Utilization of Escherichia coli Outer-Membrane Endoprotease OmpT Variants as Processing Enzymes for Production of Peptides from Designer Fusion Proteins
Utilization of Escherichia coli Outer-Membrane Endoprotease OmpT Variants as Processing Enzymes for Production of Peptides from Designer Fusion Proteins
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利用大肠杆菌外膜内切蛋白酶 OmpT 变体作为加工酶从设计融合蛋白生产肽
DOI：
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发表时间：
2004
影响因子：
4.4
通讯作者：
S. Kinoshita
中科院分区：
文献类型：
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作者：
K. Okuno;M. Yabuta;T. Ooi;S. Kinoshita
ABSTRACT Escherichia coli outer-membrane endoprotease OmpT has suitable properties for processing fusion proteins to produce peptides and proteins. However, utilization of this protease for such production has been restricted due to its generally low cleavage efficiency at Arg (or Lys)-Xaa, where Xaa is a nonbasic N-terminal amino acid of a target polypeptide. The objective of this study was to generate a specific and efficient OmpT protease and to utilize it as a processing enzyme for producing various peptides and proteins by converting its substrate specificity. Since OmpT Asp97 is proposed to interact with the P1′ amino acid of its substrates, OmpT variants with variations at Asp97 were constructed by replacing this amino acid with 19 natural amino acids to alter the cleavage specificity at Arg (P1)-Xaa (P1′). The variant OmpT that had a methionine at this position, but not the wild-type OmpT, efficiently cleaved a fusion protein containing the amino acid sequence -Arg-Arg-Arg-Ala-Arg↓motilin, in which motilin is a model peptide with a phenylalanine at the N terminus. The OmpT variants with leucine and histidine at position 97 were useful in releasing human adrenocorticotropic hormone (1-24) (serine at the N terminus) and human calcitonin precursor (cysteine at the N terminus), respectively, from fusion proteins. Motilin was produced by this method and was purified up to 99.0% by two chromatographic steps; the yield was 160 mg/liter of culture. Our novel method in which the OmpT variants are used could be employed for production of various peptides and proteins.
人源大肠杆菌分离株中 ompT 的流行率。
影响因子：
2.1
作者：
Lundrigan,MD;Webb,RM
通讯作者：
Webb,RM