WT horse Mb was purchased from Sigma-Aldrich (Saint Louis, MO, USA). Its K3A2H, K3A2H-L137E, and K3A2H-L137D Mb mutant genes were constructed using primers (Eurofins Genomics) with the KOD Plus Mutagenesis Kit (Toyobo, Japan) and confirmed by DNA sequencing (ABI PRISM 310 genetic analyzer sequencing system, Applied Biosystems, Inc.). The primers used to generate the mutations are listed in Table S1† . Mb mutants were expressed in Escherichia coli LE392 cells. All Mb mutants were obtained as a mixture of monomer and dimer using a procedure similar to that described previously.37 (link) The monomers and dimers of K3A2H, K3A2H-L137E, and K3A2H-L137D Mb were purified by size exclusion chromatography (SEC; HiLoad 26/60 Superdex 75 pg, GE Healthcare) using a fast protein liquid chromatography (FPLC) system (Biologic DuoFlow 10, Bio-Rad) with 50 mM potassium phosphate buffer, pH 7.0, at 4 °C. The Mb concentration was adjusted by the Soret band intensity at 408 nm.
Abi prism 310 genetic analyzer sequencing system
Manufactured by Thermo Fisher Scientific
Sourced in Japan
The ABI PRISM 310 genetic analyzer sequencing system is a capillary electrophoresis-based DNA sequencing instrument. It is designed for automated, high-throughput DNA sequence analysis.
Automatically generated - may contain errors
Lab products found in correlation
Hiload 26 60 superdex 75 pg, by GE Healthcare (1 mentions)
Biologic duoflow 10, by Bio-Rad (1 mentions)
Kod plus mutagenesis kit, by Toyobo (1 mentions)
Forward and reverse primer, by Eurofins (1 mentions)
Bigdye terminator v3.1 cycle sequencing kit, by Thermo Fisher Scientific (1 mentions)
Primestar max dna polymerase, by Takara Bio (1 mentions)
2 protocols using abi prism 310 genetic analyzer sequencing system
1
Purification and characterization of Mb mutants
2
Point Mutation of Tyr67 to Phe in Horse Cyt c
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Point mutation of Try67 to Phe was performed by PCR-based in vitro mutagenesis of the horse cyt c expression plasmid (containing the heme lyase gene) using forward and reverse primers (Eurofins genomics) (Table S1 in Suppl. mat.). Plasmid DNAs were prepared using the PrimeSTAR max DNA polymerase (Takara Bio). DNA sequencing was carried out with the BigDye Terminator v3.1 cycle sequencing kit (Applied Biosystems, Inc., Foster City, CA) and an ABI PRISM 310 genetic analyzer sequencing system (Applied Biosystems, Inc.). The obtained plasmid was introduced into the competent cells of E. coli Rosetta2 (DE3) pLysS (Novagen). Ferric Y67F horse cyt c was purified with a similar procedure reported for wild-type human cyt c [32] . Purification of Y67F cyt c without an N-acetyl group was confirmed by a MALDI-TOF mass spectrum measured under the same conditions described above.
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