Expression of Carcinoscorpius rotundicauda factor C cDNA.
The cDNA encoding Factor C (FC) from the Singaporean horseshoe crab Carcinoscorpius rotundicauda has been studied for in vitro coupled transcription-translation (TnT) under the T7 promoter. Two species of full length cDNA, CrFC26 and CrFC21 which differ in length and nucleotide sequence at their 5' untranslated regions (UTR) were used in this study. Wild type CrFC26 with a long 5' UTR containing multiple "false" ATGs failed to generate a translated product. With a more accessible ATG codon in CrFC21, the recombinant construct gave a high yield of FC when transcribed and translated in vitro. CrFC26 deletion mutants which lack the entire 5' UTR and portions of the putative leader peptide were translatable, albeit at lower efficiency as compared to CrFC21. In vitro and in vivo expression of truncated portions of the CrFC21-T7 gene 10 fusions have been compared. In vitro reactions yielded single gene products from each of the expression constructs whereas E. coli produced three major immunoreactive bands of FC.
Roopashree
S D
SD
Department of Zoology, National University of Singapore, Kent Ridge Crescent.
Chai
C
C
Ho
B
B
Ding
J L
JL
D000818:Animals; D060829:Arthropod Proteins; D017123:Bacteriophage T7 / Q000235:genetics; D003001:Cloning, Molecular; D018387:Codon, Initiator; D018076:DNA, Complementary / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D004792:Enzyme Precursors / Q000096:biosynthesis* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D006737:Horseshoe Crabs / Q000235:genetics*; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis*; D015183:Restriction Mapping; D017384:Sequence Deletion; D012697:Serine Endopeptidases / Q000096:biosynthesis* / Q000235:genetics
D060829:Arthropod Proteins; D018387:Codon, Initiator; D018076:DNA, Complementary; D004792:Enzyme Precursors; D011993:Recombinant Fusion Proteins; D012697:Serine Endopeptidases; C057151:Limulus clotting factor C