[Expression and purification of tissue factor pathway inhibitor in Pichia pastoris].
OBJECTIVE
To generate recombinant human tissue factor pathway inhibitor (TFPI) in Pichia pastoris.


METHODS
To improve the expression of TFPI, a silent mutation was generated at the specific site of TFPI cDNA. Both wild-type TFPI cDNA and mutated TFPI cDNA were cloned into the expression vector pPic9. The constructed plasmids were subsequently transformed into Pichia pastoris cells GS115 and KM71, and the transformants were confirmed by polymerase chain reaction and DNA sequencing. The expression of recombinant protein was induced by addition of 0.5% methanol in the culture medium. The cell culture medium after induction was concentrated through ultra filtration. The recombinant protein was further purified by a three-step process (Heparin-sepharose CL-6B affinity chromatography, DEAE-Sepharose Fast Flow affinity chromatography, and Sephadex G75-gel filtration). The amount of the recombinant protein was quantified with gel imaging system. The activity of the recombinant protein was analyzed by the chromogenic substrate assay.


RESULTS
The amount of TFPI expressed in the mutated clone (1 mg/L) was much higher than that in the wild type clone (0.1 mg/L). The TFPI activity in the recombinant GS115 cells could be detected 12 hours after induction and reached the peak at 36 hours, while the TFPI activity in the recombinant KM71 cells started to show up at 24 hours after induction and reached the peak at 72 hours. The expression of recombinant protein in the silent mutant was significantly higher than those of wild type clone in both GS115 and KM71 host cells. The relative molecular mass of recombinant TFPI was approximately 42 000.


CONCLUSIONS
Introduction of the silent mutation at the specific site of TFPI cDNA can increase the recombinant protein expression in Pichia pastoris, which is much higher than that in insect cells or saccharomyces cerevisiae.
Luo
Shi-Ping
SP
Tianjin Key Laboratory of Biomedical Materials, Institute of Biomedical Engineering, CAMS and PUMC, Tianjin 300192, China.
Song
Li-Ping
LP
Yu
Bo
B
Zhang
Hai-Ling
HL
Liu
Lan-Xia
LX
Leng
Xi-Gang
XG
D006801:Humans; D008074:Lipoproteins / Q000096:biosynthesis* / Q000235:genetics; D009154:Mutation; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics
D008074:Lipoproteins; D011994:Recombinant Proteins; C051928:lipoprotein-associated coagulation inhibitor