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1 Supporting Information Wiley-VCH Weinheim, Germany
2 A Biosynthetic Route to Dehydroalanine Containing Proteins Jiangyun Wang, Stefan M. Schiller and Peter G. Schultz* Department of Chemistry and The Skaggs Institute for Chemical Biology, The Scripps Research Institute, North Torrey Pines Road, La Jolla, CA 92037, Genomics Institute of The Novartis Research Foundation, John Jay Hopkins Drive, San Diego, CA Materials and Methods General. All chemicals were obtained from commercial sources and used without further purification. 1-Thio-α-D-mannopyranose was synthesized according to literature. [17] Protein mass spectra were acquired at the Scripps Center for Mass Spectrometry (La Jolla, CA). Plasmids and cell lines used. Plasmid pbk-lib5 encodes a library of M. jannaschii tyrosyl trna synthetase (TyrRS) mutants randomized at residues Tyr32, Leu65, Phe108, Gln109, Asp158 and Leu162; His70 is mutated to Gly, and Ala is fixed as either Ala or Gly. Plasmid prep(2)/yc encodes MjtRNA Tyr CUA, the chloramphenicol acetyltransferase (CAT) gene with a TAG codon at residue 112, the GFP gene under control of the T7 promoter, and a Tet r Tyr marker; plasmid plwj17b3 encodes MjtRNA CUA under the control of the lpp promoter and rrnc terminator, the barnase gene (with three amber codons at residues 2, 44 and 65) under the control of the ara promoter, and an Amp r marker. The GFP-UV gene (Clonetech) was inserted into the pet-26b(+) vector (Novagen) to afford the pet-gfp protein expression plasmid. The Gly242TAG mutation was introduced by the quick-change site-directed mutagenesis kit (Stratagene) to afford the pet-gfpx242 protein expression plasmid. The phenylselenocysteine specific synthetase PhSeRS-K4 gene was inserted into the psup vector [18] to afford plasmid psup- PhSeRS-K4. BL21(DE3) cells from Novagen were used for protein expression. Genetic selection of the mutant synthetase specific for phenylselenocysteine 1. pbklib5 consisting of mutant TyrRS clones was constructed using standard PCR methods. Roche high fidelity polymerase was used for all PCR reactions with the manufacturer s protocol. E. coli DH10B harboring the prep(2)/yc plasmid was used as the host strain for the positive selection. Cells were transformed with the pbk-lib5 library, recovered in SOC for 1 h, washed twice in the cold with glycerol minimal media with leucine (GMML) before plating on GMML-agar plates supplemented with kanamycin, chloramphenicol, tetracycline and 1 at 50 µg/ml, 60 µg/ml, 15 µg/ml and 1 mm, respectively. The plates were incubated at 37 o C for 60 hours and surviving cells were scraped and plasmid DNA was extracted and purified by gel electrophoresis. The pbklib5 DNA was then transformed into electro-competent cells harboring the negative selection plasmid plwj17b3, recovered for 1 h in SOC and then plated on LB-agar plates containing 0.2% arabinose, 50 µg/ml ampicillin and 50 µg/ml kanamycin. The plates were then incubated at 37 o C for 8-12 hours and pbk-lib5 DNA from the surviving clones was recovered as described above. The library was then carried through a
3 subsequent round of positive selection, followed by a negative selection and a final round of positive selection (with chloramphenicol at 70 µg/ml). At this stage, 96 individual clones were selected and suspended in 50 µl of GMML in a 96-well plate, and replicaspotted on two sets of GMML plates. One set of GMML-agar plates was supplemented with tetracycline (15 µg/ml), kanamycin (50 µg/ml) and chloramphenicol at concentrations of 60, 80, 100 and 120 µg/ml with 1 mm 1. The other set of plates were identical but contained no 1; the chloramphenicol concentrations used were 0, 20, 40 and 60 µg/ml. After 60 h incubation at 37 o C, three clones survived at more than 100 µg/ml chloramphenicol in the presence of 1 mm 1 and less than 40 µg/ml chloramphenicol in the absence of 1. These clones have the following mutations : Clones wildtype Clone 1 (SD) Positions Tyr Leu Ala His Phe Gln Asp Leu Trp Glu Ala Gly Phe Gln Gln Ser Clone 2 (K4) Trp His Ala Gly Asn Ser Ser Glu Clone 3 (K5) Trp His Gly Gly Lys Ser Glu Glu Table S1: Sequences of phenyselenocysteine specific aarss.
4 28254 Figure S1. ESI-MS spectra of the TAG242 phenylselenocysteine mutant GFP. The insert shows deconvoluted spectrum. Expected mass: Da, found: Da.
5 28112 Figure S2. ESI-MS spectra of the TAG242 dehydroalanine mutant GFP. The insert shows the deconvoluted spectrum. Expected mass: Da, found: Da.
6 Figure S3. ESI-MS spectra of the TAG242 S-mannosyl-cysteine mutant GFP. The insert shows the deconvoluted spectrum. Expected mass: Da, found: Da. The peak at Da corresponds to the TAG242 dehydroalanine mutant GFP that did not react with 1-thio-α-D-mannopyranose. The relative signal intensity of the reacted and unreacted GFP indicates that 80% of GFP is converted to the TAG242 S-mannosyl-cysteine mutant.
7 [17] Z. C. Pei, R. Larsson, T. Aastrup, H. Anderson, J. M. Lehn, O. Ramstrom, Biosensors & Bioelectronics 2006, 22, 42. [18] Y. H. Ryu, P. G. Schultz, Nat. Meth. 2006, 3, 263.
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