Full-length cloning and 3'-terminal portion expression of human perforin cDNA.
BACKGROUND
Perforin (also known as pore-forming protein, PFP) is one of the main effector molecules which natural killer cells (NK) and cytotoxic T lymphocytes (CTL) utilize to kill their targets both in vivo and in vitro. We report the full length of human perforin cDNA, which was cloned from liver tissue.


RESULTS
Sequencing analysis showed that there were discrepancies of four nucleotides and three amino acids compared with previously published sequence of human PFP. The cDNA fragment was then inserted into fusion protein expressive vector pGEX-2T to construct a recombinant expressive plasmid. The C-terminal truncated 125 amino acids polypeptide (410-534aa) of human perforin (hPFP-C) was selectively expressed in a form of fusion protein. Under the induction of IPTG, GST/hPFP-C fusion protein was expressed in E. coli BL21 (DE3). The fusion protein GST/hPFP-C was purified by affinity chromatography with glutathione agarose. The recombinant hPFP-C obtained by thrombin cleavage showed a significant hemolytic activity when tested with rabbit erythrocytes.


CONCLUSIONS
These results suggest that the domain responsible for lytic function lies not only in the N-terminal portion but also in the C-terminal portion of perforin molecule. The recombinant hPFP-C protein will be useful as a highly purified biological factor for immunological, pathological and structural studies.
Li
F
F
Laboratory of Molecular Biology, Center of Medical Laboratory Sciences, Jinling Hospital, 210002, Nanjing, People's Republic of China. njlifq@jlonline.com
Zhou
X
X
Qin
W
W
Wu
J
J
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004591:Electrophoresis, Polyacrylamide Gel; D005822:Genetic Vectors; D006461:Hemolysis; D006801:Humans; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D054353:Perforin; D052899:Pore Forming Cytotoxic Proteins; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D012689:Sequence Homology, Nucleic Acid
D018076:DNA, Complementary; D008562:Membrane Glycoproteins; D052899:Pore Forming Cytotoxic Proteins; D011994:Recombinant Proteins; D054353:Perforin