[Cloning and expression of an antifreeze peptide gene of winter flounder in E. coli]

Antifreeze peptide (AFP) produced in some animals is able to lower freezing temperature to prevent serum from freezing. There is a great potential to apply AFP in various circumstances wherever tolerance to cold environment is required. cDNA copies of AFP and proAFP coding sequence were synthesized according to the published sequence from a winter flounder (Pseudopleuronectes americanus). Expression of AFP or proAFP cDNA was initially tested in E. coli using a procaryotic expression vector. Level of the expression was essentially low indicated by SDS-PAGE and protein staining procedures. In order to detect the low level expression, the AFP (or proAFP) cDNA was fused, in frame, to the 5'-end of the CAT reporter gene resulting in a chimeric AFP/CAT (or proAFP/CAT) cDNA. The product translated from such a chimeric mRNA must contain a N'-terminal AFP (or proAFP) portion and C'-terminal CAT portion. Thus, low level of expression of AFP (or proAFP) in the fused form may be detected indirectly by sensitive CAT assay as long as the chimeric CAT still remains the activity. As expected, CAT activity has been clearly detected in protein extract from induced E. coli indicating that AFP (or proAFP) cDNA clones be expressed in E. coli.