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1、422Improvements in yeast expression systems, coupled with the development of yeast surface display and refinements in two- hybrid methodology, are expanding the role of yeasts in the process of understanding and engineering eukaryotic proteins.Addresses Department of Biochemistry and Molecular Biolo
2、gy, Oregon Graduate Institute of Science and Technology, 20 000 NW Walker Road, Beaverton, Oregon 7006-8921, USA *e-mail: geoffcbmb.ogi.edu Correspondence: James M Cregg; e-mail: creggbmb.ogi.eduCurrent Opinion in Biotechnology 1999, 10:4224270958-1669/99/$ see front matter 1999 Elsevier Science Ltd
3、. All rights reserved.Abbreviations Aga-agglutinin AOX1alcohol oxidase I BPTIbovine pancreatic trypsin inhibitor ERendoplasmic reticulum GAPglyceraldehyde-3-phosphate dehydrogenase PDIprotein disulfide isomerase scFvsingle-chain antibody variable region fragmentIntroduction As we enter the millenniu
4、m, the so-called Biotech Centu- ry, scientists are continuing to engineer yeasts to produce eukaryotic proteins. Although these creations are not as appreciated as bread and beer, foreign proteins expressed in yeasts are being used to synthesize life-saving drugs for the pharmaceutical industry and
5、unravel the complex reg- ulatory phenomena at the heart of basic research. This review will describe recent advances in two broad areas: firstly, the use of yeasts to make large quantities of foreign proteins for research and therapeutic applications; second- ly, the use of yeasts to determine the f
6、unctional and regulatory dynamics of a recombinant protein, such as its interaction partners or its affinity for ligands.Yeasts are suitable for these uses for several reasons. Fore- most, yeasts offer the ease of microbial growth and gene manipulation found in bacteria along with the eukaryotic env
7、ironment and ability to perform many eukaryote-specif- ic post-translational modifications, such as proteolytic processing, folding, disulfide bridge formation, and glycosy- lation 1. Bacteria lack these capabilities and often produce eukaryotic proteins that are misfolded, insoluble, or inac- tive.
8、 Relative to more complex eukaryotic expression systems, such as Chinese hamster ovary cells and bac- ulovirus-infected cell lines, yeasts are economical, usually give higher yields, and are less demanding in terms of time and effort 2. Nevertheless, there are disadvantages to using yeasts for expre
9、ssion of some heterologous proteins, mostly related to their inability to perform certain complex post-translational modifications, such as prolyl hydroxyla-tion and amidation, as well as some types of phosphorylation and glycosylation 3. Recent findings, however, should help to alleviate some of th
10、ese problems and broaden the scope of future applications for yeasts in biotechnology.Yeasts as protein factories Yeasts have been used since the early 1980s for the large- scale production of intracellular and extracellular proteins of human, animal, and plant origin 4,5. The expression of a foreig
11、n protein in yeasts consists of four steps: firstly, cloning of a foreign protein-coding DNA sequence within an expression cassette containing a yeast promoter and transcriptional termination sequences; secondly, transfor- mation and stable maintenance of this DNA fusion in the host; thirdly, synthe
12、sis of the foreign protein under speci- fied culture conditions; and finally, purification of the heterologous protein and comparison with its native coun- terpart. Usually, a regulatable promoter is used to drive foreign protein expression because, prior to induction, the ability to maintain cultur
13、es in an expression off mode minimizes selection for non-expressing mutant cells dur- ing the cell growth phase. Such a selection can occur as a result of the added metabolic burden placed on cells expressing high levels of a foreign protein or the toxic effect of a foreign protein on the cells. Sev
14、eral yeast species have been engineered as systems for heterologous protein expression 69. This review, however, will focus on the methylotrophic yeast Pichia pastoris and the bakers yeast Saccharomyces cerevisiae, the organisms most common- ly used for this purpose.P. pastoris and other nonconventi
15、onal yeasts P. pastoris has been utilized to produce 300 foreign pro- teins since 1984 10,11,12. There are several factors that account for this systems popularity: firstly, the use of the alcohol oxidase I (AOX1) promoter, one of the strongest, most regulated promoters known; secondly, the ability
16、to stably integrate expression plasmids at specific sites in the P. pastoris genome in either single or multicopy; thirdly, the ability to culture strains in high density fermenters; and finally, its ready availability as a kit from Invitrogen Cor- poration (Carlsbad, CA, USA). The AOX1 promoter is tightly repressed by glucose and most other carbon sources but is induced 1000-fold in cells shifted to methanol as a sole carbon source 13. With this promoter, ex
