Boclys

Manufactured by Bachem

Sourced in Switzerland

BocLys is a laboratory product used in organic synthesis. It is a protected amino acid derivative, specifically N-Boc-L-lysine, which is commonly used as a building block in peptide and protein synthesis.

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Lab products found in correlation

T4 dna ligase, by New England Biolabs (4 mentions) Kod hot start dna polymerase, by Merck Group (4 mentions) Restriction enzyme, by New England Biolabs (3 mentions) Barnstead nanopure ultrapure water purification system, by Thermo Fisher Scientific (3 mentions) E coli c321 δa exp, by Addgene (2 mentions) Gibson assembly reagent, by New England Biolabs (1 mentions) Anti his6 antibody, by Santa Cruz Biotechnology (1 mentions) E coli max efficiency stbl2, by Thermo Fisher Scientific (1 mentions) Masslynx software, by Waters Corporation (1 mentions) Synapt g2 mass spectrometer, by Waters Corporation (1 mentions) Black 96 well half area microtiter plate, by Greiner (1 mentions) 7 amino 4 methylcoumarin, by Bachem (1 mentions) Pherastar plus, by BMG Labtech (1 mentions)

7 protocols using boclys

Synthesis of Lysine Derivatives

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BocLys, AlocLys, DBocLys, oClZLys, and oNiZLys were purchased from Bachem. ZLys was from Watanabe Chemical Industries (Hiroshima, Japan). AlocLysOH, ptmdZLys, mAmZLys, oAzZLys, oEtZLys, AmAzZLys, and AzNiZLys were from Shinsei Chemical Company (Osaka, Japan). ProcLys was from Synchem.

Yamaguchi A., Iraha F., Ohtake K, & Sakamoto K. (2018). Pyrrolysyl-tRNA Synthetase with a Unique Architecture Enhances the Availability of Lysine Derivatives in Synthetic Genetic Codes. Molecules : A Journal of Synthetic Chemistry and Natural Product Chemistry, 23(10), 2460.

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Recombinant Protein Expression in E. coli

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BocLys was purchased from Bachem. K-alkyne and K-alkene were synthesized by following previously reported method (22 (link)). Primers were ordered from Sigma. Restriction enzymes, Gibson Assembly reagents, Restriction enzymes, and T4 DNA ligase were purchased from New England Biolabs. KOD hot start DNA polymerase was purchased from EMD Millipore. Standard molecular biology techniques (23 ) were used throughout. Site-directed mutagenesis was carried out using overlapping PCR. Gibson Assembly or ligation was used for cloning. Escherichia coli GeneHogs (Thermo Fisher Scientific Inc) were used for routine cloning and DNA propagation. E. coli C321.ΔA.exp (Addgene) or GeneHogs were used for libraries screening and evaluation. All solutions were prepared in deionized water further treated by Barnstead Nanopure® ultrapure water purification system (Thermo Fisher Scientific Inc.). Antibiotics were added where appropriate to the following final concentrations: ampicillin, 100 mg L⁻¹; kanamycin, 50 mg L⁻¹.

Chen Y., He X., Ma B., Liu K., Gao T., Niu W, & Guo J. (2022). Noncanonical amino acid mutagenesis in response to recoding signal-enhanced quadruplet codons. Nucleic Acids Research, 50(16), e94.

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Quantifying Protein Modifications

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Anti-His₆ antibody (Santa Cruz Biotechnology, Cat# SC-804). Anti-ChloroTyr antibody (Hycult Biotech, Cat# HP5002). Anti-Halotyrosine antibody, BTK-94C (Santa Cruz Biotechnology, Cat# SC-293152). Anti-NitroTyr antibody (Millipore (Upstate), Cat# 06–284; 1:2,000). Anti-PON1 antibody (Epitomics, 2835–1; 1:10,000). NitroTyr was purchased from Alfa Aesar (#A11018). ChloroTyr, bromoTyr, and iodoTyr were purchased from Ark Pharm Inc. (#AK-50086, AK-82171 and AK-86417, respectively). BocLys was purchased from Bachem (#4000211.0005).

Jang H.S., Gu X., Cooley R.B., Porter J.J., Henson R.L., Willi T., DiDonato J.A., Hazen S.L, & Mehl R.A. (2020). Efficient site-specific prokaryotic and eukaryotic incorporation of halotyrosine amino acids into proteins. ACS chemical biology, 15(2), 562-574.

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Molecular Cloning and Mutagenesis of HIV-1 NL4-3

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Infectious molecular clone NL4-3 was obtained from the NIH AIDS Reagent Program. BocLys was purchased from Bachem. Primers were ordered from Sigma. Restriction enzymes and T4 DNA ligase were purchased from New England Biolabs. KOD hot start DNA polymerase was purchased from EMD Millipore. Standard molecular biology techniques³² were used throughout. Site-directed mutagenesis was carried out using overlapping PCR. E. coli GeneHogs were used for routine cloning and DNA propagation for none-HIV plasmids. E. coli MAX Efficiency Stbl2 (Thermo Fisher Scientific Inc) was used for routine cloning and DNA propagation for HIV plasmids. All solutions were prepared in deionized water further treated by Barnstead Nanopure® ultrapure water purification system (Thermo Fisher Scientific Inc). Antibiotics were added where appropriate to following final concentrations: ampicillin, 100 mg L^-1; kanamycin, 50 mg L^-1; tetracycline, 12.5 mg L^-1.

Chen Y., Wan Y., Wang N., Yuan Z., Niu W., Li Q, & Guo J. (2018). Controlling the replication of a genomically recoded HIV-1 with a functional quadruplet codon in mammalian cells. ACS synthetic biology, 7(6), 1612-1617.

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Quadruplet Decoding tRNA Selection

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Boc-Lys was purchased from Bachem. Primers were ordered from Sigma. Restriction enzymes, Antarctic phosphatase (AP) and T4 DNA ligase were purchased from New England Biolabs. KOD hot start DNA polymerase was purchased from EMD Millipore. Protein mass spectrometric data was collected on a Waters Synapt^® G2 mass spectrometer. Data was processed using Masslynx^TM software (Waters). Standard molecular biology techniques56 were used throughout. Site-directed mutagenesis was carried out using overlapping PCR. E. coli GeneHogs were used for routine cloning and DNA propagation. E. coli C321.ΔA.exp (Addgene) and C321.ΔA (Addgene) were used for quadruplet decoding tRNA selection and evaluation. All solutions were prepared in deionized water further treated by Barnstead Nanopure^® ultrapure water purification system (Thermo Fisher Scientific Inc). Antibiotics were added where appropriate to following final concentrations: ampicillin, 100 mg L⁻¹; kanamycin, 50 mg L⁻¹; tetracycline, 12.5 mg L⁻¹.

Wang N., Shang X., Cerny R., Niu W, & Guo J. (2016). Systematic Evolution and Study of UAGN Decoding tRNAs in a Genomically Recoded Bacteria. Scientific Reports, 6, 21898.

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Fluorescence-based HDAC4 Enzyme Activity Assay

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Enzyme activity tests were performed with 1 nM of HDAC4 wild type and 10 nM of the double mutant (Cys669Ala/His675Ala) and 20 μM Boc‐Lys(Ffa)‐7‐Amino‐4‐methylcoumarin (Bachem, Bubendorf, Switzerland) and carried out in a black 96 well half area microtiter plate (Greiner, Kremsmünster, Austria) for 1 h at 30°C in Assay‐Buffer (25 mM Tris–HCl, 75 mM KCl, 0.00001% Pluronic, pH 8.0) under shaking. The reaction was terminated by the addition of 1.7 μM SATFMK. To release the 7‐amino‐4‐methyl‐coumarin fluorophore (AMC) from the deacetylated substrate, 0.4 mg/mL trypsin was added, followed by another incubation step for 1 h at 30°C under shaking. The assay is based on the work of Wegener et al. (2003 (link)). Fluorescence intensity was measured in a PheraStar Plus (BMG Labtech, Ortenberg, Germany) fluorescence plate reader at 450 nm (Ex: 350 nm) and the blank (substrate in assay buffer without HDAC4) was subtracted from the signal. Subsequently, fluorescence intensity was converted to turned over substrate using a calibration curve. Finally, the turned over substrate was divided by the respective HDAC4 concentration for fair enzyme activity comparison.

Schweipert M., Nehls T., Frühauf A., Debarnot C., Kumar A., Knapp S., Lermyte F, & Meyer‐Almes F. (2024). The catalytic domain of free or ligand bound histone deacetylase 4 occurs in solution predominantly in closed conformation. Protein Science : A Publication of the Protein Society, 33(3), e4917.

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Synthesis and Cloning of Modified Amino Acids

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All primers were purchased from Sigma-Aldrich. AcPhe was purchased from Santa Cruz Biotechnology and BocLys was purchased from Bachem. AlkyneLys was synthesized by following a literature report.^{42 (link)} KOD hot start DNA polymerase was purchased from EMD Millipore. Restriction enzymes and T4 DNA ligase were purchased from New England Biolabs and Thermo Scientific. Standard molecular biology techniques were employed in all cloning experiments. The E. coli GeneHogs and C321.ΔA.exp strains were used for cloning and selection, respectively. All solutions were prepared with water that was purified by the Barnstead Nanopure ultrapure water purification system. Antibiotics were added where appropriate to the following final concentrations: ampicillin, 100 mg L⁻¹; kanamycin, 50 mg L⁻¹; tetracycline, 12.5 mg L⁻¹; chloramphenicol, 34–100 mg L⁻¹.

Hankore E.D., Zhang L., Chen Y., Liu K., Niu W, & Guo J. (2019). Genetic Incorporation of Noncanonical Amino Acids Using Two Mutually Orthogonal Quadruplet Codons. ACS synthetic biology, 8(5), 1168-1174.

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