Physicochemical, functional and structural properties of the major protein fractions extracted from Cordyceps militaris fruit body.

The physicochemical and functional as well as structural properties of major protein fractions (albumin, globulin, glutelin) sequentially extracted in water, salt, alkaline solution respectively from Cordyceps militaris Minfu20 fruit body were investigated. The glutelin (43.11%, w/w) was the predominant protein component of C. militaris fruit body followed by albumin (36.47%) and globulin (17.94%). The three proteins extracted from different solvents showed different characteristics, which were related to the alternation of amino acid composition, surface hydrophobicity, and structural feature. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that the albumin and globulin mainly consisted of polypeptides with size < 20 kDa. The glutelin showed serious staining on the lane which may have a relatively bigger molecular weight. Intrinsic fluorescence intensity (FI) suggested glutelin contained more unfolding conformations (highest FI) which were probably resulted in a better foaming capacity of 151% and emulsion formation with the smallest size oil droplets (10.410 µm). The protein fractions showed great nutritional quality since they satisfied all recommended essential amino acid allowances for adults of Food & Agriculture Organization (FAO)/World Health Organization (WHO). Therefore, Cordyceps militaris Minfu20 fruit body proteins have potential alternative renewable edible fungi (mushroom) protein and could be used effectively as a food ingredient to improve food nutrition and product diversification compared with plant proteins.

[Construction and expression of the eukaryotic expression plasmids of human TSHR extracellular domain].

AIM: To construct the eukaryotic expression plasmids of hTSHR extracellular domain and study their expression in CHO cells. METHODS: The human TSHR extracellular domain cDNAs, which were 188-403 bp and 407-904 bp, were amplified from human normal thyroid by RT-PCR. Two fragments were inserted into pcDNA3.1(D)/V5-His-TOPO.Then the recombinant plasmids pcDNA3.1-hTSHRf and pcDNA3.1-hTSHRe were transfected into CHO cells by Lipofectin after they were identified by restricting enzyme HindIII digestion analysis, PCR amplifying and DNA sequencing. RT-PCR and Western blot analysis were used to analyse hTSHR expression on mRNA and at protein levels. RESULTS: Two bands of 220 bp and 540 bp were amplified from CHO cells transfected by the recombinant plasmids pcDNA3.1-hTSHRf and pcDNA3.1-hTSHRe, respectively. Western blot analysis revealed that CHO cells transfected by pcDNA3.1-hTSHRf and pcDNA3.1-hTSHRe had strong bands with molecular weight of about 11 900 and 23 600, respectively. CONCLUSION: The recombinant plasmids have been successfully constructed. The transcription on CHO cells transfected by the recombinant plasmids has been proved by RT-PCR and eukaryotic expression has been confirmed by Western blot analysis. Our research will contribute to further study on gene expression in vivo and the establishment of animal models of Graves' disease.