Sandman K, Grayling R A, Reeve J N
Department of Microbiology, Ohio State University, Columbus 43210, USA.
Biotechnology (N Y). 1995 May;13(5):504-6. doi: 10.1038/nbt0595-504.
Preparations of rHMfA (recombinant histone A from Methanothermus fervidus) synthesized in E. coli by the heterologous expression of the hmfA gene were found to contain a mixture of rHMfA molecules, approximately 40% that retained the N-terminal formyl-methionyl residue (f-met-rHMfA), approximately 50% that lacked the formyl moiety but retained the methionyl residue (met-rHMfA), and only approximately 10% that had lost both components of the protein synthesis initiating amino acid residue and therefore had the same N-terminal sequence as native HMfA molecules synthesized in Mt. fervidus. Expression of the hmfA gene in E. coli cells grown in the presence of trimethoprim and thymidine, coupled with the concurrent over-expression of a methionine aminopeptidase-encoding map gene, has been shown to overcome this N-terminal heterogeneity problem and to result in rHMfA preparations in which > 85% of the molecules have the fully processed, native N-terminal sequence. This procedure should be generally useful for ensuring N-terminal processing of recombinant proteins synthesized in E. coli.
通过在大肠杆菌中异源表达hmfA基因合成的rHMfA（来自嗜热栖热甲烷菌的重组组蛋白A）制剂被发现含有rHMfA分子的混合物，约40%保留N端甲酰甲硫氨酰残基（f-met-rHMfA），约50%缺乏甲酰基部分但保留甲硫氨酰残基（met-rHMfA），只有约10%失去了蛋白质合成起始氨基酸残基的两个组分，因此具有与在嗜热栖热甲烷菌中合成的天然HMfA分子相同的N端序列。已证明，在三甲氧苄氨嘧啶和胸苷存在下生长的大肠杆菌细胞中hmfA基因的表达，与同时过量表达编码甲硫氨酸氨肽酶的map基因相结合，可克服这种N端异质性问题，并产生rHMfA制剂，其中>85%的分子具有完全加工的天然N端序列。该方法通常可用于确保在大肠杆菌中合成的重组蛋白的N端加工。
