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Kex2p在甲醇酵母中的分泌表达 被引量：3

Expression of Kex2p in Pichia pastoris
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摘要 通过 PCR方法得到去除 C端 2 0 1个氨基酸残基 (胞浆区 ,跨膜区和 Ser/Thr丰富区 )的Kex2 p的 DNA片段 ,并将该片段装载到质粒 p PICZ- C中构建成表达质粒 p PICZ- C- KEX2 ΔCP。经过大肠杆菌 ( DH5 α)的扩增 ,表达质粒被转入甲醇酵母 ( GS1 1 5 )中 ,并得以分泌表达。发酵上清液中存在切割肽链中双碱性氨基酸 C端的酶活力。SDS- PAGE电泳的结果证实了 Kex2 p的分泌表达 ,以及 Kex2 A DNA segment, which encodes truncated Kex2p with a deletion of 201 residues on C terminus (cytosolic domain, transmembrane domain and Ser/Thr rich domain), was obtained by PCR method. An expression plasmid (pPICZ C KEX2ΔCP) was made by cloning this segment into pPICZ C. After being amplified in E.coli (DH5α), the constructed plasmid was transformed into Pichia pastoris (GS115) and was expressed. The supernatant showed an enzymatic activity of hydrolyzing on the carboxyl side of paired basic residues in peptides. Result of SDS PAGE confirmed the secreted expression and the auto processing of Kex2p in aqueous solution.
作者 艾敏榕 杨曜中
机构地区 华东理工大学生物化学系
出处 《华东理工大学学报（自然科学版）》 CAS CSCD 北大核心 2002年第2期161-163,215,共4页 Journal of East China University of Science and Technology
关键词 Kex2p 丝氨酸蛋白酶 前体加工酶 甲醇酵母 Kex2p serine protease proprotein convertase Pichia pastoris
分类号 Q556.3 [生物学—生物化学]
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参考文献9

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同被引文献15

  • 1王秀青,张素芳,曹瑞兵,周斌,陈溥言.抗菌肽天蚕素B基因及其串联体在毕赤酵母中的表达[J].南京农业大学学报,2007,30(3):120-123. 被引量：20
  • 2Holyoak T, Wilson MA, Fenn TD. 2. 4 Resolution Crystal Structure of the Prototypical Hormone-Processing Protease Kex2 in Complex with an Ma-Lys-Arg Boronic Acid inhibitor [ J ] . Biochemistry, 2003, ,2 (22) : 6709-6718.
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  • 4Fuller RS, Sleme RE, Thomer J. Errne required for yeast prohonnone processing [ J ]. Annual Reviews Physiol, 1988,50 ( 1 ) : 345-362.
  • 5Sreenivas S, Krishnaiah S, Govindappa N. Enhancement in production of recombinant two-chain Insulin Glargine by over-expression of Kex2 protease in Piehia past0ris [ J ] . Applied Microbiology and Biotechnology, 2014, 5:178-199.
  • 6Kensaku M, Tomoko N. Yeast Kex2 gen encodes an endopeptldase homologous to subtilisin-llke seine pteases [J ] .Biochencal and Biophysical reseamh conmamicafiom, 1988, 156:246-254.
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华东理工大学学报（自然科学版）
2002年 第2期

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