公开(公告)号：US06423538B1

公开(公告)日：2002-07-23

申请号：US09724297

申请日：2000-11-28

申请人： K. Dane Wittrup ,  David M. Kranz ,  Michele Keike ,  Eric T. Boder

发明人： K. Dane Wittrup ,  David M. Kranz ,  Michele Keike ,  Eric T. Boder

IPC分类号： C12N1581

CPC分类号： C12N15/1037 ,  C40B40/02 ,  G01N33/68 ,  G01N33/6854 ,  G01N2333/39

摘要： The present invention provides a genetic method for tethering polypeptides to the yeast cell wall in a form accessible for binding to macromolecules. Combining this method with fluorescence-activated cell sorting provides a means of selecting proteins with increased or decreased affinity for another molecule, altered specificity, or conditional binding. Also provided is a method for genetic fusion of the N terminus of a polypeptide of interest to the C-terminus of the yeast Aga2p cell wall protein. The outer wall of each yeast cell can display approximately 104 protein agglutinins. The native agglutinins serve as specific adhesion contacts to fuse yeast cells of opposite mating type during mating. If effect, yeast has evolved a platform for protein-protein binding without steric hindrance from cell wall components. As one embodiment, attaching an scFv antibody fragment to the Aga2p agglutinin effectively mimics the cell surface display of antibodies by B cells in the immune system for affinity maturation in vivo. As another embodiment, T cell receptor mutants can be isolated by this method that are efficiently displayed on the yeast cell surface, providing a means of altering T cell receptor binding affinity and specificity by library screening.

摘要翻译： 本发明提供了一种遗传方法，用于以能够与大分子结合的形式将多肽连接到酵母细胞壁。 将该方法与荧光激活细胞分选相结合提供了选择具有增加或降低的对另一分子的亲和力，改变的特异性或条件结合的蛋白质的方法。 还提供了目的多肽的N末端与酵母Aga2p细胞壁蛋白的C末端的遗传融合的方法。 每个酵母细胞的外壁可以显示大约104个蛋白质凝集素。 天然凝集素用作特异性粘附接触，以在交配期间融合相反交配型的酵母细胞。 如果发挥作用，酵母已经发展出蛋白质 - 蛋白质结合的平台，没有细胞壁组分的空间位阻。 作为一个实施方案，将scFv抗体片段连接到Aga2p凝集素上有效地模拟了免疫系统中B细胞的抗体的细胞表面显示，用于体内的亲和力成熟。 作为另一个实施方案，可以通过这种方法分离T细胞受体突变体，其有效地显示在酵母细胞表面上，提供通过文库筛选来改变T细胞受体结合亲和力和特异性的方法。

公开(公告)号：US06670148B2

公开(公告)日：2003-12-30

申请号：US09368834

申请日：1999-08-05

申请人： David D. Mundschenk ,  Leonard A. Smith

发明人： David D. Mundschenk ,  Leonard A. Smith

IPC分类号： C12N1581

CPC分类号： C07K1/1133 ,  C07K14/46

摘要： A method of preparing a bioactive polypeptide in a stable, inactivated form, the method comprising the step of treating the polypeptide with ozonated water in order to oxidize and/or stabilize the cysteine residues, and in turn, prevent the formation of disulfide bridges necessary for bioactivity. The method can involve the use of ozonated water to both oxidize the disulfide bridges in a bioactive polypeptide, and to then stabilize the resultant cysteine residues. Optionally, and preferably, the method can involve the use of ozonated water to stabilize the cysteine residues, and thereby prevent the formation of disulfide bridges, in a polypeptide produced by recombinant means in a manner that allows the polypeptide to be recovered with the disulfide bridges unformed.

摘要翻译： 一种以稳定的灭活形式制备生物活性多肽的方法，所述方法包括用臭氧水处理多肽以使氧化和/或稳定半胱氨酸残基的方法，从而防止形成二硫键形成 生物活性。 该方法可以涉及使用臭氧化水来氧化生物活性多肽中的二硫键，然后稳定所得的半胱氨酸残基。 并且优选地，优选地，该方法可以包括使用臭氧水来稳定半胱氨酸残基，从而防止在通过重组方法产生的多肽中形成二硫键，以允许多肽用二硫键回收 未成形

公开(公告)号：US06534315B1

公开(公告)日：2003-03-18

申请号：US09415216

申请日：1999-10-12

申请人： Jürgen Bauer ,  Valérie Nacken ,  Annie Loiez

发明人： Jürgen Bauer ,  Valérie Nacken ,  Annie Loiez

IPC分类号： C12N1581

CPC分类号： C12N15/81

摘要： The present invention relates to a DNA cassette intended for the transformation of yeast, leaving no useless exogenous DNA but the gene(s) of interest comprising at least one negative dominant marker, two direct repeat sequences (DRS) which are non exogenous and non recombinogenic with the genome of the host strain, these two direct repeat sequences flanking the negative dominant marker and optionally at least one gene of interest containing, if necessary, the elements necessary for its expression in the host cell. The invention relates as well to a method of integration of gene(s) of interest or inactivation of a gene in yeast, and of transformation of yeast with the DNA cassette, and to yeast strains thus obtained.

摘要翻译： 本发明涉及用于转化酵母的DNA盒，不留下无​​用的外源DNA，但目的基因包括至少一个阴性显性标志物，两种非外源性和非重组型直接重复序列（DRS） 与宿主菌株的基因组相反，这两个直接重复序列位于负主要标记和任选的至少一个感兴趣的基因侧翼，如果需要，其含有在宿主细胞中表达所必需的元件。 本发明还涉及将感兴趣的基因或酵母中的基因失活以及酵母与DNA盒的转化以及由此得到的酵母菌株整合的方法。

公开(公告)号：US06284534B1

公开(公告)日：2001-09-04

申请号：US09242690

申请日：1999-02-23

申请人： Keiji Kondo ,  Yutaka Miura

发明人： Keiji Kondo ,  Yutaka Miura

IPC分类号： C12N1581

CPC分类号： C12N9/2411 ,  C07K14/43 ,  C12N9/2408 ,  C12N15/815 ,  C12N15/905 ,  C12Y302/01001

摘要： An object of the present invention is to provide a vector which can be integrated into a yeast chromosome in a high number of copies. Another object of the present invention is to provide a modified vector which can be integrated into the yeast chromosome in a high number of copies and of which expression units stably maintain on the chromosome. The vector according to the present invention comprises a marker gene for selecting transformants, a shortened promoter sequence which is operably linked to the marker gene and a sequence homologous to the chromosomal DNA of Candida utilis, and optionally a heterologous gene or a gene derived from C. utilis, wherein the vector is linearized by cleaving within said homologous DNA sequence or at both ends of the homologous DNA sequence with restriction enzymes, and wherein the heterologous gene or the gene derived from C. utilis can be integrated into the chromosomal DNA of C. utilis by homologous recombination.

摘要翻译： 本发明的目的是提供一种能够以大量拷贝结合到酵母染色体中的载体。 本发明的另一个目的是提供一种修饰的载体，其能够以大量的拷贝整合到酵母染色体中，并且其表达单位稳定地保持在染色体上。 根据本发明的载体包含用于选择转化体的标记基因，可操作地连接到标记基因的缩短的启动子序列和与产朊假丝酵母的染色体DNA同源的序列，以及任选的异源基因或衍生自C的基因 其中载体通过在所述同源DNA序列内切割或用限制性内切酶在同源DNA序列的两端进行线性化，其中异源基因或源自产朊酵母的基因可以整合入C的染色体DNA中 通过同源重组产生。

公开(公告)号：US06197545B1

公开(公告)日：2001-03-06

申请号：US09269345

申请日：1999-03-25

申请人： Hong Fang ,  Neil Green ,  Luc Van Kaer

发明人： Hong Fang ,  Neil Green ,  Luc Van Kaer

IPC分类号： C12N1581

CPC分类号： C12N9/52 ,  C12N15/81

摘要： The present invention provides a yeast cell comprising a nucleic acid functionally encoding a eukaryotic (e.g., mammalian) protein homologue of a subunit (Sec11p, Spc1p, Spc2p and/or Spc3p) of the yeast signal peptidase complex. The yeast cell of this invention can be, for example, of the genus Saccharomyces, Schizosaccharomyces, Pichia, Hansenula, Kluyveromyces and/or Yarrowia. Furthermore, the yeast cell can lack one or more functional subunits (Sec11p, Spc1p, Spc2p and/or Spc3p) of the yeast signal peptidase complex. The present invention further provides a method for producing a protein heterologous to a yeast cell comprising expressing, in the yeast cell of this invention, a nucleic acid functionally encoding the heterologous protein under conditions which permit the expression of the nucleic acid as a precursor protein having a signal peptide and processing of the precursor protein to a signal peptide-cleaved form of the protein.

摘要翻译： 本发明提供了酵母细胞，其包含功能性编码酵母信号肽酶复合物的亚基（Sec11p，Spc1p，Spc2p和/或Spc3p）的真核（例如哺乳动物）蛋白质同源物的核酸。 本发明的酵母细胞可以是例如酵母属，裂殖酵母属，毕赤酵母，汉逊酵母，克鲁维酵母和/或耶氏酵母属。 此外，酵母细胞可以缺乏酵母信号肽酶复合物的一个或多个功能性亚基（Sec11p，Spc1p，Spc2p和/或Spc3p）。 本发明还提供了用于产生与酵母细胞异源的蛋白质的方法，包括在本发明的酵母细胞中表达在允许以表达核酸作为前体蛋白的条件下功能性编码异源蛋白质的核酸， 信号肽和前体蛋白加工成信号肽切割形式的蛋白质。

公开(公告)号：US06673613B2

公开(公告)日：2004-01-06

申请号：US09911781

申请日：2001-07-24

申请人： David L. Craft ,  C. Ron Wilson ,  Dudley Eirich ,  Yeyan Zhang

发明人： David L. Craft ,  C. Ron Wilson ,  Dudley Eirich ,  Yeyan Zhang

IPC分类号： C12N1581

CPC分类号： C12N15/81

摘要： A nucleic acid sequence including a CYP promoter operably linked to nucleic acid encoding a heterologous protein is provided to increase transcription of the nucleic acid. Expression vectors and host cells containing the nucleic acid sequence are also provided. The methods and compositions described herein are especially useful in the production of polycarboxylic acids by yeast cells.

摘要翻译： 提供了包含与编码异源蛋白质的核酸可操作地连接的CYP启动子的核酸序列，以增加核酸的转录。 还提供了含有核酸序列的表达载体和宿主细胞。 本文所述的方法和组合物特别可用于通过酵母细胞生产多元羧酸。

公开(公告)号：US06573090B1

公开(公告)日：2003-06-03

申请号：US09458068

申请日：1999-12-09

申请人： Xandra O. Breakefield ,  E. Antonio Chiocca ,  Yoshinaga Saeki ,  Cornel Fraefel ,  Kurt Tobler ,  Mathias Ackermann ,  Mark Suter ,  Gosse J. Adema ,  Ken Shortman

发明人： Xandra O. Breakefield ,  E. Antonio Chiocca ,  Yoshinaga Saeki ,  Cornel Fraefel ,  Kurt Tobler ,  Mathias Ackermann ,  Mark Suter ,  Gosse J. Adema ,  Ken Shortman

IPC分类号： C12N1581

CPC分类号： C12N15/86 ,  A61K39/00 ,  A61K39/12 ,  A61K39/245 ,  A61K48/00 ,  A61K2039/5256 ,  A61K2039/53 ,  A61K2039/545 ,  C12N2710/16634 ,  C12N2710/16643 ,  C12N2800/204

摘要： The present invention relates to an enhanced and simplified herpes virus amplicon packaging system. The packaging system comprises a herpes virus amplicon vector and a packaging vector. In one embodiment, the packaging vector comprises a bacterial artificial chromosome (BAC) containing the HSV-1 genome. The packaging vector contains an intact pac site but is otherwise rendered packaging defective. The packaging vector can be rendered packaging defective by inserting nucleotides into the pac site, or by otherwise interfering with the capsid's ability to close, for example, by increasing the size of the DNA fragment upon which the herpes virus genome is cloned. This system can be used to package a wide range of nucleotide sequences (e.g., a therapeutic or antigenic gene) into an empty herpes virus particle taking advantage of the large transgene capacity of herpes viruses. This system can also be used as a vaccine to induce protective immunity against HSV-1, or other complex pathogens.

摘要翻译： 本发明涉及增强和简化的疱疹病毒扩增子包装系统。 包装系统包括疱疹病毒扩增子载体和包装载体。 在一个实施方案中，包装载体包含含有HSV-1基因组的细菌人造染色体（BAC）。 包装载体包含完整的pac位点，否则会导致包装有缺陷。 包装载体可以通过将核苷酸插入pac位点而被包装有缺陷，或以其它方式干扰衣壳关闭的能力，例如通过增加克隆疱疹病毒基因组的DNA片段的大小。 该系统可用于利用疱疹病毒的大转基因能力将大范围的核苷酸序列（例如，治疗或抗原基因）包装到空疱疹病毒颗粒中。 该系统也可以用作疫苗来诱导针对HSV-1或其他复杂病原体的保护性免疫。

公开(公告)号：US06531289B1

公开(公告)日：2003-03-11

申请号：US09573322

申请日：2000-05-18

申请人： John D. Bradley ,  Craig M. Thompson ,  Jeffrey B. Moore ,  C. Richard Wobbe ,  David A. Bailey

发明人： John D. Bradley ,  Craig M. Thompson ,  Jeffrey B. Moore ,  C. Richard Wobbe ,  David A. Bailey

IPC分类号： C12N1581

CPC分类号： C07K14/395 ,  C12N15/81 ,  C12Q1/18

摘要： The invention provides novel yeast cells comprising genes whose expression can be modulated by growth in the presence or absence of metal ions, methods for making such yeast cells, and methods of using such yeast cells for determining the requirement for expression of particular genes for the growth or viability of the yeast cells. The invention also provides methods of using such yeast cells in the isolation, screening and analysis of candidate antifungal compounds.

摘要翻译： 本发明提供了包含可在金属离子存在或不存在下通过生长调节表达的基因的新型酵母细胞，以及用于制备这种酵母细胞的方法，以及使用这种酵母细胞确定特定生长基因表达需求的方法 或酵母细胞的活力。 本发明还提供了在候选抗真菌化合物的分离，筛选和分析中使用这种酵母细胞的方法。