EXPRESSION IN BACTERIA OF FUNCTIONAL INHIBITORY SUBUNIT OF RETINAL ROD CGMP PHOSPHODIESTERASE
EXPRESSION IN BACTERIA OF FUNCTIONAL INHIBITORY SUBUNIT OF RETINAL ROD CGMP PHOSPHODIESTERASE
复制标题
DOI：
10.1073/pnas.86.13.4922
复制
发表时间：
1989-07-01
影响因子：
11.1
通讯作者：
STRYER, L
中科院分区：
文献类型：
--
作者：
BROWN, RL;STRYER, L
脊椎动物视网膜杆外节的 cGMP 磷酸二酯酶在视觉转导中起着关键作用。抑制性γ的功能活性形式。磷酸二酯酶亚基使酶在黑暗中保持失活，已从大肠杆菌中表达的合成基因中以高产率获得。编码87个残基的牛γ的DNA序列。亚基是由 10 个寡核苷酸化学合成和组装而成。将合成基因克隆到使用λ的启动子PL的表达载体中。噬菌体。用该载体转化大肠杆菌，该载体编码由 lambda 的前 31 个残基组成的融合蛋白。 cII 蛋白，一种 7 个残基的连接序列，在其 C 末端被凝血蛋白酶因子 Xa 特异性切割，以及 87 个残基 .gamma。亚基。将融合蛋白溶解在 6 M 尿素中，并通过 CM-Sephadex 柱上的离子交换层析进行纯化。典型产量为每升细菌培养物 1 mg 融合蛋白，这对应于 γ 的量。大约 2500 个牛视网膜中。 Xa因子对融合蛋白的蛋白水解裂解释放出合成的γ。具有与天然γ相同的氨基酸序列。融合蛋白和合成γ。以高亲和力 (Kd < 100 pM) 抑制胰蛋白酶激活的磷酸二酯酶。同样，两者都与原生 .lambda 一样有效。抑制杆外节膜中转导蛋白激活的磷酸二酯酶。这种抑制可通过激活额外的转导蛋白而逆转。因此，γ的N末端。不密切参与与磷酸二酯酶的催化亚基或转导蛋白的活化形式的相互作用。相反，C端缺失突变体终止于λ的残基74。刺激而不是抑制胰蛋白酶激活酶。因此，γ的C末端区域。对于磷酸二酯酶的抑制至关重要。
The cGMP phosphodiesterase of vertebrate retinal rod outer segments plays a key role in visual transduction. A functionally active form of the inhibitory .gamma. subunit of the phosphodiesterase, which keeps the enzyme inactive in the dark, has been obtained in high yield from a synthetic gene expressed in Escherichia coli. A DNA sequence encoding the 87-residue bovine .gamma. subunit was chemically synthesized and assembled from 10 oligonucleotides. The synthetic gene was cloned into an expression vector that uses the promoter PL of .lambda. phage. E. coli was transformed with this vector, which encodes a fusion protein consisting of the first 31 residues of the .lambda. cII protein, a 7-residue joining sequence that is specifically cleaved at its C-terminal end by clotting protease factor Xa, and the 87-residue .gamma. subunit. The fusion protein was solubilized in 6 M urea and purified by ion-exchange chromatography on a CM-Sephadex column. The typical yield was 1 mg of fusion protein per liter of bacterial culture, which corresponds to the amount of .gamma. in about 2500 bovine retinas. Proteolytic cleavage of the fusion protein by factor Xa released a synthetic .gamma. with the same amino acid sequence as that of native .gamma.. Both fusion protein and synthetic .gamma. inhibited trypsin-activated phosphodiesterase with high affinity (Kd < 100 pM). Likewise, both were as effective as native .lambda. in inhibiting transducin-activated phosphodiesterase in rod outer segment membranes. This inhibition was reversed by the activation of additional transducin. Thus, the N terminus of .gamma. is not intimately involved in interactions with either the catalytic subunits of the phosphodiesterase or the activated form of transducin. In contrast, a C-terminal deletion mutant terminating at residue 74 of .lambda. stimulated rather than inhibited the trypsin-activated enzyme. Thus, the C-terminal region of .gamma. is critical for inhibition of the phosphodiesterase.