Expression and bioactivity of recombinant segments of human perforin.
The aim of the study was to prepare an active recombinant human perforin by comparing 5 candidate segments of human perforin. Full-length perforin, MAC1 (28-349 aa), MAC2 (166-369 aa), C-100, and N-60 of human perforin were selected as candidate active segments and designated, respectively, HP1, HP2, HP3, HP4, and HP5. The target genes were amplified by PCR and the products were individually subcloned into pGEM-T. The genes for HP1, HP2, HP3, and HP5 were subcloned into pET-DsbA, whereas pET-41a (+) was used as the expression vector of HP4. The fusion proteins were expressed in Escherichia coli BL21pLysS(DE3) and purified using nickel nitrilotriacetic acid (NTA) agarose affinity chromatography. The hemolysis microassay was used as an activity assay of fusion protein. From this study, we obtained the recombinant plasmids pGEM-T-HP1, -HP2, -HP3, -HP4 and -HP5, consisting of 1600, 960, 600, 300bp, and 180, respectively. From these recombinant plasmids, expression plasmids were successfully constructed and expressed in E. coli BL21pLysS(DE3). The resultant fusion proteins, affinity purified using Ni-NTA, were approximately 80, 58, 45, 44, and 30 kDa, respectively. The recombinant proteins were assayed for activity on hemolysis. HP2 and HP5 were the only recombinant proteins that were active in hemolysis, and the hemolytic function was concentration dependent. These results demonstrate that active recombinant forms of perforin can be synthesized in a prokaryote model. The recombinant N-60 and MAC1 (28-349 aa) of human perforin have the function of forming pores. Our study provides the experimental basis for further investigation on the application of perforin.
Dong
Hongmei
H
Department of Forensic Medicine, Shantou University Medical College, 22 Xinling Road, Shantou 0754, Guangdong province, China. hongmeidong1@hotmail.com
Xu
Xiaohu
X
Deng
Mohong
M
Yu
Xiaojun
X
Zhao
Hu
H
Song
Hui
H
Geng
Yiqun
Y
D004912:Erythrocytes / Q000166:cytology; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006461:Hemolysis / Q000187:drug effects; D006801:Humans; D008562:Membrane Glycoproteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D054353:Perforin; D052899:Pore Forming Cytotoxic Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology
D008562:Membrane Glycoproteins; D052899:Pore Forming Cytotoxic Proteins; D011993:Recombinant Fusion Proteins; D054353:Perforin