888</td><td></td></tr><tr><td>Concentrator 5301</td><td>Eppendorf, Hamburg, Germany</td><td>5301 000.210</td><td></td></tr><tr><td>2xYT</td><td>MP biomedicals, Santa Ana, USA</td><td>113012032</td><td></td></tr><tr><td>Bacto-Agar</td><td>BD Diagnostics, Franklin Lakes, USA</td><td>214010</td><td></td></tr><tr><td>Bead-beating tubes (polypropylene microvials)</td><td>Biospec, Bartlesville, USA</td><td>522S</td><td></td></tr><tr><td>Beads, 0.1&amp;nbsp;mm dia.</td><td>Biospec, Bartlesville, USA</td><td>11079101</td><td></td></tr><tr><td>BL21 Rosetta 2 &lt;em&gt;E. coli&lt;/em&gt; strain</td><td>Merck, Darmstadt, Germany</td><td>71402</td><td></td></tr><tr><td>Bradford BSA protein assay Kit</td><td>Bio-Rad, M&amp;uuml;nchen, Germany</td><td>500-0201</td><td></td></tr><tr><td>Chloramphenicol</td><td>Sigma-Aldrich, St. Louis, USA</td><td>C1919</td><td></td></tr><tr><td>Cuvettes, 1.5&amp;nbsp;ml</td><td>Thermo Fisher Scientific, Waltham, USA</td><td>14-955-127</td><td></td></tr><tr><td>DTT</td><td>Sigma-Aldrich, St. Louis, USA</td><td>D0632</td><td></td></tr><tr><td>Micro Bio-Spin Chromatography Columns</td><td>Bio-Rad, M&amp;uuml;nchen, Germany</td><td>732-6204</td><td></td></tr><tr><td>Nunc 384-well optical bottom plates</td><td>Thermo Fisher Scientific, Waltham, USA</td><td>142761</td><td></td></tr><tr><td>Nunc sealing tape</td><td>Thermo Fisher Scientific, Waltham, USA</td><td>232701</td><td></td></tr><tr><td>PEG-8000</td><td>Promega, Madison, USA</td><td>V3011</td><td></td></tr><tr><td>Potassium phosphate dibasic solution</td><td>Sigma-Aldrich, St. Louis, USA</td><td>P8584</td><td></td></tr><tr><td>Potassium phosphate monobasic solution</td><td>Sigma-Aldrich, St. Louis, USA</td><td>P8709</td><td></td></tr><tr><td>Slide-A-Lyzer dialysis cassettes, 10k MWCO (Kit)</td><td>Thermo Fisher Scientific, Waltham, USA</td><td>66382</td><td></td></tr><tr><td>Spermidine</td><td>Sigma-Aldrich, St. Louis, USA</td><td>85558</td><td></td></tr><tr><td>1&amp;nbsp;L centrifuge bottle</td><td>Beckman-Coulter, Brea, USA</td><td>A98813</td><td></td></tr><tr><td>4&amp;nbsp;L Erlenmeyer flask</td><td>Kimble Chase, Vineland (NJ), USA</td><td>26500-4000</td><td></td></tr><tr><td>Avanti J-26XP centrifuge</td><td>Beckman-Coulter, Brea, USA</td><td>393127</td><td>Or equivalent centrifuge able to centrifuge 1 L bottles.</td></tr><tr><td>Forma 480 orbital shaker</td><td>Thermo Fisher Scientific, Waltham, USA</td><td>480</td><td>Or equivalent shaker able to shake chest-size 6 x 4 L .</td></tr><tr><td>JLA-8.1000 rotor</td><td>Beckman-Coulter, Brea, USA</td><td>363688</td><td>Or equivalent 5,000 x g rotor for the centrifuge above, able to centrifuge 1 L bottles.</td></tr><tr><td>Mini-Beadbeater-1</td><td>Biospec, Bartlesville, USA</td><td>3110BX</td><td></td></tr><tr><td>Microfuge 22R refrigerated microcentrifuge</td><td>Beckman-Coulter, Brea, USA</td><td>368831</td><td>Or equivalent centrifuge able to centrifuge 2 ml reaction tubes.</td></tr><tr><td>Heating block HLC HBT 130</td><td>Labexchange, Burladingen, Germany</td><td>24465</td><td>Or equivalent heating block able to heat samples in reaction tubes up to 100 &amp;deg;C.</td></tr><tr><td>Eppendorf MiniSpin centrifuge</td><td>Eppendorf, Hamburg, Germany</td><td>5452000018</td><td>Or equivalent centrifuge able to centrifuge 2 ml reaction tubes.</td></tr><tr><td>IKA Vortex 3 (4 mm orbital shaker diameter, 0 - 2,500 rpm)</td><td>Sigma-Aldrich, St. Louis, USA</td><td>Z654760</td><td>Or equivalent vortex.</td></tr><tr><td>Scotsman AF103 ice flaker machine</td><td>K&amp;auml;lte-Berlin, Berlin, Germany</td><td>AF103</td><td>Or equivalent ice flaker machine.</td></tr><tr><td>MyTemp mini digital incubator</td><td>Sigma-Aldrich, St. Louis, USA</td><td>Z763314</td><td>Or equivalent incubator able to heat samples at 29 &amp;deg;C.</td></tr><tr><td>EcoCell electrophoresis cell / chamber</td><td>Anamed Elektrophorese, Gro&amp;szlig;-Bieberau / Rodau, Germany</td><td>AN12005</td><td>Or equivalent electrophoresis chamber able to perform vertical gel electrophoresis with the above precast gels or other gels used.</td></tr><tr><td>Power-phor power supply for electrophoresis cell / chamber</td><td>Anamed Elektrophorese, Gro&amp;szlig;-Bieberau / Rodau, Germany</td><td>AN12001</td><td>Or equivalent power supply able to supply above used electrophoresis cell / chamber with power</td></tr><tr><td>VWR Signature Ergonomic High Performance Single-Channel Variable Volume Pipettors Starter Kit 1</td><td>VWR International, Darmstadt, Germany</td><td>613-5278</td><td>Or equivalent micropipettes enabling to pipette similar volumes with the same precision</td></tr><tr><td>VWR Signature Ergonomic High Performance Single-Channel Variable Volume Pipettors Starter Kit 2</td><td>VWR International, Darmstadt, Germany</td><td>613-5279</td><td>Or equivalent micropipettes enabling to pipette similar volumes with the same precision</td></tr><tr><td>Pipetman Tips Diamond D10ST (0.1 - 10 &amp;micro;l)</td><td>Gilson, Middleton, USA</td><td>F171101</td><td>Or equivalent low-binding pipette tips enabling to pipette similar volumes with the same precision</td></tr><tr><td>Pipetman Tips Diamond D200ST (2 - 200 &amp;micro;l)</td><td>Gilson, Middleton, USA</td><td>F171301</td><td>Or equivalent low-binding pipette tips enabling to pipette similar volumes with the same precision</td></tr><tr><td>Pipetman Tips Diamond D1000ST (100 - 1,000&amp;nbsp;&amp;micro;l)</td><td>Gilson, Middleton, USA</td><td>F171501</td><td>Or equivalent low-binding pipette tips enabling to pipette similar volumes with the same precision</td></tr><tr><td>50 ml centrifuge tubes with screw caps (sterile)</td><td>VWR International, Darmstadt, Germany</td><td>50-0156</td><td></td></tr><tr><td>15 ml centrifuge tubes with screw caps (sterile)</td><td>VWR International, Darmstadt, Germany</td><td>525-0150</td><td></td></tr><tr><td>14 ml polypropylene tubes (round bottom, two-position vent stopper, sterile)</td><td>Greiner Bio-One, Frickenhausen, Germany</td><td>187262</td><td></td></tr><tr><td>Discovery BIO Wide Pore C5 HPLC Column (3 &amp;micro;m particle size, L x I.D. 10 cm x 2.1 mm)</td><td>Sigma-Aldrich, St. Louis, USA</td><td>567227-U</td><td></td></tr><tr><td>Agilent 1260 HPLC machine</td><td>Agilent Technologies, Santa Clara, USA</td><td>G1312B</td><td></td></tr><tr><td>6500 Series Accurate-Mass Quadrupole Time-of-Flight (Q-TOF) LC/MS</td><td>Agilent Technologies, Santa Clara, USA</td><td>G6530BA</td><td></td></tr><tr><td>Acetonitrile&amp;nbsp;</td><td>Sigma-Aldrich, St. Louis, USA</td><td>270717</td><td></td></tr><tr><td>FLUOstar Omega microplate reader</td><td>BMG Labtech, Ortenberg, Germany</td><td>415-101</td><td>Or equivalent microplate reader able to measure the fluorescence of the expressed model protein</td></tr><tr><td>Hanna Checker pH meter</td><td>Sigma-Aldrich, St. Louis, USA</td><td>Z351091&amp;nbsp;</td><td></td></tr><tr><td>Formic acid eluent additive for LC-MS</td><td>Sigma-Aldrich, St. Louis, USA</td><td>56302</td><td></td></tr></tbody></table>

residue-specific incorporation of noncanonical amino acids into model proteins using an escherichia coli cell-free transcription-translation system

The canonical set of amino acids leads to an exceptionally wide range of protein functionality. Nevertheless, the set of residues still imposes limitations on potential protein applications. The incorporation of noncanonical amino acids can enlarge this scope. There are two complementary approaches for the incorporation of noncanonical amino acids. For site-specific incorporation, in addition to the endogenous canonical translational machineries, an orthogonal aminoacyl-tRNA-synthetase-tRNA pair must be provided that does not interact with the canonical ones. Consequently, a codon that is not assigned to a canonical amino acid, usually a stop codon, is also required. This genetic code expansion enables the incorporation of a noncanonical amino acid at a single, given site within the protein. The here presented work describes residue-specific incorporation where the genetic code is reassigned within the endogenous translational system. The translation machinery accepts the noncanonical amino acid as a surrogate to incorporate it at canonically prescribed locations, i.e., all occurrences of a canonical amino acid in the protein are replaced by the noncanonical one. The incorporation of noncanonical amino acids can change the protein structure, causing considerably modified physical and chemical properties. Noncanonical amino acid analogs often act as cell growth inhibitors for expression hosts since they modify endogenous proteins, limiting in vivo protein production. In vivo incorporation of toxic noncanonical amino acids into proteins remains particularly challenging. Here, a cell-free approach for a complete replacement of L-arginine by the noncanonical amino acid L-canavanine is presented. It circumvents the inherent difficulties of in vivo expression. Additionally, a protocol to prepare target proteins for mass spectral analysis is included. It is shown that L-lysine can be replaced by L-hydroxy-lysine, albeit with lower efficiency. In principle, any noncanonical amino acid analog can be incorporated using the presented method as long as the endogenous in vitro translation system recognizes it.

This protocol guides through the cell-free residue-specific incorporation of ncAAs into model proteins. It proposes SDS-PAGE for a preliminary evaluation of the incorporation experiment and further steps to prepare the model proteins for an appropriate HPLC-ESI mass spectroscopic analysis.
Here, representative results of the cell-free residue-specific incorporation of the Arg analog Can, as well as the Lys analog L-hydroxy-lysine (Hyl) are presented. The different amino acid solutions, the energy buffer, the vector DNA coding for the model protein and cell-free reactions are prepared as described above. The reference cell-free reaction is provided with the amino acid solution consisting of the 20 cAAs. For each experiment, a negative control cell-free reaction is supplied with the amino acid solution that lacks the canonical analog of the ncAA in question. For each approach, one cell-free reaction expresses the model protein in the presence of the amino acid solution, where the cAA is substituted by the noncanonical analog. His-tag purification, buffer exchange and HPLC-ESI mass spectrometric analysis are executed according to the above described protocol.
The model protein is the C-terminal His-tagged deGFP32, a truncated version of EGFP53. Its one letter amino acid sequence can be found in (Supplemental Material 2). This model protein contains 6 Arg and 18 Lys positions, respectively. The expression vector is pBEST-OR2-OR1-Pr-UTR1-deGFP-T500.
In the case of complete incorporation of the ncAA, one can assume that the negative control reaction does not express deGFP, since one of the 20 cAAs is missing. Contrary, deGFP must be detectable in the other two reactions: the native one in the reference cell-free reaction and the modified protein in the cell-free reaction that is provided with the ncAA.
Figure 1A shows the preliminary SDS-PAGE evaluation of the Can incorporation experiment. The reference cell-free reaction has the highest deGFP expression level. In the cell-free reaction that is provided with Can, deGFP is expressed at slightly lower concentration. No deGFP expression can be detected in the negative control. This SDS-PAGE result is a good indication for a successful incorporation of Can into the target protein deGFP.
To prove the hypothesized complete incorporation of Can into deGFP, both purified model proteins, visualized in Figure 1B, are analyzed through HPLC-ESI mass spectroscopy. Figure 1C shows the deconvoluted mass spectra of the purified deGFP molecules. The deconvoluted mass of deGFP that is expressed in the reference cell-free reaction is 26,192.8 Da. For deGFP expressed in the Can containing cell-free reaction a mass of 26202.5 Da appears. The expected masses for the native deGFP6Arg and the modified deGFP6Can with Arg being fully replaced by Can are 26,193 Da and 26,204 Da, respectively. The mass difference of 1.5 Da for deGFP6Can is within the error of spectrum deconvolution. Thus, the full incorporation of Can into deGFP at all 6 Arg positions is confirmed.
The two peaks of reduced intensity correspond to the native deGFP6Arg and modified deGFP6Can that did not attain their mature fluorophore. The fluorophore is autocatalytically generated by elimination of a H2O molecule, followed by oxidation. This leads to a mass increased by 20 Da if this process does not proceed.
<img alt="Figure 1" src="/files/ftp_upload/54273/54273fig1.jpg" /> 
Figure 1. SDS-PAGE evaluation of the Can incorporation experiment and HPLC-ESI mass spectroscopy of the cell-free expressed and purified deGFP molecules. (A) Preliminary evaluation of the Can incorporation experiment using SDS-PAGE. From left to right: Protein standard, reference cell-free reaction, negative control and cell-free reaction providing Can instead of Arg. (B) SDS-PAGE after His-tag purification and buffer exchange of the expressed deGFP molecules. From left to right: Protein standard, purified deGFP from the reference reaction, purified deGFP from the Can containing reaction. (C) Confirmation of full incorporation of Can by HPLC-ESI mass spectroscopy. The expected masses of the native deGFP and the modified deGFP with Arg fully replaced by Can are 26,193 Da and 26,204 Da, respectively. Each spectrum is normalized to its highest intensity (counts). Peak positions are indicated in Da. For visualization purposes, the gel lanes are extracted from the gel pictures, joined together, are converted into gray scale format, size is optimized and contrast as well as brightness are enhanced. Original gel lanes are presented in Supplemental Material 3. <a href="https://www.jove.com/files/ftp_upload/54273/54273fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Figure 2A shows the preliminary SDS-PAGE evaluation of the Hyl incorporation experiment. In the cell-free reaction that is provided with Hyl, deGFP is expressed. Contrary to the first experiment, a weak deGFP band can be observed in the negative control reaction. This might be due to Lys residues in the cell-free reactions. This enables a faint deGFP expression in the negative control reaction, where neither Lys nor Hyl are added.
For HPLC-ESI mass spectroscopy, the deGFP molecules of the cell-free reaction provided with Hyl are purified and the buffer is exchanged (Figure 2B).
<img alt="Figure 2" src="/files/ftp_upload/54273/54273fig2.jpg" /> 
Figure 2. SDS-PAGE evaluation of the Hyl incorporation experiment. (A) From left to right: Negative control cell-free reaction, cell-free reaction containing Hyl and protein standard. (B) SDS-PAGE after His-tag purification and buffer exchange of the expressed deGFP molecules. From left to right: Purified deGFP from the Hyl containing reaction and protein standard. For visualization purposes, the gel lanes are extracted from the gel pictures, joined together, are converted into gray scale format, size is optimized and contrast as well as brightness are enhanced. Original gel lanes are presented in Supplemental Material 3. <a href="https://www.jove.com/files/ftp_upload/54273/54273fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Figure 3 shows the deconvoluted mass spectrum of purified deGFP molecules. Figure 3A confirms the hypothesis of already present Lys residues in the cell-free reactions. The predominant peak of the spectrum corresponds to the native deGFP (expected mass: 26,193 Da). Again, deGFP molecules of a 20 Da higher mass that did not develop their fluorophore can be detected. The Lys residues are preferentially loaded onto the tRNALys by lysyl-tRNA-synthetase leading to a high expression level of the native deGFP18Lys.
The mass difference between Hyl and Lys is 16 Da. Due to the presence of Hyl that is in competition to the Lys residues all possible deGFP species are generated (deGFP18Hyl, deGFP17Hly+1Lys, &#8230;, deGFP16Hyl+2Lys) (Figure 3B). Admittedly, the peak of deGFP1Hyl+17Lys overlaps with the peak of the native deGFP that did not produce its fluorophore (Figure 3A) and the mass of some peaks differs more than 2 Da from the expected mass. These mass differences can be attributed to high noise due to low amounts of the deGFP species. However, Hyl is generally incorporated by the cell-free system. Further improvements have to be done to abolish Lys residues in the cell-free reactions.
<img alt="Figure 3" src="/files/ftp_upload/54273/54273fig3.jpg" /> 
Figure 3. HPLC-ESI mass spectroscopy of the purified deGFP molecules of the cell-free reaction containing Hyl. (A) The native deGFP18Lys is predominantly detected (expected masses: with fluorophore 26,193 Da, without mature fluorophore 26,213 Da). (B) Magnification reveals the existence of all possible deGFP species (deGFP18Hyl, deGFP17Hly+1Lys, deGFP16Hyl+2Lys,&#8230;, deGFP1Hyl+17Lys). Their expected masses are 26,193 Da + N x 16 Da (N = 1, &#8230;, 18). The spectrum is normalized to its highest intensity (counts). Peak positions are indicated in Da. <a href="https://www.jove.com/files/ftp_upload/54273/54273fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 1" src="/files/ftp_upload/54273/54273supfig1.jpg" />
Supplemental Material 1. Calculation template. In section 2, the preparation of the energy buffer master mix is exemplified using crude extract aliquots of 30 &#181;l and optimal Mg- and K-glutamate concentrations of respectively 3 mM and 30 mM. This example leads to a master mix volume that yields 3 energy buffer aliquots. In section 3, the preparation of the cell-free reaction is exemplified using a 90 nM DNA stock solution leading to an optimal vector DNA concentration of 10 nM in the cell-free reaction. <a href="https://www.jove.com/files/ftp_upload/54273/cell-free_calculations_JoVE_11.03.2016_final.xlsx" target="_blank">Please click here to download this file.</a>
For an appropriate traceability, the use of the calculation template is exemplified by insertion of these typical values that differ from the above example: Crude extract volume: 28 &#181;l, optimal Mg-glutamate: 2 mM, optimal K-glutamate: 40 mM, number of desired buffer aliquots: 100, optimal vector concentration in cell-free reaction: 8 nM, DNA vector stock solution: 150 nM.
First enter 28 &#181;l as crude extract volume into the orange field of the first template section. Then, enter into the second template section 2 mM and 40 mM as optimal Mg- and K-glutamate concentrations into the orange fields. Taking into account the optimal Mg- and K-concentrations, the composition of a 15 &#181;l energy buffer, as well as a corresponding, scaled up 16 &#181;l aliquot is calculated. Below, accordingly enter 100 as desired number of energy buffer aliquots (16 &#181;l). The template adapts the volumes of the different buffer components for the 1,700 &#181;l master mix as follows: 204 &#181;l of 100 mM Mg-glutamate stock solution, 136 &#181;l of 3 M K-glutamate stock solution, 728.73 &#181;l of 14x energy solution, 510 &#181;l of 40% PEG-8000 and 121.27 &#181;l sterile ddH2O. Finally, in the third template section, enter 8 nM and 150 nM as optimal vector concentration in the cell-free reaction and respectively, vector DNA stock solution concentration. The template adapts the volumes of the different components that must be added to the 28 &#181;l of crude extract to finalize the preparation of a 90 &#181;l cell-free reaction as follows: 15 &#181;l of energy buffer, 15 &#181;l of one of the 3 differently composed amino acid solutions, 4.80 &#181;l of vector DNA solution (150 nM), and 27.20 &#181;l of sterile ddH2O.
<img alt="Figure 2" src="/files/ftp_upload/54273/54273supfig2.jpg" /> 
Supplemental&#160;Material 2. One letter amino acid sequence of the model protein deGFP. This model protein contains 6 Arg and 18 Lys positions. <a href="https://www.jove.com/files/ftp_upload/54273/54273supfig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54273/54273supfig3.jpg" /> 
Supplemental Material 3. Full length and unmodified gel lanes that correspond to the gel pictures presented Figure 1 and 2. The gel lanes presented in each individual subfigure are extracted from the same SDS polyacrylamide gel. In Figure 1 and 2 these lanes were joined together for presentation purposes. Molecular weights of the protein standard bands are indicated beside the figures. (A.1) Uncropped gel lanes of Figure 1A. From left to right: Protein standard, reference cell-free reaction, negative control and cell-free reaction providing Can instead of Arg. (A.2) Uncropped gel lanes of Figure 1B. From left to right: Protein standard, purified deGFP from the reference reaction, purified deGFP from the Can containing reaction. (B.1) Uncropped gel lanes of Figure 2A. From left to right: Negative control cell-free reaction, cell-free reaction containing Hyl and protein standard. (B.2) Uncropped gel lanes of Figure 2B. From left to right: Purified deGFP from the Hyl containing reaction and protein standard. <a href="https://www.jove.com/files/ftp_upload/54273/54273supfig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Residue-specific Incorporation of Noncanonical Amino Acids into Model Proteins Using an Escherichia coli Cell-free Transcription-translation System at JoVE.co

Residue-specific Incorporation of Noncanonical Amino Acids into Model Proteins Using an Escherichia coli Cell-free Transcription-translation System

An easy-to-use, cell-free expression protocol for the residue-specific incorporation of noncanonical amino acid analogs into proteins, including downstream analysis, is presented for medical, pharmaceutic, structural and functional studies.

Residue-specific Incorporation of Noncanonical Amino Acids into Model Proteins Using an Escherichia coli Cell-free Transcription-translation System

The canonical set of amino acids leads to an exceptionally wide range of protein functionality. Nevertheless, the set of residues still imposes ...

residue-specific-incorporation-noncanonical-amino-acids-into-model

Research

JoVE Journal

Biology

15.9K Views.  Saarland University. An easy-to-use, cell-free expression protocol for the residue-specific incorporation of noncanonical amino acid analogs into proteins, including downstream analysis, is presented for medical, pharmaceutic, structural and functional studies.

Video: Residue-specific Incorporation of Noncanonical Amino Acids into Model Proteins Using an Escherichia coli Cell-free Transcription-translation System

Optimizing the Genetic Incorporation of Chemical Probes into GPCRs for Photo-crosslinking Mapping and Bioorthogonal Chemistry in Live Mammalian Cells

The genetic incorporation of non-canonical amino acids (ncAAs) via amber stop codon suppression is a powerful technique to install artificial probes and reactive moieties onto proteins directly in the live cell. Each ncAA is incorporated by a dedicated orthogonal suppressor-tRNA/amino-acyl-tRNA-synthetase (AARS) pair that is imported into the host organism. The incorporation efficiency of different ncAAs can greatly differ, and be unsatisfactory in some cases. Orthogonal pairs can be improved by manipulating either the AARS or the tRNA. However, directed evolution of tRNA or AARS using large libraries and dead/alive selection methods are not feasible in mammalian cells. Here, a facile and robust fluorescence-based assay to evaluate the efficiency of orthogonal pairs in mammalian cells is presented. The assay allows screening tens to hundreds of AARS/tRNA variants with a moderate effort and within a reasonable time. Use of this assay to generate new tRNAs that significantly improve the efficiency of the pyrrolysine orthogonal system is described, along with the application of ncAAs to the study of G-protein coupled receptors (GPCRs), which are challenging objects for ncAA mutagenesis. First, by systematically incorporating a photo-crosslinking ncAA throughout the extracellular surface of a receptor, binding sites of different ligands on the intact receptor are mapped directly in the live cell. Second, by incorporating last-generation ncAAs into a GPCR, ultrafast catalyst-free receptor labeling with a fluorescent dye is demonstrated, which exploits bioorthogonal strain-promoted inverse Diels Alder cycloaddition (SPIEDAC) on the live cell. As ncAAs can be generally applied to any protein independently on its size, the method is of general interest for a number of applications. In addition, ncAA incorporation does not require any special equipment and is easily performed in standard biochemistry labs.

A facile fluorescence assay is presented to evaluate the efficiency of amino-acyl-tRNA-synthetase/tRNA pairs incorporating non-canonical amino-acids (ncAAs) into proteins expressed in mammalian cells. The application of ncAAs to study G-protein coupled receptors (GPCRs) is described, including photo-crosslinking mapping of binding sites and bioorthogonal GPCR labeling on live cells.

The genetic incorporation of non-canonical amino acids (ncAAs) via amber stop codon suppression is a powerful technique to install artificial probes and ...

Chemistry

Escherichia coli-Based Cell-Free Protein Synthesis: Protocols for a robust, flexible, and accessible platform technology

Over the last 50 years, Cell-Free Protein Synthesis (CFPS) has emerged as a powerful technology to harness the transcriptional and translational capacity of cells within a test tube. By obviating the need to maintain the viability of the cell, and by eliminating the cellular barrier, CFPS has been foundational to emerging applications in biomanufacturing of traditionally challenging proteins, as well as applications in rapid prototyping for metabolic engineering, and functional genomics. Our methods for implementing an E. coli-based CFPS platform allow new users to access many of these applications. Here, we describe methods to prepare extract through the use of enriched media, baffled flasks, and a reproducible method of tunable sonication-based cell lysis. This extract can then be used for protein expression capable of producing 900 &#181;g/mL or more of super folder green fluorescent protein (sfGFP) in just 5 h from experimental setup to data analysis, given that appropriate reagent stocks have been prepared beforehand. The estimated startup cost of obtaining reagents is $4,500 which will sustain thousands of reactions at an estimated cost of $0.021 per &#181;g of protein produced or $0.019 per &#181;L of reaction. Additionally, the protein expression methods mirror the ease of the reaction setup seen in commercially available systems due to optimization of reagent pre-mixes, at a fraction of the cost. In order to enable the user to leverage the flexible nature of the CFPS platform for broad applications, we have identified a variety of aspects of the platform that can be tuned and optimized depending on the resources available and the protein expression outcomes desired.

Escherichia coli-Based Cell-Free Protein Synthesis: Protocols for a robust, flexible, and accessible platform technology

This protocol details the steps, costs, and equipment necessary to generate E. coli-based cell extracts and implement in vitro protein synthesis reactions within 4 days or less. To leverage the flexible nature of this platform for broad applications, we discuss reaction conditions that can be adapted and optimized.

Over the last 50 years, Cell-Free Protein Synthesis (CFPS) has emerged as a powerful technology to harness the transcriptional and translational capacity ...

Site Specific Lysine Acetylation of Histones for Nucleosome Reconstitution using Genetic Code Expansion in Escherichia coli

Acetylated histone proteins can be easily expressed in Escherichia coli encoding a mutant, N&#949;-acetyl-lysine (AcK)-specific Methanosarcina mazi pyrrolysine tRNA-synthetase (MmAcKRS1) and its cognate tRNA (tRNAPyl) to assemble reconstituted mononucleosomes with site specific acetylated histones. MmAcKRS1 and tRNAPyl deliver AcK at an amber mutation site in the mRNA of choice during translation in Escherichia coli. This technique has been used extensively to incorporate AcK at H3 lysine sites. Pyrrolysyl-tRNA synthetase (PylRS) can also be easily evolved to incorporate other noncanonical amino acids (ncAAs) for site specific protein modification or functionalization. Here we detail a method to incorporate AcK using the MmAcKRS1 system into histone H3 and integrate acetylated H3 proteins into reconstituted mononucleosomes. Acetylated reconstituted mononucleosomes can be used in biochemical and binding assays, structure determination, and more. Obtaining modified mononucleosomes is crucial for designing experiments related to discovering new interactions and understanding epigenetics.

Site Specific Lysine Acetylation of Histones for Nucleosome Reconstitution using Genetic Code Expansion in Escherichia coli

Here we present a method to express acetylated histone proteins using genetic code expansion and assemble reconstituted nucleosomes in vitro.

Acetylated histone proteins can be easily expressed in Escherichia coli encoding a mutant, N&#949;-acetyl-lysine (AcK)-specific Methanosarcina mazi ...

Biochemistry

Engineering 'Golden' Fluorescence by Selective Pressure Incorporation of Non-canonical Amino Acids and Protein Analysis by Mass Spectrometry and Fluorescence

Fluorescent proteins are fundamental tools for the life sciences, in particular for fluorescence microscopy of living cells. While wild-type and engineered variants of the green fluorescent protein from Aequorea victoria (avGFP) as well as homologs from other species already cover large parts of the optical spectrum, a spectral gap remains in the near-infrared region, for which avGFP-based fluorophores are not available. Red-shifted fluorescent protein (FP) variants would substantially expand the toolkit for spectral unmixing of multiple molecular species, but the naturally occurring red-shifted FPs derived from corals or sea anemones have lower fluorescence quantum yield and inferior photo-stability compared to the avGFP variants. Further manipulation and possible expansion of the chromophore&#39;s conjugated system towards the far-red spectral region is also limited by the repertoire of 20 canonical amino acids prescribed by the genetic code. To overcome these limitations, synthetic biology can achieve further spectral red-shifting via insertion of non-canonical amino acids into the chromophore triad. We describe the application of SPI to engineer avGFP variants with novel spectral properties. Protein expression is performed in a tryptophan-auxotrophic E. coli strain and by supplementing growth media with suitable indole precursors. Inside the cells, these precursors are converted to the corresponding tryptophan analogs and incorporated into proteins by the ribosomal machinery in response to UGG codons. The replacement of Trp-66 in the enhanced &#34;cyan&#34; variant of avGFP (ECFP) by an electron-donating 4-aminotryptophan results in GdFP featuring a 108 nm Stokes shift and a strongly red-shifted emission maximum (574 nm), while being thermodynamically more stable than its predecessor ECFP. Residue-specific incorporation of the non-canonical amino acid is analyzed by mass spectrometry. The spectroscopic properties of GdFP are characterized by time-resolved fluorescence spectroscopy as one of the valuable applications of genetically encoded FPs in life sciences.

Synthetic biology enables the engineering of proteins with unprecedented properties using the co-translational insertion of non-canonical amino acids. Here, we presented how a spectrally red-shifted variant of a GFP-type fluorophore with novel fluorescence spectroscopic properties, termed &#34;gold&#34; fluorescent protein (GdFP), is produced in E. coli via selective pressure incorporation (SPI).

Fluorescent proteins are fundamental tools for the life sciences, in particular for fluorescence microscopy of living cells. While wild-type and ...

Bioengineering

Antimicrobial Peptides Produced by Selective Pressure Incorporation of Non-canonical Amino Acids

Nature has a variety of possibilities to create new protein functions by modifying the sequence of the individual amino acid building blocks. However, all variations are based on the 20 canonical amino acids (cAAs). As a way to introduce additional physicochemical properties into polypeptides, the incorporation of non-canonical amino acids (ncAAs) is increasingly used in protein engineering. Due to their relatively short length, the modification of ribosomally synthesized and post-translationally modified peptides by ncAAs is particularly attractive. New functionalities and chemical handles can be generated by specific modifications of individual residues. The selective pressure incorporation (SPI) method utilizes auxotrophic host strains that are deprived of an essential amino acid in chemically defined growth media. Several structurally and chemically similar amino acid analogs can then be activated by the corresponding aminoacyl-tRNA synthetase and provide residue-specific cAA(s) &#8594; ncAA(s) substitutions in the target peptide or protein sequence. Although, in the context of the SPI method, ncAAs are also incorporated into the host proteome during the phase of recombinant gene expression, the majority of the cell&#39;s resources are assigned to the expression of the target gene. This enables efficient residue-specific incorporation of ncAAs often accompanied with high amounts of modified target. The presented work describes the in vivo incorporation of six proline analogs into the antimicrobial peptide nisin, a lantibiotic naturally produced by Lactococcus lactis. Antimicrobial properties of nisin can be changed and further expanded during its fermentation and expression in auxotrophic Escherichia coli strains in defined growth media. Thereby, the effects of residue-specific replacement of cAAs with ncAAs can deliver changes in antimicrobial activity and specificity. Antimicrobial activity assays and fluorescence microscopy are used to test the new nisin variants for growth inhibition of a Gram-positive Lactococcus lactis indicator strain. Mass spectroscopy is used to confirm ncAA incorporation in bioactive nisin variants.

The protocol presents the Escherichia coli-based selective pressure incorporation of non-canonical amino acids (ncAAs) into the lactococcal antimicrobial peptide nisin. Its properties can be changed during recombinant expression via substitution with desired ncAAs in defined growth media. Resulting changes in bioactivity are mapped by growth inhibition assays and fluorescence microscopy.

Nature has a variety of possibilities to create new protein functions by modifying the sequence of the individual amino acid building blocks. However, all ...

Targeting Cysteine Thiols for in Vitro Site-specific Glycosylation of Recombinant Proteins

Stromal interaction molecule-1 (STIM1) is a type-I transmembrane protein located on the endoplasmic reticulum (ER) and plasma membranes (PM). ER-resident STIM1 regulates the activity of PM Orai1 channels in a process known as store operated calcium (Ca2+) entry which is the principal Ca2+ signaling process that drives the immune response. STIM1 undergoes post-translational N-glycosylation at two luminal Asn sites within the Ca2+ sensing domain of the molecule. However, the biochemical, biophysical, and structure biological effects of N-glycosylated STIM1 were poorly understood until recently due to an inability to readily obtain high levels of homogeneous N-glycosylated protein. Here, we describe the implementation of an in vitro chemical approach which attaches glucose moieties to specific protein sites applicable to understanding the underlying effects of N-glycosylation on protein structure and mechanism. Using solution nuclear magnetic resonance spectroscopy we assess both efficiency of the modification as well as the structural consequences of the glucose attachment with a single sample. This approach can readily be adapted to study the myriad glycosylated proteins found in nature.

Targeting Cysteine Thiols for in Vitro Site-specific Glycosylation of Recombinant Proteins

Biochemical and structural analyses of glycosylated proteins require relatively large amounts of homogeneous samples. Here, we present an efficient chemical method for site-specific glycosylation of recombinant proteins purified from bacteria by targeting reactive Cys thiols.

Stromal interaction molecule-1 (STIM1) is a type-I transmembrane protein located on the endoplasmic reticulum (ER) and plasma membranes (PM). ER-resident ...

Immunology and Infection

A Facile Protocol to Generate Site-Specifically Acetylated Proteins in Escherichia Coli

Post-translational modifications that occur at specific positions of proteins have been shown to play important roles in a variety of cellular processes. Among them, reversible lysine acetylation is one of the most widely distributed in all domains of life. Although numerous mass spectrometry-based acetylome studies have been performed, further characterization of these putative acetylation targets has been limited. One possible reason is that it is difficult to generate purely acetylated proteins at desired positions by most classic biochemical approaches. To overcome this challenge, the genetic code expansion technique has been applied to use the pair of an engineered pyrrolysyl-tRNA synthetase variant, and its cognate tRNA from Methanosarcinaceae species, to direct the cotranslational incorporation of acetyllysine at the specific site in the protein of interest. After first application in the study of histone acetylation, this approach has facilitated acetylation studies on a variety of proteins. In this work, we demonstrated a facile protocol to produce site-specifically acetylated proteins by using the model bacterium Escherichia coli as the host. Malate dehydrogenase was used as a demonstration example in this work.

A Facile Protocol to Generate Site-Specifically Acetylated Proteins in Escherichia Coli

Genetic code expansion serves as a powerful tool to study a wide range of biological processes, including protein acetylation. Here we demonstrate a facile protocol to exploit this technique for generating homogeneously acetylated proteins at specific sites in Escherichia coli cells.

Post-translational modifications that occur at specific positions of proteins have been shown to play important roles in a variety of cellular processes. ...

Residue-Specific Exchange of Proline by Proline Analogs in Fluorescent Proteins: How "Molecular Surgery" of the Backbone Affects Folding and Stability

Replacement of proline (Pro) residues in proteins by the traditional site-directed mutagenesis by any of the remaining 19 canonical amino acids is often detrimental to protein folding and, in particular, chromophore maturation in green fluorescent proteins and related variants. A reasonable alternative is to manipulate the translation of the protein so that all Pro residues are replaced residue-specifically by analogs, a method known as selective pressure incorporation (SPI). The built-in chemical modifications can be used as a kind of &#34;molecular surgery&#34; to finely dissect measurable changes or even rationally manipulate different protein properties. Here, the study demonstrates the usefulness of the SPI method to study the role of prolines in the organization of the typical &#946;-barrel structure of spectral variants of the green fluorescent protein (GFP) with 10-15 prolines in their sequence: enhanced green fluorescent protein (EGFP), NowGFP, and KillerOrange. Pro residues are present in connecting sections between individual &#946;-strands and constitute the closing lids of the barrel scaffold, thus being responsible for insulation of the chromophore from water, i.e., fluorescence properties. Selective pressure incorporation experiments with (4R)-fluoroproline (R-Flp), (4S)-fluoroproline (S-Flp), 4,4-difluoroproline (Dfp), and 3,4-dehydroproline (Dhp) were performed using a proline-auxotrophic E. coli strain as expression host. We found that fluorescent proteins with S-Flp and Dhp are active (i.e., fluorescent), while the other two analogs (Dfp and R-Flp) produced dysfunctional, misfolded proteins. Inspection of UV-Vis absorption and fluorescence emission profiles showed few characteristic alterations in the proteins containing Pro analogs. Examination of the folding kinetic profiles in EGFP variants showed an accelerated refolding process in the presence of S-Flp, while the process was similar to wild-type in the protein containing Dhp. This study showcases the capacity of the SPI method to produce subtle modifications of protein residues at an atomic level (&#34;molecular surgery&#34;), which can be adopted for the study of other proteins of interest. It illustrates the outcomes of proline replacements with close chemical analogs on the folding and spectroscopic properties in the class of &#946;-barrel fluorescent proteins.

To overcome the limitations of classical site-directed mutagenesis, proline analogs with specific modifications were incorporated into several fluorescent proteins. We show how the replacement of hydrogen by fluorine or of the single by double bonds in proline residues ("molecular surgery") affects fundamental protein properties, including their folding and interaction with light.

Replacement of proline (Pro) residues in proteins by the traditional site-directed mutagenesis by any of the remaining 19 canonical amino acids is often ...

Cell-Type Specific Protein Purification and Identification from Complex Tissues Using a Mutant Methionine tRNA Synthetase Mouse Line

Understanding protein homeostasis in vivo is key to knowing how the cells work in both physiological and disease conditions. The present protocol describes in vivo labeling and subsequent purification of newly synthesized proteins using an engineered mouse line to direct protein labeling to specific cellular populations. It is an inducible line by Cre recombinase expression of L274G-Methionine tRNA synthetase (MetRS*), enabling azidonorleucine (ANL) incorporation to the proteins, which otherwise will not occur. Using the method described here, it is possible to purify cell-type-specific proteomes labeled in vivo and detect subtle changes in protein content due to sample complexity reduction.

This protocol describes how to perform cell-type-specific protein labeling with azidonorleucine (ANL) using a mouse line expressing a mutant L274G-Methionine tRNA synthetase (MetRS*) and the necessary steps for labeled cell-type-specific proteins isolation. We outline two possible ANL administration routes in live mice by (1) drinking water and (2) intraperitoneal injections.

Understanding protein homeostasis in vivo is key to knowing how the cells work in both physiological and disease conditions. The present protocol ...

Neuroscience

CAPRRESI: Chimera Assembly by Plasmid Recovery and Restriction Enzyme Site Insertion

Here, we present chimera assembly by plasmid recovery and restriction enzyme site insertion (CAPRRESI). CAPRRESI benefits from many strengths of the original plasmid recovery method and introduces restriction enzyme digestion to ease DNA ligation reactions (required for chimera assembly). For this protocol, users clone wildtype genes into the same plasmid (pUC18 or pUC19). After the in silico selection of amino acid sequence regions where chimeras should be assembled, users obtain all the synonym DNA sequences that encode them. Ad hoc Perl scripts enable users to determine all synonym DNA sequences. After this step, another Perl script searches for restriction enzyme sites on all synonym DNA sequences. This in silico analysis is also performed using the ampicillin resistance gene (ampR) found on pUC18/19 plasmids. Users design oligonucleotides inside synonym regions to disrupt wildtype and ampR genes by PCR. After obtaining and purifying complementary DNA fragments, restriction enzyme digestion is accomplished. Chimera assembly is achieved by ligating appropriate complementary DNA fragments. pUC18/19 vectors are selected for CAPRRESI because they offer technical advantages, such as small size (2,686 base pairs), high copy number, advantageous sequencing reaction features, and commercial availability. The usage of restriction enzymes for chimera assembly eliminates the need for DNA polymerases yielding blunt-ended products. CAPRRESI is a fast and low-cost method for fusing protein-coding genes.

Here, we present chimera assembly by plasmid recovery and restriction enzyme site insertion (CAPRRESI), a protocol based on the insertion of restriction enzyme sites into synonym DNA sequences and functional plasmid recovery. This protocol is a fast and low-cost method for fusing protein-coding genes.

Here, we present chimera assembly by plasmid recovery and restriction enzyme site insertion (CAPRRESI). CAPRRESI benefits from many strengths of the ...

Residue-specific Incorporation of Noncanonical Amino Acids into Model Proteins Using an Escherichia coli Cell-free Transcription-translation System

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