美洲拟鲽抗冻肽基因在E.coli中的表达

CLONING AND EXPRESSION OF AN ANTIFREEZE PEPTIDE GENE OF WINTER FLOUNDER IN E. COLI

动物抗冻肽(AFP)能降低动物体液冰点而使其能在寒冷的环境中生活,因而AFP在防寒抗冻方面有较大的应用潜力。根据美洲拟鲽AFP基因的cDNA序列,人工合成了AFP及其含前导序列的proAFP的cDNA,并构建成pAFP及pPAFP原核表达载体。由于AFP表达量少且蛋白分子很小,用SDS-蛋白电泳难以检测。为了提高检测的灵敏度,把报道基因CAT的cDNA按读码框连接到AFP的3′末端,构建成融合基因表达载体pAFP/CAT和pPAFP/CAT。在大肠杆菌表达系统JM109(DE 3)中诱导表达。结果表明,融合蛋白AFP/CAT和proAFP/CAT具有明显CAT活力。这表明AFP和proAFP可以在大肠杆菌中表达。

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 (Pseudo-pleuronectes americanus). Expression of AFP or proAFP cDNA was initially tested in B. 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.