Full-length cloning and 3 '-terminal portion expression of human perforin cDNA

Citation
Fq. Li et al., Full-length cloning and 3 '-terminal portion expression of human perforin cDNA, CLIN CHIM A, 313(1-2), 2001, pp. 125-131
Citations number
17
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Medical Research Diagnosis & Treatment
Journal title
CLINICA CHIMICA ACTA
ISSN journal
0009-8981 → ACNP
Volume
313
Issue
1-2
Year of publication
2001
Pages
125 - 131
Database
ISI
SICI code
0009-8981(200111)313:1-2<125:FCA3'P>2.0.ZU;2-K
Abstract
Background: Perforin (also known as pore-forming protein, PFP) is one of th e main effector molecules which natural killer cells (NK) and cytotoxic T l ymphocytes (CTL) utilize to kill their targets both in vivo and in vitro. W e report the full length of human perforin cDNA, which was cloned from live r tissue. Results: Sequencing analysis showed that there were discrepancies of four nucleotides and three amino acids compared with previously publish ed sequence of human PFP. The cDNA fragment was then inserted into fusion p rotein expressive vector pGEX-2T to construct a recombinant expressive plas mid. The C-terminal truncated 125 amino acids polypeptide (410-534aa) of hu man 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 c hromatography with glutathione agarose. The recombinant hPFP-C obtained by thrombin cleavage showed a significant hemolytic activity when tested with rabbit erythrocytes. Conclusion: These results suggest that the domain resp onsible for lytic function lies not only in the N-terminal portion but also in the C-terminal portion of perform molecule. The recombinant hPFP-C prot ein will be useful as a highly purified biological factor for immunological , pathological and structural studies. (C) 2001 Elsevier Science B.V. All r ights reserved.