SECRETION OF HETEROLOGOUS PROTEINS BY ASPERGILLUS-NIGER - PRODUCTION OF ACTIVE HUMAN INTERLEUKIN-6 IN A PROTEASE-DEFICIENT MUTANT BY KEX2-LIKE PROCESSING OF A GLUCOAMYLASE-HIL6 FUSION PROTEIN
SECRETION OF HETEROLOGOUS PROTEINS BY ASPERGILLUS-NIGER - PRODUCTION OF ACTIVE HUMAN INTERLEUKIN-6 IN A PROTEASE-DEFICIENT MUTANT BY KEX2-LIKE PROCESSING OF A GLUCOAMYLASE-HIL6 FUSION PROTEIN
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DOI：
10.1016/0168-1656(93)90156-h
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发表时间：
1993-11-01
影响因子：
4.1
通讯作者：
VANDENHONDEL, CAMJJ
中科院分区：
文献类型：
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作者：
BROEKHUIJSEN, MP;MATTERN, IE;VANDENHONDEL, CAMJJ
为了研究尼日尔生产异源蛋白的方法，我们研究了人白细胞介素-6（hIL-6）的分泌。由于在体外培养基实验中发现hIL 6被迅速降解，几种蛋白酶缺陷的A.分离尼日尔，并测试与野生型菌株相比hIL 6降解的降低。用几种hIL-6表达质粒转化对hIL-6降解作用最小的突变株（命名为AB 1.13）。最初，使用融合到成熟hIL 6序列的各种信号序列表达hIL 6。所得转化体不产生可检测量的hIL 6，尽管在一个PCR中具有高转录水平。我们假设hIL 6在沿着分泌途径传代期间没有被有效加工。因此，hIL 6表达为与葡糖淀粉酶的融合蛋白，葡糖淀粉酶是由A.尼日尔，并且其表达可以容易地通过酶法测量。为了获得成熟的hIL 6，将编码KEX 2切割位点（Lys-Arg）的序列插入葡糖淀粉酶和hIL 6序列之间。发现成熟的活性hIL 6分泌在细胞外培养基中。使用这种组合的方法转化蛋白酶缺陷型菌株与融合构建体含有KEX 2网站，高达15毫克升-1活性hIL 6在摇瓶培养中获得。没有KEX 2位点的融合构建体导致融合蛋白的显著更高的产量，但是hIL 6在融合形式中没有活性。这些结果表明A.尼日尔含有一种蛋白酶，其特异性与酵母的KEX 2蛋白酶相似。
To develop improved methods for heterologous protein production in Aspergillus niger, we studied the secretion of human interleukin-6 (hIL6). Since in vitro experiments with culture medium revealed that hIL6 was rapidly degraded, several protease-deficient strains of A. niger were isolated and tested for reduced degradation of hIL6 compared with the wild-type strain. The mutant strain giving the least degradative effect on hIL6 (designated AB1.13) was transformed with several hIL6-expression plasmids. Initially, hIL6 was expressed using various signal sequences fused to the sequence of mature hIL6. The resulting transformants did not produce detectable amounts of hIL6, despite high transcription levels in one transformant. We hypothesized that hIL6 was not efficiently processed during passage along the secretion pathway. Therefore, hIL6 was expressed as a fusion protein with glucoamylase, a protein which is efficiently secreted by A. niger and expression of which can easily be measured enzymatically. To obtain mature hIL6, a sequence encoding the KEX2 cleavage-site (Lys-Arg) was inserted between glucoamylase and hIL6 sequences. Mature active hIL6 was found to be secreted in the extracellular medium. Using this combined approach of transforming a protease-deficient strain with a fusion construct containing the KEX2 site, up to 15 mg l-1 active hIL6 was obtained in shake-flask culture. A fusion construct without the KEX2 site resulted in substantially higher production of the fusion protein, but hIL6 was not active in the fused form. These results indicate that A. niger contains a protease with similar specificity as the KEX2 protease from yeast.