Mikosch T, Klemm P, Gassen H G, van den Hondel C A, Kemme M
Institut für Biochemie, Technische Hochschule Darmstadt, Germany.
J Biotechnol. 1996 Dec 10;52(2):97-106. doi: 10.1016/s0168-1656(96)01634-3.
We report the production of human mucus proteinase inhibitor (MPI) by the filamentous fungus Aspergillus niger to test the ability of this host organism to secrete low molecular weight, highly disulfide-bonded proteins in biologically active conformation. Fungal transformants have been obtained with an expression cassette consisting of a chimeric gene founded on a mpi cDNA, encoding mature MPI, fused in frame to sequences encoding A. niger glucoamylase (glaA), separated by a KEX2-like processing sequence. Expression of the glucoamylase fusion gene in these transformants resulted in secretion of MPI into the growth medium with yields up to 3 mg 1-1. N-terminal sequence analysis of the purified inhibitor confirmed that the glucoamylase-MPI fusion protein was correctly processed to mature MPI by a KEX2-type endopeptidase present in A. niger. Furthermore, recombinant MPI retains full inhibitory activity against chymotrypsin and leukocyte elastase indicating that the protein was folded properly.
我们报道了丝状真菌黑曲霉产生人黏液蛋白酶抑制剂（MPI）的情况，以测试这种宿主生物体分泌具有生物活性构象的低分子量、高度二硫键连接蛋白的能力。通过一个表达盒获得了真菌转化体，该表达盒由一个基于mpi cDNA构建的嵌合基因组成，该基因编码成熟的MPI，与编码黑曲霉糖化酶（glaA）的序列读框融合，并由一个类似KEX2的加工序列隔开。这些转化体中糖化酶融合基因的表达导致MPI分泌到生长培养基中，产量高达3 mg·L⁻¹。对纯化抑制剂的N端序列分析证实，糖化酶-MPI融合蛋白被黑曲霉中存在的KEX2型内肽酶正确加工成成熟的MPI。此外，重组MPI对胰凝乳蛋白酶和白细胞弹性蛋白酶保留了完全的抑制活性，表明该蛋白折叠正确。
