Krohn B M, Lindsay J A
Food Science and Human Nutrition Department, University of Florida, Gainesville 32611.
Curr Microbiol. 1993 Apr;26(4):217-22. doi: 10.1007/BF01577379.
The cyclomaltodextrinase gene from Bacillus subtilis high-temperature growth transformant H-17 was cloned on separate PstI, BamHI, and EcoRI fragments into the plasmid vector pUC18, but was expressed in an inactive form in the host, Escherichia coli DH5 alpha. High level constitutive expression of the gene product was also detrimental to the E. coli host, which led to structural instability of the recombinant plasmid. The cyclomaltodextrinase gene was cloned on a 3-kb EcoRI fragment into the plasmid vector pPL708, and the fragment was structurally maintained in the host B. subtilis YB886. The cloned gene product was synthesized in an enzymatically active form in the B. subtilis host; however, expression was at a low level. Subcloning of the 3-kb EcoRI fragment into pUC18 and transformation into E. coli XL1-Blue (F' lacIq) indicated that the cyclomaltodextrinase gene was cloned with its own promoter, since expression of the gene occurred in the absence of IPTG. Subcloning of the cyclomaltodextrinase gene downstream from the Bacillus temperature phage SPO2 promoter of pPL708 may increase expression of this gene.
将枯草芽孢杆菌高温生长转化体H-17的环麦芽糊精酶基因分别以PstI、BamHI和EcoRI片段的形式克隆到质粒载体pUC18中，但其在宿主大肠杆菌DH5α中以无活性形式表达。该基因产物的高水平组成型表达对大肠杆菌宿主也有害，这导致重组质粒的结构不稳定。将环麦芽糊精酶基因以3 kb的EcoRI片段形式克隆到质粒载体pPL708中，该片段在宿主枯草芽孢杆菌YB886中结构稳定。克隆的基因产物在枯草芽孢杆菌宿主中以酶活性形式合成；然而，表达水平较低。将3 kb的EcoRI片段亚克隆到pUC18中并转化到大肠杆菌XL1-Blue（F'lacIq）中表明，环麦芽糊精酶基因是与其自身启动子一起克隆的，因为该基因在没有IPTG的情况下也能表达。将环麦芽糊精酶基因亚克隆到pPL708的芽孢杆菌温度噬菌体SPO2启动子下游可能会增加该基因的表达。
