科研之友
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摘要
A fungal immunomodulatory protein (Fip-gts) was purified from Ganoderma tsugae, The DNA encoding Fip-gts was isolated from a cDNA library of G. tsugae by reverse transcriptase-polymerase chain reaction, The complete amino acid sequence of Fip-gts, deduced from the nucleotide sequence of the cDNA, was the same as LZ-8 isolated from Ganodermn lucidum, Recombinant Fip-gts was expressed as a glutathione S-transferase fusion protein in Escherichia coli with a yield of 20 mg/liter of culture, Recombinant Fip-gts, purified to homogeneity, had the same blast formation stimulatory activity to human peripheral blood lymphocytes as native Fip-gts.
The yeast two-hybrid system and site directed mutagenesis were used to determine whether dimerization of Fip-gts occurred, Deletion analysis of the N-terminal amphipathic alpha-helix domain of Fip-gts identified a sequence of about 10 amino acids responsible for inducing immunomodulatory activity. Non-functional Fip-gts deletion mutants did not form dimers, whereas wild type Fip-gts did as determined by gel filtration, A mutant with deletions at Leu-5, Phe-7, and Leu-9 lost the amphipathic characteristics of the N-terminal domain and the ability to form dimers as well as its immunomodulatory activity.
Fusion of Fip-gts with the DNA binding and the transactivation domains of GAL4 resulted in the activation of the lacZ activator gene, indicating the interaction of Fip-gts with it itself, The dimerization domain was further defined by analyzing the ability of the N-terminal 13 amino acids or Leu-5, Phe-7, and Leu-9 deletion mutants of Fip-gts to interact with the wild type Fip-gts. These experiments confirmed the N-terminal amphipathic alpha-helix as the dimerization domain and suggest that the dimerization of Fip-gts may play an important role in Fip-gts immunomodulatory activity.
- 出版日期1997-8-8
