Libby R T, Braedt G, Kronheim S R, March C J, Urdal D L, Chiaverotti T A, Tushinski R J, Mochizuki D Y, Hopp T P, Cosman D
DNA. 1987 Jun;6(3):221-9. doi: 10.1089/dna.1987.6.221.
The human granulocyte-macrophage colony stimulating factor (GM-CSF) was expressed and purified from a high-level Escherichia coli secretion vector. A cDNA fragment encoding mature GM-CSF was fused with the aid of a synthetic oligonucleotide to the E. coli outer membrane signal peptide (ompA) of the secretion expression vector pIN-III-ompA3. The primary construction, designated pLB5001, is under transcriptional control of the tandem lipoprotein promoter (lppP) lactose promoter-operator (lacPO), and is regulated by the lactose repressor. Upon induction, a polypeptide of MW = 14,600 was produced which had GM-CSF activity in a human bone marrow colony assay. The linker sequence between the ompA signal peptide and the amino terminus of the mature GM-CSF was removed by oligonucleotide-directed site-specific mutagenesis to produce GM-CSF with an authentic amino terminus. The resulting construct, designated pLB5001-4, expressed authentic GM-CSF with a specific activity similar to that observed for the pLB5001 specified GM-CSF. Both versions of GM-CSF were associated with the membrane fraction after osmotic shock, and were purified to homogeneity by DEAE-Sephacel chromatography, followed by reversed-phase HPLC. Amino acid sequencing from the amino terminus of the purified GM-CSF established that the ompA signal peptide was cleaved at its normal processing site in both cases.
人粒细胞巨噬细胞集落刺激因子（GM-CSF）是从一种高效大肠杆菌分泌载体中表达并纯化得到的。编码成熟GM-CSF的cDNA片段借助合成寡核苷酸与分泌表达载体pIN-III-ompA3的大肠杆菌外膜信号肽（ompA）融合。最初构建的载体命名为pLB5001，受串联脂蛋白启动子（lppP）-乳糖启动子-操纵子（lacPO）的转录控制，并由乳糖阻遏物调控。诱导后，产生了一种分子量为14,600的多肽，其在人骨髓集落测定中具有GM-CSF活性。通过寡核苷酸定向位点特异性诱变去除了ompA信号肽与成熟GM-CSF氨基末端之间的连接序列，以产生具有真实氨基末端的GM-CSF。所得构建体命名为pLB5001-4，表达的真实GM-CSF的比活性与pLB5001指定的GM-CSF相似。两种版本的GM-CSF在渗透休克后都与膜部分相关联，并通过DEAE-琼脂糖凝胶色谱法，随后进行反相高效液相色谱法纯化至均一。从纯化的GM-CSF氨基末端进行的氨基酸测序确定，在两种情况下ompA信号肽都在其正常加工位点被切割。
