Name: residue-specific incorporation of noncanonical amino acids into model proteins using an escherichia coli cell-free transcription-translation system
Uploaded: 2016-08-01T15:00:00
Duration: 11 min 47 s
Description: 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

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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 protocol35 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)&#160; (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 E. coli, 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&#160;</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&#160;</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&#223;-Bieberau / Rodau, Germany</td>
			<td>TG 81610</td>
			<td></td>
		</tr>
		<tr>
			<td>SDS running buffer (10 x concentrate, 5000 ml)</td>
			<td>Anamed Elektrophorese, Gro&#223;-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&#223;-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&#160;</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, 0.1mm dia.</td>
			<td>Biospec, Bartlesville, USA</td>
			<td>11079101</td>
			<td></td>
		</tr>
		<tr>
			<td>BL21 Rosetta 2 E. coli 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&#252;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, 1.5ml</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&#252;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>1L centrifuge bottle</td>
			<td>Beckman-Coulter, Brea, USA</td>
			<td>A98813</td>
			<td></td>
		</tr>
		<tr>
			<td>4L 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 centrifuge equivalent being 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 shaker equivalent being 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 5000 x g rotor equivalent for above centrifuge equivalent being able to centrifuge 1L-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 centrifuge equivalent being 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 heating block equivalent being able to heat samples in reaction tubes up to 100 &#176;C</td>
		</tr>
		<tr>
			<td>Eppendorf MiniSpin centrifuge</td>
			<td>Eppendorf, Hamburg, Germany</td>
			<td>5452000018</td>
			<td>Or centrifuge equivalent being able to centrifuge 2 ml reaction tubes.</td>
		</tr>
		<tr>
			<td>IKA Vortex 3 (4 mm orbital shaker diameter, 0 - 2500 rpm)</td>
			<td>Sigma-Aldrich, St. Louis, USA</td>
			<td>Z654760</td>
			<td>Or vortex equivalent</td>
		</tr>
		<tr>
			<td>Scotsman AF103 ice flaker machine</td>
			<td>K&#228;lte-Berlin, Berlin, Germany</td>
			<td>AF103</td>
			<td>Or ice flaker machine equivalent</td>
		</tr>
		<tr>
			<td>MyTemp mini digital incubator</td>
			<td>Sigma-Aldrich, St. Louis, USA</td>
			<td>Z763314</td>
			<td>Or incubator equivalent being able to heat samples at 29 &#176;C</td>
		</tr>
		<tr>
			<td>EcoCell electrophoresis cell / chamber</td>
			<td>Anamed Elektrophorese, Gro&#223;-Bieberau / Rodau, Germany</td>
			<td>AN12005</td>
			<td>Or electrophoresis chamber equivalent being able to perform vertical gel electrophoresis with above precast gels or other used gels</td>
		</tr>
		<tr>
			<td>Power-phor power supply for electrophoresis cell / chamber</td>
			<td>Anamed Elektrophorese, Gro&#223;-Bieberau / Rodau, Germany</td>
			<td>AN12001</td>
			<td>Or power supply equivalent being 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 &#181;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 &#181;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 - 1000 &#181;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 &#181;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&#160;</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&#160;</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 microplate reader equivalent being 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&#160;</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 ...

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