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Published on: June 10, 2015
Expression and bioactivity of recombinant segments of human perforin
Hongmei Dong1, Xiaohu Xu, Mohong Deng
1Department of Forensic Medicine, Shantou University Medical College, 22 Xinling Road, Shantou 0754, Guangdong province, China. hongmeidong1@hotmail.com
Abstract:
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.
Insights
This study successfully produced active recombinant human perforin segments in E. coli. The N-60 (HP5) and MAC1 (HP2) segments showed concentration-dependent hemolytic activity, forming pores.
Area of Science:
- Biochemistry
- Molecular Biology
- Immunology
Background:
- Human perforin is a key cytotoxic protein involved in immune responses.
- Understanding perforin's functional domains is crucial for therapeutic applications.
- Recombinant protein expression in prokaryotic systems offers a scalable production method.
Purpose of the Study:
- To prepare active recombinant human perforin by evaluating five candidate segments.
- To identify which perforin fragments retain hemolytic activity.
- To establish a basis for the application of perforin.
Main Methods:
- Five human perforin segments (HP1-HP5) were cloned and expressed as fusion proteins in Escherichia coli.
- Nickel nitrilotriacetic acid (NTA) agarose affinity chromatography was used for purification.
- Hemolysis microassays assessed the activity of the purified recombinant proteins.
Main Results:
- Recombinant plasmids and expression in E. coli were successful for all five segments.
- Purified fusion proteins ranged from 30 to 80 kDa.
- Only HP2 (MAC1) and HP5 (N-60) demonstrated concentration-dependent hemolytic activity and pore formation.
Conclusions:
- Active recombinant human perforin segments can be synthesized in a prokaryotic system.
- The N-60 and MAC1 domains of human perforin are essential for its hemolytic function.
- This research provides a foundation for further studies on perforin applications.
