Expression and purification of recombinant human coagulation factor VII fused to a histidine tag using Gateway technology.
BACKGROUND
Factor VII (FVII) is a plasma glycoprotein that participates in the coagulation process leading to the generation of fibrin. The aim of this study was to construct, express and purify recombinant FVII fused to a polyhistidine (his) tag using Gateway technology.


METHODS
To construct the entry clone, blunt-end FVII cDNA and subsequent polymerase chain reaction (PCR) product isolated from a HepG2 cell line was TOPO-cloned into a pENTR TOPO vector. To construct the expression clone, a LR recombination reaction was carried out between the entry clone and destination vector, pDEST26. Chinese hamster ovary (CHO) cells were transfected with 1 microg of DNA of PDEST26-FVII using the FuGENE HD transfection reagent. Two cell lines that permanently expressed recombinant FVII were established. The expression of recombinant FVII was confirmed by reverse transcriptase PCR and enzyme-linked immunosorbent assay. Culture medium containing his-tagged FVII was added to the nickel-nitrilotriacetic acid resin column and bound protein was eluted. The purified protein was detected by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis. The biological activity of the recombinant FVII was determined by a prothrombin time assay using FVII-depleted plasma.


RESULTS
The results showed that human recombinant FVII was successfully cloned and the accuracy of the nucleotide sequence of the gene and its frame in the vector were confirmed by DNA sequencing. Stable clones transfected with the construct expressed FVII mRNA and related protein but no expression was detected in the CHO cells containing an empty vector. A protein of about 52 KDa was detected in SDS-PAGE and was further confirmed by western blot analysis. A three-fold decrease in clotting time was observed using this recombinant FVII.


CONCLUSIONS
As far as we are aware, this is the first report of expression of recombinant FVII fused with a his-tag through Gateway technology. The next steps, including large scale expression, purification, activation and stabilisation, are underway.
Halabian
Raheleh
R
Research Centre, Iranian Blood Transfusion Organisation, Tehran, Iran.
Fathabad
Mahdi Edalati
ME
Masroori
Nasser
N
Roushandeh
Amaneh Mohammadi
AM
Saki
Sasan
S
Amirizadeh
Nasser
N
Najafabadi
Ali Jahanian
AJ
Gharehbaghian
Ahmad
A
Roudkenar
Mehryar Habibi
MH
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D005167:Factor VII / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D056945:Hep G2 Cells; D006639:Histidine / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*
D011993:Recombinant Fusion Proteins; C033223:polyhistidine; D006639:Histidine; D005167:Factor VII