Allen S J, Holbrook J J
Molecular Recognition Centre, School of Medical Sciences, University of Bristol, UK.
Gene. 1995 Aug 30;162(1):99-104. doi: 10.1016/0378-1119(95)00347-9.
NAD-dependent formate dehydrogenase (FDH) was isolated from Candida methylica (Cm) grown on 0.5% methanol. Its N-terminal amino acid (aa) sequence was determined, as was that of a commercial FDH from Candida boidinii. Degenerate oligodeoxyribonucleotides were made to the 5' region of the fdh gene from Cm using this information and to the 3' region using C-terminal aa sequence data from the methylotropic yeast, Hansenula polymorpha. An almost complete 1.1-kb fragment was amplified from Cm genomic DNA via the polymerase chain reaction (PCR). This fragment was cloned, sequenced and used to probe a Southern blot, from which a 3.4-kb EcoRI fragment containing the fdh open reading frame (ORF) was isolated. The complete nucleotide sequence of this fdh ORF was determined and corresponds to a protein of 364 aa (40,343 Da). The ORF of fdh was cloned into pKK223-3 using PCR and transformed into Escherichia coli. Enzymatically active FDH was produced to 15% of soluble E. coli protein. The deduced aa sequence of this FDH is compared to the aa sequences of four known FDH, from bacteria, yeast, fungi and plant mitochondria.
从在0.5%甲醇上生长的甲基营养假丝酵母（Cm）中分离出依赖烟酰胺腺嘌呤二核苷酸（NAD）的甲酸脱氢酶（FDH）。测定了其N端氨基酸（aa）序列，以及来自博伊丁假丝酵母的市售FDH的N端氨基酸序列。利用这些信息针对Cm的fdh基因的5'区域制备简并寡脱氧核糖核苷酸，并利用嗜甲基酵母多形汉逊酵母的C端aa序列数据针对3'区域制备简并寡脱氧核糖核苷酸。通过聚合酶链反应（PCR）从Cm基因组DNA中扩增出一个几乎完整的1.1 kb片段。将该片段克隆、测序并用于探测Southern印迹，从中分离出一个包含fdh开放阅读框（ORF）的3.4 kb EcoRI片段。确定了该fdh ORF的完整核苷酸序列，其对应于一个由364个aa组成的蛋白质（40343 Da）。利用PCR将fdh的ORF克隆到pKK223-3中并转化到大肠杆菌中。产生的具有酶活性的FDH占可溶性大肠杆菌蛋白质的15%。将该FDH推导的aa序列与来自细菌、酵母、真菌和植物线粒体的四种已知FDH的aa序列进行比较。
