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    Process for producing peptides in E. coli
    1.
    发明授权
    Process for producing peptides in E. coli 失效
    在大肠杆菌中生产肽的方法

    公开(公告)号：US5670340A

    公开(公告)日：1997-09-23

    申请号：US352179

    申请日：1994-12-05

    申请人： Masayuki Yabuta ,  Yuji Suzuki ,  Kazuhiro Ohsuye ,  Takehiro Oshima ,  Seiko Onai ,  Koji Magota ,  Shoji Tanaka

    发明人： Masayuki Yabuta ,  Yuji Suzuki ,  Kazuhiro Ohsuye ,  Takehiro Oshima ,  Seiko Onai ,  Koji Magota ,  Shoji Tanaka

    IPC分类号： C12N15/09 ,  C07K14/58 ,  C07K14/585 ,  C12N9/38 ,  C12N15/16 ,  C12N15/62 ,  C12N15/67 ,  C12N15/70 ,  C12P21/02 ,  C12P21/06 ,  C12R1/19 ,  C12P21/00

    CPC分类号： C07K14/58 ,  C07K14/585 ,  C12N15/62 ,  C12N9/2471 ,  C12Y302/01023 ,  C07K2319/00 ,  C07K2319/23 ,  C07K2319/43 ,  C07K2319/50 ,  C07K2319/75 ,  C07K2319/90

    摘要： The present invention is a process to express a target peptide in a large amount and accumulate the target peptide in host cells in the form of inclusion bodies. The process comprises: A) culturing host cells transformed with a plasmid able to express a gene coding for a fusion protein represented in the formula A--L--B, wherein B is a target peptide, A is a protective peptide comprising a 90-210 amino acid fragment E. coli .beta.-galactosidase, and L is a linker peptide positioned between the C-terminus of the protective peptide and the N-terminus of the target peptide and selected so that when the fusion protein is treated by an enzyme or chemical substance, the target peptide is separated, and wherein the protective peptide and linker peptide are selected so that the isoelectric point of the fusion protein in between 4.9 and 6.9; B) obtaining an insoluble fraction comprising inclusion bodies by homogenization of th cultured transformed cells; C) solubilizing the fusion protein in the inclusion bodies by treatment of the insoluble fraction with a solubilizing agent; and, D) cleaving the peptide bond between the C-terminus of the linker peptide and the N-terminus of the target peptide of the solubilized fusion protein to release the target peptide from the other peptides followed by purification of the target peptide.

    摘要翻译： 本发明是大量表达目标肽并以包涵体的形式在宿主细胞中聚集靶肽的方法。 该方法包括：A）培养用能够表达编码式ALB中表示的融合蛋白的基因的质粒转化的宿主细胞，其中B是靶肽，A是包含90-210个氨基酸片段E的保护肽 大肠杆菌β-半乳糖苷酶，L是位于保护肽的C-末端和靶肽的N末端之间的连接肽，并且被选择使得当融合蛋白被酶或化学物质处理时，靶 分离肽，并且其中选择保护性肽和接头肽，使得融合蛋白的等电点在4.9和6.9之间; B）通过均质培养的转化细胞获得包含包含体的不溶性级分; C）通过用增溶剂处理不溶性级分将融合蛋白溶解在包涵体中; 并且D）切割接头肽的C末端和溶解的融合蛋白的靶肽的N末端之间的肽键，从其他肽释放靶肽，然后纯化目标肽。

    Process for production of protein
    2.
    发明授权
    Process for production of protein 失效
    蛋白质生产工艺

    公开(公告)号：US6037145A

    公开(公告)日：2000-03-14

    申请号：US523373

    申请日：1995-09-05

    申请人： Masayuki Yabuta ,  Kazuhiro Ohsuye

    发明人： Masayuki Yabuta ,  Kazuhiro Ohsuye

    IPC分类号： C12N1/21 ,  C12N9/52 ,  C12N15/62 ,  C12N15/63

    CPC分类号： C12N9/52 ,  C12N15/62 ,  C07K2319/00

    摘要： A process for the production of a desired polypeptide comprising the steps of: (1) transforming host cells with an expression vector comprising a gene coding for a fusion protein comprising a desired polypeptide and a protective polypeptide; (2) culturing the transformed host cells so as to express said gene to produce a fusion protein; and (3) excising the desired polypeptide from the fusion protein with a protease intrinsic to the host cells. According to the present invention, a large amount of a desired polypeptide can be produced at a low cost. Especially according to the present invention, a large amount of S. aureus V8 protease can be efficiently produced at low cost using a safe host such as E. coli according to gene recombination procedures.

    摘要翻译： 一种制备所需多肽的方法，包括以下步骤：（1）用包含编码包含所需多肽和保护性多肽的融合蛋白的基因的表达载体转化宿主细胞; （2）培养转化的宿主细胞，以表达所述基因以产生融合蛋白; 和（3）用融合蛋白切割所需的多肽与宿主细胞固有的蛋白酶。 根据本发明，可以低成本生产大量所需的多肽。 特别是根据本发明，可以使用安全宿主如大肠杆菌根据基因重组方法以低成本有效地生产大量的金黄色葡萄球菌V8蛋白酶。

    Mutant Staphylococcus aureus V8 proteases
    3.
    发明授权
    Mutant Staphylococcus aureus V8 proteases 失效
    突变型金黄色葡萄球菌V8蛋白酶

    公开(公告)号：US5747321A

    公开(公告)日：1998-05-05

    申请号：US657192

    申请日：1996-06-03

    申请人： Masayuki Yabuta ,  Kazuhiro Ohsuye

    发明人： Masayuki Yabuta ,  Kazuhiro Ohsuye

    IPC分类号： C07K14/58 ,  C12N9/52 ,  C07H21/04 ,  C12N1/20 ,  C12N15/00

    CPC分类号： C07K14/58 ,  C12N9/52 ,  C07K2319/00

    摘要： Mutant proteases are obtained with one or more mutation sites in the natural V8 protease protein, and with enzyme activities even in the presence of high urea concentrations. Inactivation of enzyme activity is minimized even in the presence of high concentrations of urea, to thus allow lower amounts of enzyme to be added to urea-containing reaction systems and shorten reaction times. As an additional advantage, the ability to cleave proteins in the presence of high urea concentrations makes it possible to obtain hitherto unobtainable peptide fragments.

    摘要翻译： 用天然V8蛋白酶蛋白质中的一个或多个突变位点获得突变蛋白酶，即使在高尿素浓度的存在下也可获得酶活性。 即使在高浓度的尿素存在下，酶活性的失活最小化，从而允许较少量的酶加入到含脲反应体系中并缩短反应时间。 作为另外的优点，在高尿素浓度存在下切割蛋白质的能力使得可能获得迄今无法获得的肽片段。

    Method of controlling cleavage by OmpT protease
    5.
    发明授权
    Method of controlling cleavage by OmpT protease 失效
    通过OmpT蛋白酶控制切割的方法

    公开(公告)号：US07344856B1

    公开(公告)日：2008-03-18

    申请号：US09674777

    申请日：2000-03-03

    申请人： Kazuaki Okuno ,  Masayuki Yabuta ,  Kazuhiro Ohsuye

    发明人： Kazuaki Okuno ,  Masayuki Yabuta ,  Kazuhiro Ohsuye

    IPC分类号： C12P21/06 ,  C12P21/00 ,  C12N9/48 ,  C12N1/20 ,  C12N15/00

    CPC分类号： C07K14/585 ,  C07K14/58 ,  C07K14/605 ,  C07K2319/50 ,  C07K2319/75 ,  C12N15/62 ,  C12N15/63

    摘要： Methods of using OmpT protease are provided. In particular, the invention provides for a method of controlling cleavage of a polypeptide by OmpT protease comprising, converting a sequence site comprising two arbitrary consecutive amino acids and/or amino acid(s) in the vicinity of the site in the polypeptide into other amino acids; which method comprises setting lysine or arginine as the amino acid at the −1-position concerning the site and setting a specific amino acid as the amino acid at the +1-position; and/or setting specific amino acid(s) as the amino acid(s) at the −4-position and/or the −6-position relative to the site; so that a desired part of the polypeptide is cleaved by OmpT protease and/or an undesired part of the polypeptide is not (or hardly) cleaved by OmpT protease. A method of producing a target polypeptide is also provided.

    摘要翻译： 提供了使用OmpT蛋白酶的方法。 特别地，本发明提供了一种控制OmpT蛋白质切割多肽的方法，其包括：将包含多肽中位点附近的两个任意连续氨基酸和/或氨基酸的序列位点转化成其他氨基 酸; 该方法包括将赖氨酸或精氨酸设置为与位点相关的-1位的氨基酸，并将特定的氨基酸设置为+ 1位的氨基酸; 和/或将特异性氨基酸设置为相对于位点的-4位和/或-6位的氨基酸; 使得所需部分的多肽被OmpT蛋白酶切割和/或多余的不需要的部分不被OmpT蛋白酶切割（或几乎不被切割）。 还提供了产生靶多肽的方法。

    Methods for reducing the formation of by-products in the production of recombinant polypeptides
    7.
    发明授权
    Methods for reducing the formation of by-products in the production of recombinant polypeptides 有权
    减少重组多肽生产中副产物形成的方法

    公开(公告)号：US07547529B1

    公开(公告)日：2009-06-16

    申请号：US10030452

    申请日：2001-05-10

    申请人： Masayuki Yabuta ,  Toshihiro Sawano ,  Yumiko Masuda ,  Kazuhiro Ohsuye

    发明人： Masayuki Yabuta ,  Toshihiro Sawano ,  Yumiko Masuda ,  Kazuhiro Ohsuye

    IPC分类号： C12P21/02

    CPC分类号： C12P21/02 ,  C07K14/58

    摘要： A method for reducing the formation of a byproduct polypeptide containing an O-acetylserine residue in place of a serine residue by adding at least one of histidine, methionine or glycine to the medium in a method for producing a polypeptide containing a serine residue by culturing transformed cells, and a method for producing a polypeptide containing a serine residue by culturing transformed cells, characterized by reducing the formation of a byproduct polypeptide containing an O-acetylserine residue in place of a serine residue by adding at least one of histidine, methionine or glycine to the medium.

    摘要翻译： 一种通过在生产含有丝氨酸残基的多肽的方法中加入至少一种组氨酸，甲硫氨酸或甘氨酸来减少含有O-乙酰丝氨酸残基的副产物多肽代替丝氨酸残基的方法，该方法是通过培养转化的 细胞和通过培养转化细胞产生含有丝氨酸残基的多肽的方法，其特征在于通过加入组氨酸，甲硫氨酸或甘氨酸中的至少一种来减少含有O-乙酰丝氨酸残基的副产物多肽代替丝氨酸残基的形成 到中等

    Method for controlling gene expression
    8.
    发明授权
    Method for controlling gene expression 失效
    控制基因表达的方法

    公开(公告)号：US5622840A

    公开(公告)日：1997-04-22

    申请号：US456923

    申请日：1995-06-01

    申请人： Masayuki Yabuta ,  Seiko Miura ,  Kazuhiro Ohsuye

    发明人： Masayuki Yabuta ,  Seiko Miura ,  Kazuhiro Ohsuye

    IPC分类号： C12N15/09 ,  C07K14/00 ,  C07K14/245 ,  C07K14/575 ,  C07K14/58 ,  C07K14/585 ,  C07K14/61 ,  C07K14/62 ,  C12N15/72 ,  C12P21/00 ,  C12R1/19 ,  C12P21/02 ,  C12N15/31

    CPC分类号： C12N15/72 ,  C07K14/245

    摘要： A lactose repressor protein wherein at least one amino acid at the position of 94, 241, 265 or 300 in the wild lactose repressor is replaced with an amino acid other than that of the wild lactose repressor, and the use thereof.The present mutant lactose repressor represses the expression of a desired gene at 30.degree. C. or lower temperature, and induces the expression of a desired gene at 37.degree. C. or higher temperature, and therefor can control the expression by change of a culture temperature without using an expensive inducer such as IPTG.

    摘要翻译： 野生型乳糖阻遏物中94,241,265或300位的至少一个氨基酸被除了野生型乳糖阻遏物以外的氨基酸所取代的乳糖阻遏蛋白及其用途。 本发明的突变型乳糖阻遏物在30℃或更低温度下抑制所需基因的表达，并在37℃或更高的温度下诱导所需基因的表达，因此可通过培养温度的变化来控制表达 而不使用昂贵的诱导剂如IPTG。