THE EXPRESSION OF ESCHERICHIA-COLI DIAMINOPIMELATE DECARBOXYLASE IN MOUSE 3T3 CELLS
THE EXPRESSION OF ESCHERICHIA-COLI DIAMINOPIMELATE DECARBOXYLASE IN MOUSE 3T3 CELLS
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DOI：
10.1016/0167-4781(94)90064-7
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发表时间：
1994-10-18
期刊：
影响因子：
--
通讯作者：
REES, WD
中科院分区：
文献类型：
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作者：
SAQIB, KM;HAY, SM;REES, WD
我们已经亚克隆了编码二氨基庚二酸脱羧酶（DAP脱羧酶）的大肠杆菌lysA基因的编码序列到基于SV 40早期启动子的真核表达载体中。通过测定小鼠3 T3细胞中二氨基庚二酸（DAP）合成赖氨酸的能力，比较了在编码区5'和3'端具有不同长度非编码DNA的一系列构建体的活性。在3'端的短的非编码序列降低酶活性的表达。通过将嵌合基因与编码G-418抗性的质粒共转染，已经产生了3 T3细胞的稳定系。使细胞在含有G-418的培养基中生长，并筛选抗性克隆从DAP合成赖氨酸的能力。将由[H-3]DAP产生的[H-3]赖氨酸掺入细胞蛋白中。从已经掺入两个拷贝的基因的细胞系中提取的酶提取物每mg蛋白质合成0.082 nmol赖氨酸/min。在完整细胞中，赖氨酸合成的速率受到DAP摄取的限制，DAP的摄取仅为赖氨酸速率的5%。lysA在哺乳动物细胞基因表达的研究中具有作为报告基因的潜力。
We have subcloned the coding sequence for the Escherichia coli lysA gene coding for diaminopimelic acid decarboxylase (DAP decarboxylase) into a eukaryotic expression vector based on the SV40 early promoter. The activities of a series of constructs with different lengths of non-coding DNA at the 5' and 3' ends of the coding region have been compared by measuring the synthesis of lysine from diaminopimelic acid (DAP) in mouse 3T3 cells. A short non-coding sequence at the 3' end reduced the expression of enzyme activity. Stable lines of 3T3 cells have been produced by co-transfection of the chimeric gene with a plasmid coding for G-418 resistance. Cells were grown in medium containing G-418 and resistant clones were screened for an ability to synthesise lysine from DAP. [H-3]Lysine produced from [H-3]DAP was incorporated into cell proteins. An enzyme extract from a cell line which had incorporated two copies of the gene synthesised 0.082 nmol of lysine/min per mg protein. In the intact cell the rate of lysine synthesis is limited by the uptake of DAP which is taken up at only 5% of the rate of lysine. lysA has a potential as a reporter gene in studies of gene expression in mammalian cells.