Background
Methods
Cloning of PfTRAP and its subdomains in mammalian expression vector pHL
Expression analysis of PfTRAP subdomains in HEK 293 cells
Purification of PfTRAP subdomains from HEK-293 cells
Binding of PfTRAP ECD and subdomains to HepG2 cells
Binding assay of PfTRAP ECD with hepatocytes (HepG2 cells and Huh-7 cells) and kidney cells (HEK 293 cells)
Site-directed mutagenesis of PfTRAP ECD
P18 | gTTgTTATATTAACAgCTggAATTCCAgATAgT |
---|---|
P19 | ACTATCTggAATTCCAgCTgTTAATATAACAAC |
P20 | AgATTTCTTgTAggTggTCATCCATCAgATggT |
P21 | ACCATCTgATggATgACCACCTACAAgAAATCT |
P22 | CCATCAgATggTAAAggTAACTTgTATgCTgAT |
P23 | ATCAgCATACAAgTTACCTTTACCTTTACCATCTgATgg |
P24 | gTTTgggACgAATgggCTCCATgTAgTgTAACT |
P25 | AgTTACACTACATggAgCCCATTCgTCCCAAAC |
P26 | TgTggAgAAgAAAgAgg TCCTCCAAAATgggAA |
P27 | TTCCCATTTTggAggACCTCTTTCTTCTCCACA |
P28 | gATCAACCTAgACCAggAggAgATAATTCTTCT |
P29 | gACAgAAgAATTATCTCCTCCTggTCTAggTTg |
P30 | AgAAATATTCCATATgCACCATTACCTCCAAAA |
P31 | TTTTggAggTAATggTgCATATggAATTCT |
P32 | AgAAAATATAACgATgCTCCAAAACATCCTgAAAgg |
P33 | CCTTTCAggATgTTTTggAgCATCgTTATATTTTCT |
Forward Primer | Reverse Primer | Residue mutated |
---|---|---|
P12 | P13 | C42G |
P14 | P15 | C55G |
P16 | P17 | T131A |
P18 | P19 | D162A |
P20 | P21 | C205G |
P22 | P23 | C212G |
P24 | P25 | S251A |
P26 | P27 | S285G |
P28 | P29 | R307G |
P30 | P31 | S416A |
P32 | P33 | T470A |
HepG2 cell binding of PfTRAP ECD and its mutants
Preparation of HepG2 and HEK 293 extracts
Phosphorylation of PfTRAP and its fragments by HepG2 cell extracts
Detection of PfTRAP phosphorylation using phosphospecific antibodies
Binding of PfTRAP to tyrosine kinases
Phosphorylation of PfTRAP with tyrosine kinases proteins
Results
PfTRAP binding to hepatocytes
Asp162, Cys205 and Arg307 are important for PfTRAP-HepG2 binding
PfTRAP undergoes phosphorylation in presence of hepatocyte extract
Tyrosine kinases from hepatocytes bind and mediate phosphorylation of PfTRAP
Discussion
PfTRAP | RNIPYSPLPP; PKHPER |
---|---|
PbTRAP | RDCPPKP; PVIP IK; PDVP VK; PILP IK; PEIP SK |
PyTRAP | PSNP NK; RDCPQIP; PVIP NK; RRNPNKP; PIIP QK; KDEPEIP |
PvTRAP | PWDP; PLPP |
PkTRAP | PVPPTVP; PENPENP |
PcTRAP | RNSPMNP |