Recombinant Human Adaptor-Related Protein Complex 1, Sigma 2 Subunit, His-tagged

• Cat.No.: AP1S2-4967H
• Product Overview: Recombinant human AP1S2 protein, fused to His-tag at N-terminus, was expressed in E. coli and purified by using conventional chromatography.

Specification

• Species: Human
• Source: Human
• Tag: His
• Description: AP1S2, also known as AP-1 complex subunit sigma-2, serves as the small subunit of AP-1 complex 1 and is a member of the adaptin protein family. Adaptor protein complex 1 is found at the cytoplasmic face of coated vesicles located at the Golgi complex, where it mediates both the recruitment of clathrin to the membrane and the recognition of sorting signals within the cytosolic tails of transmembrane receptors.
• Concentration: 0.5 mg/ml
• Form: Supplied as a liquid in 20mM Tris-HCl buffer, pH 8.0, containing 40% glycerol, 0.1M NaCl and 2mM DTT.
• Purity: > 85% by SDS - PAGE
• Sequence: 1-157 amino acids: MGSSHHHHHH SSGLVPRGSH MQFMLLFSRQ GKLRLQKWYV PLSDKEKKKI TRELVQTVLA RKPKMCSFLE WRDLKIVYKR YASLYFCCAI EDQDNELITL EIIHRYVELL DKYFGSVCEL DIIFNFEKAY FILDEFLLGG EVQETSKKNV LKAIEQADLL QEEAETPRSV LEEIGLT
• Molecular Mass: 20.7 kDa (177aa), confirmed by MALDI-TOF
• Applications: SDS-PAGE
• Storage: Store at 4 deg C for short term storage (1/2 weeks). Aliquot and store at -20 deg C or - 70 deg C for long term storage. Avoid repeated freeze/thaw cycles.

Gene Information

• Gene Name: AP1S2 adaptor-related protein complex 1, sigma 2 subunit [ Homo sapiens ]
• Official Symbol: AP1S2
• Synonyms: AP1S2; DC22; MRX59; SIGMA1B; MGC:1902; AP1S2; AP-1 complex subunit sigma-2; sigma1B-adaptin; sigma-adaptin 1B; OTTHUMP00000022979; OTTHUMP00000022980; OTTHUMP00000022981; sigma 1B subunit of AP-1 clathrin; adaptor protein complex AP-1 sigma-1B subunit; clathrin adaptor complex AP1 sigma 1B subunit; golgi adaptor HA1/AP1 adaptin sigma 1B subunit; golgi adaptor HA1/AP1 adaptin sigma-1B subunit; adapter-related protein complex 1 sigma-1B subunit; clathrin assembly protein complex 1 sigma-1B small chain;adaptor-related protein complex 1, sigma 2 subunit; Sigma-adaptin 1B; Golgi adaptor HA1/AP1 adaptin sigma-1B subunit; Adaptor protein complex AP-1 sigma-1B subunit; Sigma 1B subunit of AP-1 clathrin; Clathrin assembly protein complex 1 sigma-1B small chain; mental retardation, X-linked 59; OTTHUMP00000022982; Sigma1B-adaptin; clathrin-associated/assembly/adaptor protein small 1-like
• Gene ID: 8905
• mRNA Refseq: NM_003916
• Protein Refseq: NP_003907
• MIM: 300629
• UniProt ID: P56377
• Chromosome Location: Xp22
• Pathway: Clathrin derived vesicle budding; Golgi Associated Vesicle Biogenesis; HIV Infection; Host Interactions of HIV factors; Lysosome; Lysosome Vesicle Biogenesis; Membrane Trafficking; Nef mediated downregulation of MHC class I complex cell surface expression; Nef-mediates down modulation of cell surface receptors by recruiting them to clathrin adapters; The role of Nef in HIV-1 replication and disease pathogenesis; trans-Golgi Network Vesicle Budding
• Function: protein transporter activity

Citation

Human dihydrofolate reductase influences the sensitivity of the malaria parasite Plasmodium falciparum to ketotifen – A cautionary tale in screening transgenic parasites

Journal: International Journal for Parasitology: Drugs and Drug Resistance    PubMed ID: 27705841    Data: 2016/9/22

Authors: Phuong N. Tran, Cameron J. Tate, Alexander G. Maier

Article Snippet:The effect of ketotifen on the conversion of DHF to THF by recombinant hDHFR was investigated using an in vitro assay ( , ).The effect of ketotifen on the conversion of DHF to THF by recombinant hDHFR was investigated using an in vitro assay ( , ).. Reactions were carried out at 27 °C in a flat bottom 96-well plate containing 0.1 M K 3 PO 4 , 0.1 M NaCl, pH 7.0; 0.1 mM NADPH 2 (Sigma), 50 mM 2-mercaptoethanol, 100 nM purified recombinant hDHFR (Creative BioMart) and a range of concentrations of ketotifen fumarate (Sigma).. The reduction of NADPH 2 to NADP + was measured at OD 340 .The reduction of NADPH 2 to NADP + was measured at OD 340 .

Expression of the selectable marker hDHFR antagonises the inhibition of P. falciparum parasite proliferation by ketotifen . The proliferation of asexual blood-stage parasites in the presence of a range of ketotifen concentrations was assessed over 72 h. Selectable marker cassettes integrated into the genome are indicated by (i), selectable marker cassettes located on episomal plasmids are indicated by (e). A) Sensitivity of parasites to ketotifen is independent of the presence of PfgABCG2. Wild-type: 3D7 parental line; ΔPfgABCG2-hDHFR (i): PfgABCG2 knock-out parasites expressing the hDHFR selectable marker. ΔPFD1170c-hDHFR (i): PFD1170c knock-out parasites expressing the hDHFR selectable marker. ΔPfgABCG2-hDHFR (i)/PfgABCG2-BSD (e): PfgABCG2 knock-out parasites expressing the hDHFR selectable marker and PfgABCG2 expressed from an episomal plasmid, under the control of the PfgABCG2 promoter, using blasticidin-S deaminase (BSD) as the selectable marker. B) Sensitivity to ketotifen is modulated by the presence of hDHFR. BSD (e): parasites expressing actin II (tagged by red fluorescent protein) and the blasticidin-S deaminase (BSD) selectable marker from an episomal plasmid. hDHFR (e): parasites expressing the hDHFR selectable marker from an episomal plasmid. Mean and standard deviation (SD) values of biological triplicates are shown.

Recombinant hDHFR does not modify ketotifen in an in vitro enzyme assay . DHF or ketotifen fumarate (100 μM) was incubated in the presence or absence of recombinant hDHFR (100 nM) for 10 min at 27 °C. The LC/MS trace shown is representative of those obtained in three independent experiments. A) In the presence of recombinant hDHFR, the substrate 7,8-dihydrofolate (DHF), was broken down. B) Ketotifen is not broken down in the presence of recombinant hDHFR.

Ketotifen increases the activity of hDHFR . A) Schematic outline of the conversion of 7,8-dihydrofolate (DHF) to 5,6,7,8-tetrahydrofolate (THF) through the enzymatic action of human dihydrofolate-reductase (hDHFR). The reaction involves the oxidation of NADPH 2, which can be measured at OD 340 . B) Ketotifen (KETO) caused a significant concentration-dependent increase in the conversion of DHF to THF (as measured by oxidation of NADPH 2 over 10 min) by recombinant hDHFR. The NADPH 2 oxidation is indicative of hDHFR activity. The bars indicate mean values at equilibrium of biological triplicates and are shown ± standard deviation. *** ( p < 0.001) and ** ( p < 0.01) indicate a statistically significant difference in hDHFR activity in the presence of ketotifen, relative to the activity measured in the absence of ketotifen (Student's unpaired t -test). Time courses for the conversion of DHF to THF are shown in Supplementary Fig. S3 .