Gene expression in yeast: protein secretion Jeffrey R. Shuster Chiron Corporation, Emeryville, California, USA Maximizing efficiency for the secretion of proteins from yeast requires an understanding of the rate limiting stages in secretion that can result from high levels of gene expression. Recent progress in this area has produced a number of improvements in yeast expression systems for protein secretion. Current Opinion in Biotechnology 1991, 2:685-690

Introduction

The secretion of proteins from yeast

The export of protein from the cytoplasm of a cell to an extracellular location is a key biological process in all organisms. A detailed description of protein secretion in yeast is developing concurrently with the fidd of yeast biotechnology that seeks to use the secretory capacity of yeast for the expression of heterologous proteins. Proteins produced using yeast secretion systems are being applied successfully in areas that range in diversity from the treatment of diabetics with human insulin produced from the yeast, Saccharomyces cerevisiae, to the manufacture of cheese with chymosin produced from the yeast, Kluyveromyces lactis. In addition, yeast secretion systems are being used to produce other proteins that are unavailable from traditional sources either because they cannot be isolated in sufficient amounts from natural sources, or because they are specially designed mutant proteins that are not found in nature.

In the search for a universal system for heterologous protein secretion in yeast, many questions have arisen, the first of which is, naturally, what characterizes efficient protein secretion? This is not a trivial issue as there does not appear to be any one model heterologous protein or standard set of yeast strains and growth conditions that is tried for developing secretion systems. Most frequently, the amount of protein secreted is reported as grams (or often milligrams) of protein produced per liter of culture broth. A more accurate measure of secretion efficiency would be the percentage of a protein that is secreted relative to the total amount synthesized. In addition, it is often difficult to compare the secretion efficiencies of many different proteins in terms of grams per liter. Because the basic mechanism of secretion involves transport between cellular compartments, it is likely that a number of cellular functions act in a stoichiometric manner. For example, the binding of nascent signal peptides to the signal recognition particle and the subsequent binding of this complex to its receptor on the endoplasmic reticulum (ER) would be stages expected to act in a stoichiometric fashion. Therefore, instead of assaying secretion efficiencies of different proteins on a weight basis, it is perhaps more appropriate to compare the production of extracellular protein in terms of the concentration of the molecules secreted. Table 1 shows an example of this type of analysis. For example, if both yeast ~-factor and glucose oxidase were secreted at the same concentration (0.5 g/l) on a weight basis, the concentration of 0t-factor in the culture broth would be 290 ~tM and that of glucose oxidase would be about sixty4bld lower at 5 ~tM.

In common with all heterologous gene expression systems, the function of a yeast secretion system is to produce substantial amounts of biologically active proteins. Invariably, this involves supra-physiological levels of gene expression. Not surprisingly, the biological secretory mechanisms normally present in the host organism can easily be over-taxed by the very high levels of gene expression: when more is demanded of a biological system than that system can process, intermediates in the pathway will often accumulate. These intermediates are the reporters that indicate the rate limiting stage(s) in the process. This review will address the recent advances in identifying and ameliorating the rate limiting stages of yeast protein secretion systems. Unless otherwise stated, 'yeast' will refer to Saccharomycescerevisiae, because the majority of the studies in protein secretion have been performed with this organism, and 'secretion' will refer to the production of periplasmic or extracellular proteins.

In addition to the lack of a model protein for study, an accurate description of an 'optimal' yeast expression system for the secretion of protein has been difficult partly as a result of the effects on secretion of a wide variety of system components. Many significant variables that affect

Abbreviations ER~ndoplasmic reticulum; IGF~insulin-like growth factor.

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Expressionsystems secretion have been identified. One set of variables conceres the level of gene expression of the protein to be secreted. If promoter strength can impart an effect on the production of extracellular proteins, it is not surprising that variations in gene copy number should also have an effect. This is of particular relevance as multi-copy plasmid vectors are normally employed as expression vehicles and plasmid copy numbers can vary as a function of gene expression and growth conditions. Janes et a l [1 °°] present a very good description of the interplay of many of these variables and their effects on the secretion of himdin from yeast. Finally, different host strain backgrounds can have tremendous effects on the amount of secreted protein produced (JR Shuster unpublished data) [2.°,3°]. More important, perhaps, than the level of gene expression, is the fact that secretion of specific proteins from yeast is highly dependent upon the specific protein involved. When the secretion levels of different proteins have been compared using carefully controlled expression conditions and one host yeast strain, a very wide range of protein secretion effciencies has been observed (JR Shuster, unpublished data). Thus, there is not one expression system that is universal for all secreted proteins. In general, the main stages of protein secretion are as follows. Most proteins destined to be secreted contain a signal peptide encoded at the amino terminus of the protein. Translation of RNA encoding this signal peptide is followed by the direction of the nascent polypeptide to the ER. In the case of glycoproteins, a primary oligosaccharide addition is made to the protein upon translocation, creating a core glycosylated protein. The protein is then transported, probably via a vesicle intermediate, to the Golgi compartment where it can obtain secondary carbohydrate addition to the core oligosaccharide. From the Golgi compartment, the protein is translocated into secretory vesicles and finally past the cellular plasma membrane and through the cell wall.

Table 1. Relative concentrations of different proteins assuming 'equivalent secretion efficiencies by a weight basis of 0.5g/l.

Secreted protein Yeast m-factor Human [3-endorphin Human insulin (B-Lys-Arg-A) Human IGF-I Human lysozyme Human interferon Bovine prochymosin Human serum albumin A. niger glucose oxidase Epstein-Barr virus gp350

Molecular weight 1 700 3 500 6100 7 700 14 700 20000 40 000 66 500 gplO0 000 gp400000

Concentration (p.M) 290 140 82 65 34 25 13 8 5 I

Two basic classes of leader sequences are used in yeast secretion systems. The first is exemplified by the presequence of the sucrose cleaving enzyme, invertase. In-

vertase is found in yeast either as a non-glycosylated protein in the cytoplasm or as a secreted glycoprotein. The secreted form is produced by an RNA encoding invertase with a 20 amino acid signal pre-sequence. The cytoplasmic form is encoded with no signal peptide sequence. Thus, the signal peptide is both necessary and sufficient for the secretion of invertase. An early model for secretion suggested that fusion of an amino acid signal peptide, such as that found in invertase, to any protein would be sufficient to result in direction of that protein into the ER, after which point the remainder of the yeast secretion machinery would operate in processing and secreting the protein. The second class of secretion leader is best represented by the prepro-leader of the yeast a-factor mating pheromone. The a-factor gene encodes, in addition to a canonical amino-terminal signal peptide, a 'pro' sequence between the signal peptide and the mature a-factor. This pro-region is glycosylated and may act as a chaperone for the a-factor until it is received by the Golgi compartment. In the Golgi, the pro-region is cleaved from a-factor precursor by the Kex2 protease. Because the glycosylated a-factor pro-region may act as a chaperone peptide for secreted proteins, it is possible that some heterologous proteins could be secreted more efficientlywith a preproleader than with a pre-peptide alone. Both classes of leader sequences have been investigated in the development of yeast protein-secretion systems. Many different proteins have been encoded following either a pre- or prepro-leader sequence. Some heterologous proteins are efficiently secreted by yeast when fused to these sequences while others are secreted only at low or non-detectable levels. These studies have revealed that efficient secretion of proteins from yeast is not simply a function of attaching a secretion signal peptide. Therefore, other rate limiting variables must exist to account for the difference in secretion efffciencies. Some of these rate limiting stages are discussed below.

Translocation from the cytoplasm to ER The basic structure of a functional signal peptide has been described [4.,5..], and although there is not a strict sequence requirement for directing proteins to the ER, there can be a very large difference in the efficiency of different leaders to promote secretion, Kaiser et aL [6] showed that many different random amino acid sequences could function as secretion leaders for invertase. It is interesting to note that none of the sequences isolated in this manner performed as well as the natural signal. In general, signal peptides derived from yeast proteins function as well or better than the heterologous signal peptides for the secretion of a heterologous protein, although some exceptions have been reported [7"]. How, then, does one select an optimal presequence for a specific protein? One example is provided by Hofmarm and Schultz [2-] who noted a block in the secretion of human plasminogen activator inhibitor at the stage when the protein is inserted into the ER. They have shown that a pre-sequence derived from the yeast a-galactosldase gene can be mutated to be more el-

Gene expression in yeast: protein secretion Shuster ficient for the secretion of human plasminogen activator inhibitor-1. Mutations in the same pre-sequence can also enhance secretion of the protein, echistatin. Interestingly, the enhancement of secretion resulting from the mutations was partially specific for each protein and also for the different yeast host strains that were tested. At present, the problem presented when proteins are not delivered effectively into the ER may be solved by screening many different secretion leader sequences for positive ER delivery activity. Once the best leaders are identified, they may be subsequently mutagenized and the result ing novel sequences screened for improvements. Further hopes of alleviating problems concerning the insertion of proteins into the ER lie in studies that have identified a number of key gene products involved in cytoplasm to ER transport (see [8] for a recent review). Recently, interesting reports have shown that the ER resident binding protein (BiP/GRP78/Kar2) is required for transport of yeast proteins into the ER [9"',10..]. It will be interesting to see if the over-expression of yeast BiP or a number of the other genes involved in cytoplasm to ER translocation [8,11-] can alleviate the block in this early stage of secretion.

Translocation from the ER to Golgi Many of the natural proteins secreted from yeast are glycoproteins. Their progress from the ER to the Golgi can be easily monitored by the degree of carbohydrate addition; core glycosylation occurs in the ER and outer chains are added in the Golgi. The process of protein translocation from the ER to the Golgi compartment via a vesicle intermediate has been the subject of much recent work [12,13.,14-,,15-]. A number of studies [16.*,17.*,18-] have reported the effects of glycosylation on protein intracellular transport by mutating the coding sequences of yeast glycoproteins so that they vcill only accept reduced, or even no, carbohydrate addition. In all three studies hypo- or non-glycosylation of the protein resulted in an increase in the intracellular inter-compartment transit time. In the case of the prepro-ct-factor protein and acid phosphatase, non-glycosylated proteins were found to accumulate at a stage after insertion into the ER and before Golgi compartmentalization. In a similar manner, the secretion of the Mucorpusillus glycoprotein rennin (directed by its native prepro-sequence) was severely inhibited when the carbohydrate addition sites were rendered non-functional by mutation [19o]. The secretion of an analog of human tissue plasminogen activator was similarly affected when the residues required for the addition of carbohydrate were altered [20..]. Thus it appears that glycosylation may be a signiticant factor in the efficient secretion of proteins from yeast. It would be interesting to determine if a carbohydrate receptor exists that facilitates movement of glycoproteins from the ER to the GolD. In view of these considerations, it is highly desirable to use the glycosylated yeast prepro-~-factor (which is processed off in the Golgi) for the secretion of heterologous proteins. This

may be especially important when one is attempting to secrete proteins that, themselves, would not be glycosylated.

Golgi transport to extracellular compartments Not as much is known about Golgi transport and final secretion out of the cells [21 ° ] as is known about earlier stages. The Golgi is, however, an extremely important organelle within yeast secretion systems. A vast majority of the successes in secreting heterologous proteins from yeast involve use of the or-factor prepro-leader up to the ... Lys-Arg processing site (see [22], and particularly chapters 10, 12, 14 and 15, for reviews). The Golgi is the location of the Kex2 protease [23"] that cleaves fusion proteins following this dipeptide at the end of the leader. One rate limiting step resulting from high-level gene expression of a secreted protein can be endoproteolysis at this site. The over-expression of the KEX2 gene has, in some cases, relieved this rate limiting step (see chapter 14 in [22];JR Shuster, unpublished data). The processing of a protein from the pro-a-leader indicates that the protein has probably been transported to the Golgi. Intracellular accumulation of a Kex2 processed protein is indicative of a late block in secretion at, or past, the GolD. This has been seen in the expression of a prepro-a-factor/human insulin-like growth factor (IGF)-I fusion in yeast [24..]. Pulse-chase studies indicated that the IGF-I was translocated through the ER and processed by Kex2 in the Golgi within about two minutes. The processed IGF-I, however, could still be found inside the cells after an additional 24 hours.

Other data also suggest that some heterologous proteins have difficulty exiting from the Golgi. Because the Golgi is the place where complex outer chain carbohydrate addition occurs, long resident times in this cellular compartment might be expected to result in hyperglycosylation of proteins. The fact that many glycoproteins secreted from yeast receive an abnormally high quantity of carbohydrate as compared with that seen for the natural protein [7o.,19",25",26--] supports this idea. Some glycoproteins secreted from yeast have a degree of carbohydrate addition similar to that seen in the natural protein [27°]. The requirements for ef~cient transport of proteins through the yeast cell wall have not been elucidated in detail, but a large amount of relevant data has accumulated [28,,]. It is dear that size alone does not determine whether a protein can easily pass through the cell w a r

Avoiding heterogeneity The majority of heterologous proteins successfully secreted from yeast are secretory proteins in their native organisms. A wide variety of analytical tools and bioassays has been used to characterize many of them. In general, and unlike proteins produced in the cytoplasm of cells, proteins secreted from yeast are correctly folded and bi-

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Expressionsystems ologically active molecules. In some cases, however, the secreted protein is heterogeneous largely as a result of proteolytic degradation in the culture broth. This is often controlled by mutating a variety of the yeast protease genes [3"] or by altering the growth medium or fermentation conditions [3-,29.o]. Other heterogeneities have been shown to arise from incorrect proteolytic processhag of leader sequences [30 °] or incorrect protein folding or disulfide formation [24°']. Manipulation of the level of yeast protein disulfide isomerase [31] may rectify some of the problems observed in proper protein folding and disulfide formation.

Conclusions and future directions Proteins produced from yeast are being used in many capacities ranging from human therapeutical agents to industrial enzymes in the food industry. The development of successful yeast expression systems for the secretion of proteins has progressed rapidly. A number of studies have identified key rate limiting stages in this biological process. The challenge for the future will be to design systems that alleviate these specific bottlenecks in the secretion pathway. Remediation of any one specific rate limiting step will, by definition, define the next rate limiting stage in the process. By addressing each of these issues in tum, the efficiency of the systems used for protein manufacture by secretion from yeast will continue to improve. Unlike many heterologous protein secretion systems, yeast can be easily grown in large scale with a wide range of environmental conditions. This is extremely useful in optimizing and fine-tuning growth conditions for the production of a variety of different proteins. In addition, the yeast genetic system is very well characterized. This knowledge, along with the stable haploid nature of the organism, makes it an ideal choice for classical genetic techniques of host strain improvement for the secretion of heterologous proteins [20.-,32..]. It is clear that the synergism between the research on cellular transport of homologous proteins, and that involved with the expression of high levels of heterologous proteins in yeast, will produce many new insights into the general mechanisms of protein secretion.

1. to

JANES M, MEYHACKB, ZIMMERMANNW, HINNEN & T h e Influe n c e of GAP Promoter Variants on Hirudin Production, Average Plasmid Copy N u m b e r and Cell Growth in Sac. c h a r o m y c e s cerevisiae. Curr Genet 1990, 18:97-103. This paper presents a nice study of the interactions of plasmid copy number, cell growth, and heterologous protein secretion. Because it discusses these effects relative to different glyceraldehyde-3-phosphate dehydrogenase promoter 'strengths', it would have been a more complete study had it also measured promoter strength directly by also measuring mRNA levels. HOFMANNKJ, SCHULTZLD: Mutations of t h e cz-Galactosidase Signal Peptide W h i c h Greatly Enhance Secretion of Heterologous Proteins by Yeast. Gene 1991, 101:105-111. A number of key points are described in this work: a pre-sequence can be optimized for secretion of a protein by mutagenesis: different proteins require different pre-sequences for optimal secretion; one rate limiting stage for secretion of proteins appears to be the insertion through the ER; and the e~ciency of secretion of a protein is dependent u p o n the host genetic background. 2. to

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GABRIEl.SENOS, REPPE S, SAETHERO, BLINGSMOOR, SLETrEN K, GORDELADZE JO, HOGSETA, GAUTVIKVT, ALESTROMP, OYEN TB, GAUTVlKKM: Efficient Secretion of H u m a n Parathyroid Horm o n e by Saccharomyces cerevisiae. Gene 1990, 90:255-262. Describes many methods of solving problems with heterologous protein secretion, in particular the combination of a protease-deficient strain and a change of growth conditions to both limit proteolysis and increase secreted protein titer. BOYD D, BECKWrrHJ: The Role of Charged Amino Acids in t h e Localization of Secreted and Membrane Proteins. Cell 1990, 62:1031-1033. This article reviews the effects of charged amino acid residues u p o n signal peptide function. 4. •

NOTHWEHR SF, GORDON JI: Targeting of Proteins into t h e Eukaryotic Secretory Pathway: Signal Peptide Struct u r e / F u n c t i o n Relationships. BioEssays 1990, 12:479-484. A good review of general signal peptide structure, its interaction with signal recognition particles, and signal peptidase activity. 5. °.

KAISERCA, PRUESSD, GPaSAFIP, BOTSTEIN D: Many Random Seq u e n c e s Functionally Replace t h e Secretion Signal of Yeast Invertase. Science 1987, 235:312-317. HIRAMATSUR, HORINOUCHIS, BEPPU T: Isolation and Characterization of H u m a n Pro-urokinase and its Mutants Accumulated within t h e Yeast Secretory Pathway. Gene 1991, 99:235-241. This paper compares the etliciency of the yeast invertase leader with a leader derived from Mucor rennin. It also is an excellent example of how to find the block in secretion of a heterologous gene using a combination of immunological and biochemical techniques. In this case, the block occurred after insertion into the ER and before the Golgi. 7. •°

DESHAIES RJ, KEPES F, BOHNI PC: Genetic Dissection of the Early Stages of Protein Secretion in Yeast. Trends Genet 1989, 5:87-93.

Acknowledgements I would like to thank Donna Moyer and Helen Lee for their technical assistance in performh3g some of the experiments cited as unpublished results. I also thank Phil Barr and Randy Schekman for their helpful comments on this review.

References and recommended reading Papers of special interest, published within the annual period of review, have been highlighted as: ,, of interest •. of outstanding interest

VOGELJP, MASRALM, ROSE MD: LOSS of BiP/GRP78 Function Blocks Translocation o f Secretory Proteins in Yeast. J Cell Biol 1990, 110:1885-1895. An excellent study demonstrating dependence on the binding protein BiP for the translocation of invertase, carboxypeptidase-Y, and preproc~-factor into the ER. 9. •.

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NGUYENT, LAW DTS, WILLIAMSDB: Binding Protein BiP is Required for Translocation of Secretory Proteins into the Endoplasmic Reticulum in Saccharomyces cerevisiae. Proc Natl Acad Sci USA 1991, 88:1565-1569. This paper demonstrates a dependence on BiP for the translocation of invertase and prepro-c~-factor into the ER. In contrast to [9 o" ] prepro-

G e n e e x p r e s s i o n in yeast." p r o t e i n s e c r e t i o n S h u s t e r or-factor translocation into the ER is reported to be more sensitive than invertase to a decrease in BiP levels.

technique for the isolation of mutations that enhance the amount of protein secretion.

AMAYAY, NAK~O & SRH1 Protein, t h e yeast Homologue of the 54 kDa Subunit o f a Signal Recognition Particle, is Involved in ER Translocation o f Secretory Proteins. FEBS Lett 1991, 283:325-328. This paper shows that one of the subunits of signal recognition particles in yeast is necessary for protein import into the EtL

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KAISERCA, SCHEKMANR: Distinct Sets of SEC Genes Govern Transport Vesicle Formation and Fusion Early in t h e Secretory Pathway. Cell 1990, 61:723-733.

NEWMANAP, SHIMJ, FERRo-NoVICKS: BET1, BOS1, and SEC22 are Members of a Group of Interacting Yeast Genes Required for Transport from t h e Endoplasmic Reticulum to the Golgi Complex. Mol Cell Biol 1990, 10:3405-3414. Shows that over-expression of one gene involved in ER to Golgi transitcation can suppress a number of other mutations also involved in this process. This may be useful in determining which proteins to overexpress in order to alleviate blockages in ER to Golgi transition for highly expressed heterologous proteins. 13. •

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HICKEL, SHEKMANR: Molecular Machinery Required for Protein Transport from the Endoplasmic Reticulum to t h e Golgi Complex. BioEssays 1990, 12:253-258. A good review of the processes involved in ER to Golgi transport in yeast concentrating on the role of the sec genes. 15. ••

NEWMANAP, FEP~O-NOVlCKS: Defining C o m p o n e n t s Required for Transport from the ER to Golgi C o m p l e x in Yeast. BioEssays 1990, 12:485--491. A good review of the processes involved in ER to Golgi transport in yeast including a description of the BETand BOS genes.

FRANZUSOFFA, REDDING K, CROSBYJ, FULLERRS, SHEKMANR: Localization of C o m p o n e n t s Involved in Protein Transport and Processing T h r o u g h the Yeast Golgi Apparatus. J Cell Biol 1991, 112:27-37. Localization of one of the proteins involved in the transition of secretory proteins through the Golgi is described. 22.

BARRPJ, BRAKE AJ, VALENZUELAP (EDS): Yeast Genetic Engineering [book]. Stoneham: Butterworths, 1989.

REDDINGK, HOLCOMB C, FULLERRS: Immunolocalization of Kex2 Protease Identifies a Putative Late Golgi Compartm e n t in the Yeast Saccharomyces cerevisiae. J Cell Biol 1991, 113:527-538. Shows that the Kex2 protease is localized to the Golgi compartment in yeast. 23. •

STEUBEK, CHAUDHUR1B, MARKIW, MERRYWEATHERJP, HELMJ: c~-Factor-leader-directed Secretion of Recombinant Humaninsulin-like Growth Factor I from Saccharomyces cerevisiae. Eur J Bi~hem 1991, 198:651~657. Reports an excellent example of a late block in the secretion of a heterologous protein. Pulse-chase data are presented to show that the majority of the IGF-I goes through the early secretory pathway rapidly and the later stages slowly. 24. o.

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WldHTINGTONH, KERRY-WILLIAMSS, BIDGOOD K, DODSWORTHN, PEBERDYJ, DOBSON M, HINCHLI~E E, BALIANCEDJ: Expression of t h e Aspergillus niger Glucose Oxidase Gene in A~ niger, A. nidulan& and Saccharomyces cerevisiae. Curr Genet 1990, 18:531-536. Shows that a heterologous fungal enzyme is hyper-glycosylated when expressed in yeast.

CAPLANS, GREEN R, ROCCO J, KURJANJ: Glycosylation and Structure of the Yeast MAT~lcz-Factor Precursor is Important for Efficient Transport T h r o u g h the Secretory Pathway. J Bacteriol 1991, 173:627-633. This is a key paper that shows that glycosylation is important for intracellular transport. All three of the glycosylation addition sites in the pro-leader of ct-factor were equally important in affecting intracellular transport.

DE BAETSELIER A, VASAVADAA~ DOHET P, HA-THI V, DE BEUKEIAERM, ERPICUMT, DE CLERCKL, HANOTIERJ, ROSENBERG S: Fermentation of a Yeast Producing A~ niger Glucose Oxidase: Scale-Up, Purification and Characterization of the Recombinant Enzyme. Biotechnology 1991, 9:559-561. This paper reports that a heterologous fungal enzyme is hyper-glycosyhted when expressed in yeast. It also demonstrates that a very high level of glucose oxidase ( > 3 g/l) can be obtained in the culture broth with appropriate fermenter conditions.

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WINTriERJR, STEVENS TH, KIELLAND-BRANDTMC: Yeast Carboxypeptidase Y Requires Glycosylation for Efficient Intracellular Transport, but n o t for Vacuolar Sorting, In Vivo Stability, or Activity. Eur J Biochem 1991, 197:681-689. This key paper describes the importance of glycosylation for intracellular transport. Unlike [ 16 *•], it demonstrates significant differential effects on protein translocation of mutations at different carbohydrate addition sites. 18. •

RIEDERERMA, HINNEN A: Removal of N-Glycosylation Sites of the Yeast Acid Phosphatase Severely Affects Protein Folding. J Bacteriol 1991, 173:3539-3546. Shows that carbohydrate addition is required, in s o m e cases, for correct protein folding. 19. •

AIKAWAJ, YAMASHITAT, NISHIYAMAM, HORINOUCHI S, BEPPU T: Effects of Glycosylation on t h e Secretion and Enzyme Activity of Mucor Rennin, an Aspartic Proteinase of Mucor pusillus, Produced by Recombinant Yeast. J Biol Cbem 1990, 265:13955-13959. This paper shows that the secretion of a heterologous glycoprotein is inhibited when the glycosylation sites are removed. In addition, it demonstrates that yeast can hyperglycosylate a protein relative to its natural state. The hyper-glycosylation is interesting in this case, as the protein is also of fimgal origin. 20. °o

GiLL GS, ZAWORSK1PG, MAROTrI KR, REHBERG EF: A Novel "Screening System for Yeast Strains Capable of Secreting Tissue Plasminogen Activator. Biotecbnology 1990, 8:956-957. This paper reports a significant differential effect on heterologous protein secretion of mutating two different carbohydrate addition sites of a tissue plasminogen activator analog. It also presents a useful screening

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KUMAGAIMH, SHAH M, TERASH1MEM, VRKLJANZ, WHITAKERJR, RODRIGUEZRL: Expression and Secretion of Rice or-Amylase by Saccharomyces cerevisiae. Gene 1990, 94:209-216. This paper demonstrates that a signal peptide from the plant kingdom can function in yeast. The amylase produced is a glycoprotein and shows a degree of glycosylation similar to the native enzyme produced in rice, Oryza sativa. •

28. DE NOBEL JG, BARNETt JA: Passage of Molecules T h r o u g h •. Yeast Cell Wails: a Brief Essay-Review. Yeast 1991, 7:313-323. An excellent review of homologous and heterologous proteins that have been secreted from yeast. 29. •0

SIEGELRS, BRIERIZ~ RA: Use of a Cell Reactor to Increase Production of a Proteolysis-susceptible Peptide Secreted from Recombinant Saccharomyces cerevisiae. Biotectmology 1990, 8:639-643. This paper demonstrates h o w biochemical engineering systems can be developed to limit proteolytic degradation of proteins secreted from yeast. 30. •

GUISEZY, TISON B, VANDEKERCKHOVJ, DEMOLDERJ, BAUW G, HAEGEMANG, FIERSW, CONTRERASR: Production and Purification o f Recombinant H u m a n Interleukin-6 Secreted by the Yeast Saccharomyces cerevisiae. Eur J Biochem 1991, 198:217-222. Aberrant proteolytic processing in the pro-c~-leader is described together with an example of how to solve the problem associated with the inability of the Kex2 p r o t ~ s e to cleave the leader peptide from a particular protein fusion. 31.

IAMANTIAM, TADASHIT, TACHIKAWAH, KAPLANHA, LENNAP,Z WJ, MIZUNAGAT: Glycosylation Site Binding Protein and Protein

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Expression systems Disulfide Isomerase are Identical and Essential for Cell Viability in Yeast. Proc Natl Acad Sci USA 1991, 88:4453-4457. SLEEP D, BELFIELDGP, BAILANCEDJ, STEVENJ, JONES S, EVANS LR, MOIR PD, GOODEY AR: Saccharomyces cerevisiae Strains that Overexpress Heterologous Proteins. Biotechnology 1991, 9:183-187. This paper describes an immuno-screening technique for the isolation of yeast mutants that secrete higher levels of human serum albumin, a

protein that is readily secreted by yeast. The technique is useful for any secreted protein to which antibodies have been raised.

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JR Shuster, Chiron Corporation, 4560 Horton Street, Emeryville, California 94608, US&

Gene expression in yeast: protein secretion.

Maximizing efficiency for the secretion of proteins from yeast requires an understanding of the rate limiting stages in secretion that can result from...
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