Lacal J C, Santos E, Notario V, Barbacid M, Yamazaki S, Kung H, Seamans C, McAndrew S, Crowl R
Proc Natl Acad Sci U S A. 1984 Sep;81(17):5305-9. doi: 10.1073/pnas.81.17.5305.
The H-ras gene of the BALB murine sarcoma virus (BALB-MSV) was placed under the transcriptional control of the tightly regulated PL promoter of bacteriophage lambda in the expression vectors pEV-vrf-1 and pRC23. Upon derepression of the PL promoter, large amounts (10-20% of total cellular protein) of the H-ras gene product p21 are synthesized in Escherichia coli. We constructed three H-ras gene expression vectors, designated pJCL-H5, pJCL-E30, and pJCL-33. pJCL-H5 directs the synthesis of p21, a fusion protein whose four amino-terminal residues are replaced by eight amino acids coded for by plasmid sequences. The 13 5' coding nucleotides of the BALB-MSV H-ras gene missing in pJCL-H5 were regenerated in pJCL-E30 by inserting a pair of complementary synthetic oligodeoxynucleotides. As a result, pJCL-E30 encodes a p21 protein, p21T, of sequence identical to that of the transforming p21 protein of BALB-MSV. pJCL-33 is a derivative of pJCL-E30 in which the 12th codon, AAA, a lysine codon, was replaced by GGA, a glycine codon. Thus, pJCL-33 directs the synthesis of a p21 protein, p21N, whose sequence corresponds to that of a normal cellular p21 protein. We report the purification of H-ras p21 proteins to apparent homogeneity by a method involving solubilization with chaotropic agents followed by reverse-phase high-performance liquid chromatography.
将BALB鼠肉瘤病毒（BALB-MSV）的H-ras基因置于表达载体pEV-vrf-1和pRC23中受噬菌体λ严格调控的PL启动子的转录控制之下。在PL启动子去阻遏后，在大肠杆菌中合成了大量（占细胞总蛋白的10%-20%）的H-ras基因产物p21。我们构建了三个H-ras基因表达载体，分别命名为pJCL-H5、pJCL-E30和pJCL-33。pJCL-H5指导合成p21，这是一种融合蛋白，其四个氨基末端残基被质粒序列编码的八个氨基酸所取代。通过插入一对互补的合成寡脱氧核苷酸，在pJCL-E30中再生了pJCL-H5中缺失的BALB-MSV H-ras基因的13个5'编码核苷酸。结果，pJCL-E30编码一种p21蛋白p21T，其序列与BALB-MSV的转化p21蛋白相同。pJCL-33是pJCL-E30的衍生物，其中第12个密码子AAA（赖氨酸密码子）被GGA（甘氨酸密码子）取代。因此，pJCL-33指导合成一种p21蛋白p21N，其序列与正常细胞p21蛋白的序列相对应。我们报告了通过一种方法将H-ras p21蛋白纯化至表观均一，该方法包括用离液剂溶解，然后进行反相高效液相色谱。
