公开(公告)号：US06838555B2

公开(公告)日：2005-01-04

申请号：US09993192

申请日：2001-11-14

申请人： Sangki Rhee ,  Euisung Choi ,  Hyunah Kang ,  Junghoon Sohn ,  Junghoon Bae ,  Moowoong Kim ,  Michael Agaphonov ,  Myungkuk Kim

发明人： Sangki Rhee ,  Euisung Choi ,  Hyunah Kang ,  Junghoon Sohn ,  Junghoon Bae ,  Moowoong Kim ,  Michael Agaphonov ,  Myungkuk Kim

IPC分类号： C12N9/48 ,  C12N15/80 ,  C12N15/81 ,  C07H21/04

CPC分类号： C12N15/815 ,  C12N9/48 ,  C12N15/80 ,  C12R1/78

摘要： Disclosed are Hansenula polymorpha mutants useful as host cells through which various proteins can be produced as being intact at high yield and a process for preparing recombinant proteins using the host cells. Using various vectors, Hansenula polymorpha is made to be a mutant which is deprived of methanol assimilating ability and incapable of utilizing methanol as a carbon source. This Hansenula polymorpha mutant is used as a high yield host to produce recombinant proteins without continuous feeding of methanol, with the aid of an expression cassette carrying a promoter capable of inducing the expression at a low concentration of methanol. Further, the mutant is also lacking in carboxypeptidase Y, protease Y and/or carboxypeptidase a activity, so the recombinant protein of interest is not degraded at its carboxyl terminal when being expressed in the cell. Thus, intact recombinant protein can be obtained. Also, there is disclosed a pop-out technique in which a recombinant protein expression cassette is inserted into a MOX gene site of the mutant and is allowed to pop out therefrom, thereby utilizing the mutant as a host for general use in producing various proteins of interest.

摘要翻译： 公开了可用作宿主细胞的汉逊酵母多形汉逊酵母突变体，通过该宿主细胞可以以高产量产生各种蛋白质完整的蛋白质，以及使用宿主细胞制备重组蛋白质的方法。 使用各种载体，多形汉逊酵母被认为是一种被剥夺了甲醇同化能力并且不能利用甲醇作为碳源的突变体。 这种汉逊酵母多形突变体被用作高产量宿主以产生重组蛋白，而不用连续喂入甲醇，借助于携带能够以低浓度甲醇诱导表达的启动子的表达盒。 此外，突变体也缺乏羧肽酶Y，蛋白酶Y和/或羧肽酶a活性，所以当在细胞中表达时，其目的重组蛋白质在其羧基末端不被降解。 因此，可以获得完整的重组蛋白。 此外，公开了一种突变技术，其中将重组蛋白表达盒插入到突变体的MOX基因位点，并从其中突出出来，从而利用突变体作为一般用于产生各种蛋白质的各种蛋白质的宿主 利益。