A CIRCULARLY PERMUTED RECOMBINANT INTERLEUKIN-4 TOXIN WITH INCREASED ACTIVITY
A CIRCULARLY PERMUTED RECOMBINANT INTERLEUKIN-4 TOXIN WITH INCREASED ACTIVITY
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DOI：
10.1073/pnas.91.15.6889
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发表时间：
1994-07-19
影响因子：
11.1
通讯作者：
PASTAN, I
中科院分区：
文献类型：
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作者：
KREITMAN, RJ;PURI, RK;PASTAN, I
配体如生长因子与其他蛋白质的融合通常显著降低配体对其受体的亲和力。利用重组DNA技术，两种蛋白质之间的连接点迄今为止仅限于配体的氨基或羧基末端。然而，如果两端都接近配体与其受体结合的位点，则结合可能会大大受损。为了构建在生长因子上的新位点处连接的单链生长因子融合蛋白，我们构建了编码环状排列的白细胞介素4（IL 4）的DNA片段，称为IL 4（38-37）。这是通过将起始密码子置于位置38之前，将密码子1和129与编码肽接头的序列连接，并将终止密码子置于IL 4的密码子37之后来实现的。IL 4（38-37）通过其新的羧基末端Lys（37）与假单胞菌外毒素的截短形式融合。纯化的环状排列的IL 4-毒素与IL 4受体的结合亲和力比其中毒素与IL 4的羧基末端融合的IL 4-毒素高10倍。生长因子的环状排列可以提高重组融合蛋白的有效性，因为连接可以移动到生长因子上的某个位点，使其能够以更高的亲和力结合。
Fusion of ligands such as growth factors to other proteins often dramatically reduces the affinity of the ligand for its receptor. With recombinant DNA techniques, the attachment point between the two proteins has until now been restricted to either the amino or the carboxyl terminus of the ligand. However, binding may be greatly compromised if both ends are close to the site at which the ligand binds to its receptor. To construct a single-chain growth factor fusion protein with the connection at a new site on the growth factor, we constructed a DNA fragment encoding circularly permuted interleukin 4 (IL4), termed IL4(38-37). This was accomplished by placing a start codon before position 38, connecting codons 1 and 129 with a sequence encoding a; peptide linker, and placing a stop codon after codon 37 of IL4. IL4(38-37) was fused via its new carboxyl terminus, Lys(37), to a truncated form of Pseudomonas exotoxin. The purified circularly permuted IL4-toxin bound to the IL4 receptor with 10-fold higher affinity than an IL4-toxin in which the toxin was fused to the carboxyl terminus of IL4. Circular permuteins of growth factors can improve the effectiveness of recombinant fusion proteins, because the junction can be moved to a site on the growth factor which allows it to bind with higher affinity.