公开(公告)号：US5547867A

公开(公告)日：1996-08-20

申请号：US218303

申请日：1994-03-28

申请人： Bhuphendra V. Kara ,  Robert C. Hockney ,  John E. Fitton

发明人： Bhuphendra V. Kara ,  Robert C. Hockney ,  John E. Fitton

IPC分类号： C07K14/415 ,  C12N15/09 ,  C12N15/29 ,  C12N15/70 ,  C12P21/02 ,  C12R1/19 ,  C12N9/22

CPC分类号： C07K14/415 ,  C12N15/70 ,  C12P21/02

摘要： Processes for preparing polypeptides such as ricin A such that soluble polypeptide may be obtained. The pH may be adjusted during the process or the temperature decreased during the terminal portion of the cultivation, and yeast extract may be added to the growth medium during the cultivation. In the case of ricin A the pH is generally adjusted by lowering the pH from a first value such as 6.7 to a second value such as a value between about 5.5 and 6.7.

摘要翻译： 用于制备多肽例如蓖麻毒蛋白A的方法，使得可以获得可溶性多肽。 在该过程中可以调节pH或在培养的末端部分温度降低，并且可以在培养期间将酵母提取物加入生长培养基中。 在蓖麻蛋白A的情况下，通常通过将pH从第一值（例如6.7）降低至第二值（例如约5.5至6.7之间的值）来调节pH。

公开(公告)号：US5439815A

公开(公告)日：1995-08-08

申请号：US912740

申请日：1992-07-13

申请人： John E. Fitton ,  David Timms

发明人： John E. Fitton ,  David Timms

IPC分类号： A61K38/46 ,  A61K38/00 ,  A61K47/48 ,  A61P35/00 ,  C07K14/37 ,  C12N9/16 ,  C12N9/22 ,  C12N15/09 ,  C12N15/31 ,  C12N15/55 ,  C12N1/15 ,  C12N1/21 ,  C12N5/10

CPC分类号： A61K47/48492 ,  C07K14/37 ,  C12N9/22 ,  A61K38/00

摘要： Ribotoxins, such as restrictocin, mitogillin and alpha-sarcin which do not contain a cysteine for linkage to antibodies for immunotoxin production need to be modified for such linkage without compromising biological activity. Protein analogues of such native ribotoxins are provided in which both the analogue and the native ribotoxin can cleave only a single phosphodiester bond of 28S rRNA in a 60S ribosomal subunit and in which the analogue comprises only one cysteine available from covalent linkage to a partner, such as an antibody, which cysteine is not present in the native ribotoxin. Especially preferred analogues are restrictocin Cys 13, Cys 82, Cys 106 and Cys 110 and restrictocin Cys 150-Gly151. Corresponding DNA sequences, vectors, host cells, immunotoxins and pharmaceutical compositions are also claimed.

摘要翻译： 需要修饰核毒素，例如不含半胱氨酸的限释片，mitogillin和α-sarcin用于与免疫毒素生产的抗体连接，而不损害生物活性。 提供这种天然核毒素的蛋白质类似物，其中类似物和天然核毒素都可以在60S核糖体亚基中仅切割28S rRNA的单个磷酸二酯键，其中类似物仅包含一种可从共价键与配偶体获得的半胱氨酸， 作为抗体，该半胱氨酸不存在于天然核毒素中。 特别优选的类似物是限释子Cys 13，Cys 82，Cys 106和Cys 110和限释片Cys 150-Gly151。 还要求保护相应的DNA序列，载体，宿主细胞，免疫毒素和药物组合物。

公开(公告)号：US5306626A

公开(公告)日：1994-04-26

申请号：US32842

申请日：1993-03-16

申请人： John E. Fitton

发明人： John E. Fitton

IPC分类号： C07K14/37 ,  C07K14/415 ,  C12N9/22 ,  C12N9/88 ,  C12N15/31 ,  C12N15/70 ,  C12P21/02

CPC分类号： C12N9/22 ,  C07K14/37 ,  C07K14/415 ,  C12N15/70 ,  C12N9/88 ,  C07K2319/034 ,  C07K2319/55 ,  Y10S435/849

摘要： Methods for recombinant production in procayotic microorganisms such as E. coli of ribotoxins such as restrictocin, alpha-sarcin and mitogillin are described. Known methods were relatively low yielding and not cost effective for commercial use such as in the pharmaceutical industry where relatively large quantities of toxin with consistent batch to batch quality may be required for immunotoxin production. Use of recombinant methods of production open up the possibility of making ribotoxin analogues. Toxicity of ribotoxins was recognised as a concern in development of a high yielding cost effective production method. Methods for high yielding intracellular accumulation or secretion of ribotoxins are described. Use of protease deficient strains and other methods of minimising breakdown of ribotoxin by protease are preferred. Vectors and host strains for use in the methods are described.

摘要翻译： 描述了在诸如限制性细胞毒素，α-丝氨酸蛋白酶和丝裂霉素的核毒素如大肠杆菌等原生微生物中重组生产的方法。 已知的方法相对低的产率并且在商业上不是成本有效的，例如在制药工业中，对于免疫毒素生产可能需要相对大量的具有一致批次质量的毒素。 使用重组生产方法开辟制作核毒素类似物的可能性。 在开发高产成本有效的生产方法中，核糖核酸的毒性被认为是关注的问题。 描述了高产细胞内积累或分泌核毒素的方法。 优选使用蛋白酶缺陷菌株和通过蛋白酶使核酸毒素最小化的其它方法。 描述了用于该方法的载体和宿主菌株。