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[The cloning and prokaryotic expression of human tumor necrosis factor like protein]
Xiao-yan Hu1, Yan Zhou, Yun Dai
1National Laboyatory of Medical Molecular Biology, Institute of Basic Medical Sciences, CAMS and PUMC, Beijing 100005, China.
Objective:
To identify and clone the gene encoding human TNFLP (tumor necrosis factor like protein) for some functional study on TNFLP.
Methods:
The full-length cDNA of TNFLP was isolated from fetal brain cDNA library. Several kinds of software were used to analyze nucleotide sequence and amino acid sequence of TNFLP. TNFLP mRNA distribution was identified by Northern blot. TNFLP-C and TNFLP-N were expressed in E. coli with GST expression system.
Results:
The cDNA of human TNFLP was 2,112 bp, which encoded protein of 208 amino acid. Hydrophobility analysis found there were two hydrophobility regions of human TNFLP. TNFLP-C (112-207 amino acid) and mouse TNF-alpha were homologous. The identity of their amino acid sequence was 42%. Moreover, both of them had a motif-TYKRL. TNFLP was located in chromosome 16. Human TNFLP was widely expressed in various human tissues. Northern blot showed TNFLP was highly expressed in heart, brain and spleen, only one transcript can be seen. GST-TNFLP-C and GST-TNFLP-N fusion proteins were obtained.
Conclusions:
Tissue expression spectrum of TNFLP and prokaryotic expression of TNFLP have been done, which establish the base for the functional analysis of TNFLP.