Additional file 1 of The recombinant l-lysine α-oxidase from the fungus Trichoderma harzianum promotes apoptosis and necrosis of leukemia CD34 + hematopoietic cells
Creators: name: Costa, Mariana do Nascimento
name: Silva, Thiago Aparecido
name: Guimarães, Dimitrius Santiago Passos Simões Fróes
name: Ricci-Azevedo, Rafael
name: Teixeira, Felipe Roberti
name: Silveira, Leonardo Reis
name: Gomes, Marcelo Damário
name: Faça, Vítor Marcel
name: de Oliveira, Eduardo Brandt
name: Calado, Rodrigo T.
name: Silva, Roberto N.
Publisher: name: figshare
Year: 2024
Subjects: subject: Biochemistry
subject: Medicine
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Pharmacology
subject: Biotechnology
subject: Chemical Sciences not elsewhere classified
subject: Immunology
subject: FOS: Clinical medicine
subject: Cancer
subject: Hematology
subject: Infectious Diseases
subject: FOS: Health sciences

description: Additional file 1: Figure S1. Purification of native rLO. A Step 1: Ion exchange chromatography. The native LO protein was purified based on its isoelectric point by the DEAE Sephadex column for cation exchange chromatography. The graph shows the enzyme activity (o) and protein profile () of the fractions collected through the salt gradient. B Step 2: Size exclusion chromatography. The LO protein was selected from its molecular size in a Sephacryl S-200 HR column. The graph shows the enzyme activity profile and the protein content of the fractions collected. The blue line shows the LO activity and the red line shows the absorbance at 280 nm. Figure S2. pGEX-4T1-LO vector map. A The pGEX-4T1 bacterial vector is used to express GST-fused proteins. The vector has a multiple cloning region that contains several restriction sites. In orange: LO gene was added between BamHI and EcoRI restriction sites. Between the GST tag and the cloning sites, there is a thrombin cleaving site that allows GST removal. It also has a lac promoter for induction with IPTG and a gene that attributes resistance to ampicillin. B GST-rLO DNA sequence cloned into a pGEX-4T1 vector with GST highlighted in blue and rLO in green. Figure S3. Purification of rLO. A Chromatographic profile of rLO purification by affinity chromatography. In blue: enzymatic activity of the fractions collected. In red: Absorbance of the samples at 280 nm. The firsts fractions The first fractions correspond to the crude extract. After washing the column, the enzyme activity falls and rises again when the elution of rLO begins. Figure S4. Enzymatic characterization of rLO. A Effect of pH on rLO in percentage of relative activity. The tested pHs were: 2, 3, 4, 5, 6, 7, 7.5, 8, 8.6, 9, and 10 with different buffers. B Effect of temperature on rLO activity. The incubation temperatures tested were 4, 10, 20, 25, 37, 50, 60, 70, 80, and 100 oC. C Effect of substrate concentration on rLO activity. The concentrations used were: 0.1, 0.5, 1, 5, 10, 50, 100, 200, and 300 mM of l-lysine. D Graph of substrate concentration versus initial velocity (V0) to determine the Michaelis-Menten constant (KM/K0.5) and Maximum velocity (Vmáx). Figure S5. Comparison of the effect in the viability of rLO, rLOGST- and GST in Jurkat cells. GST-tagged rLO (rLO) was produced in E. coli with pGEX-4T1-LO vector and the untagged protein (rLOGST-) was obtained using a thrombin cleavage site between GST and rLO. GST was produced in E. coli transformed with pGEX-4T1 empty vector. ATO was used as a positive control. A, D Effect of treatments on cell viability. B, E Percentage of positive cells for Annexin V only (cells in apoptosis). C, F Percentage of positive cells in late apoptosis (double marked for Annexin V and PI). All treatments were carried out with enzymes at a concentration of 1 mU/mL. Table S1. Purification yield of native LO. Table S2. Mass spectrometry analyses and blast p results. Table S3. Substrate specificity data of rLO for different l-amino acids. Table S4. Kinetic parameters were calculated for rLO with l-lysine as substrate. Table S5. List of signaling pathways, primers, and genes evaluated by qPCR.