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mosin, is being produced using the S. cerevisiae a-factor leader in another budding yeast, Kluyveromyces lactis. 66 Although efficient secretion using a-factor leader-based systems is commonly achieved, much more remains to be learned about the basic biology of a-factor secretion, and secretion in general, before optimization of these systems can be achieved. 66 K. Rietveld, J. G. Bakhuis, N. J. Jansen in de Wai, R. W. van Leen, A. C. M. Noordermeer, A. J. J. van Ooyen, A. Schaap, and J. A. van den Berg, Yeast 4, S163 (1988).

[35] U s e o f H e t e r o l o g o u s a n d H o m o l o g o u s S i g n a l Sequences for Secretion of Heterologous Proteins from Yeast By RONALD A. HITZEMAN, CHRISTINAY. CHEN, DONALD J. DOWBENKO, MARK E. RENZ, CnUNG LIU, ROGER PAI, NANCY J. SIMPSON,WILLIAMJ. KOHR, ARJtJN SINGH, VANESSA CHISHOLM, ROBERT HAMILTON,and CHUNG NAN CHANG Introduction The development of yeast genetic engineering has made possible the expression of heterologous genes and the secretion of their protein products from yeast. This article deals exclusively with the yeast Saccharomyces cerevisiae (bakers' yeast). The advantages of secretion (export) of heterologous gene products are clearly exemplified by human serum albumin (HSA), and are discussed in this article. This 65-kDa blood protein has no N-linked glycosylation sites but does have 35 cysteines;1 in the blood, this protein monomer has 17 disulfide linkages3 HSA is misfolded when produced intracellulady in yeast without its amino-terminal secretion peptide sequence. This conclusion is based on its insolubility, loss of greater than 90% of its antigenicity (as compared to human-derived HSA), and formation of large protein aggregates. Using its natural secretion signal to promote secretion into the media of yeast, HSA is soluble and appears to have the same disulfide linkages as the human blood-derived material. As a pharmaceutical product, which will be potentially used in gram amounts in humans, recombinant HSA will require such identity with the natural product. Other advantages of secreting a product into the growth media of yeast are proper R. M. Lawn, J. Adelman, S. C. Bock, A. E. Franke, C. M. Houck, R. C. Najarian, P. H. Seeburg, and K. L. Wion, Nucleic Acids Res. 9, 6103 ( 1981). 2 j. R. Brown, "Albumin Structure, Function, and Uses," p. 27. Pergamon, New York, 1977.

METHODS IN ENZYMOLOGY. VOL. 185

Copyright© 1990by AcademicPress,Inc. All rightsof reproductionin any formreserved.

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amino-terminal processing (no initiator methionine residue), the difficulty of preparing yeast extracts, the resistance of yeast cells to lysis, the increase in purity obtained by placing the product in an environment which contains only 0.5-1.0% of the total yeast protein, and the lack of toxic proteins in this environment. Secretion of the type of liSA described above can be accomplished with homologous (yeast) or heterologous (nonyeast) secretion signals. Processing of the signal sequence (amino-terminal peptide) during the secretion process can be accomplished at the level of signal peptidase 3 or at the level of a peptide prosequence clipped off within the secretion pathway. Examples of these various options are discussed. Many heterologous proteins have been secreted so as to give the proper amino terminus on the mature protein; however, most proteins secreted by the procedures that are described here are secreted proteins in their normal (homologous) environment. Whether cytoplasmic (normally nonsecreted) proteins can be secreted by these methods for various obvious applications remains to be extensively tried, and many may be misfolded or trapped in compartments or membranes. Materials, Strains, and Methodology Most of the materials, strains, and methodology have been previously described or referenced in Volume 119 of Methods in E n z y m o l o g y in an article by Hitzeman et al. 4 HSA amounts were assayed in media and in glass-beaded extracts of yeast cells using a standard ELISA 5 immunological technique with goat anti-HSA antiserum from Cappel. Somatostatin and gpl20 assays were done in a similar fashion using antibody solutions prepared at Genentech, Inc. Yeast strain 30-4 is described in an accompanying article in this volume by Chisholm et al. 6 Plasmid Components Used for Secretion of Heterologous Gene Products Heterologous Gene

In order for heterologous (nonyeast) gene products to be secreted from yeast, one must first produce the mRNA of the isolated heterologous gene 3 D. Perlman and H. O. Halvorson, Cell 25, 525 (1981). 4 R. A. Hitzeman, C. N. Chang, M. Matteucci, L. J. Perry, W. J. Kohr, J. J. Wulf, J. R. Swartz, C. Y. Chen, and A. Singh, this series, Vol. 119, p. 424. 5 A. Voller, D. E. Bidwell, and A. Barlett, "Enzymatic Immunoassays in Diagnostic Medicine; Theory and Practice." World Health Organization, 1976. 6 V. Chisholm, C. Y. Chen, N. J. Simpson, and R. A. Hitzeman, this volume [37].

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in yeast. This is done using yeast 5'- and 3'-flanking DNA sequences from isolated yeast genes to act as promoter and transcription termination signals. The protein-encoding portion from the heterologous gene is inserted between such control r e g i o n s . 7 Cloned cDNAs are normally used since yeast intron excision involves different signals than, for example, human cell intron excisions and even the intron excision of other f u n g i .9

Yeast Origin of Replication for High Copy Number of Plasmid The expression system described above is generally used on a plasmid as shown in Fig. 1. To obtain high expression, the 2-/tm origin is used as well as REP3 and the FLIP gene recombination site in adjacent DNA. The functional unit is 2 kilobase pairs (kb) of DNA between EcoRI and PstI in Fig. 1) ° This system, explained in a separate chapter by Rose and Broach,11 leads to high plasmid copy number (30-60 copies/cell) which is fairly evenly distributed in a population of CIR + yeast cells. CIR + yeast contain natural 2-/tm circle plasmid which is necessary for high copy number of the recombinant plasmid and contains at least two genes for necessary replication functions.

Genes for Selective Maintenance of Plasmid The plasmid also contains the TRP112 and URA313 genes for transformation and selective maintenance of the plasmid in trpl-, ura3-, or trpl-ura3- yeast strains. The URA3 gene has the added advantage that a plasmid containing it can be cured from the yeast easily using 5-fluoroorotic acid. Many other selective markers are available (e.g., LYS2, which can be cured in a similar way using a-aminoadipic acidt4). This general yeast expression-secretion plasmid also contains pBR322 functions, including an Escherichia coli origin of replication and a functional ampicillin resistance gene (AI~), which allow for transformation and maintenance of the plasmid in E. coli. 15

7 R. A. Hitzeman, F. E. Hagie, H. L. Levine, D. V. Goeddel, G. Ammerer, and B. D. Hall, Nature (London.) 293, 717 (1981). S. M. Mount, Nucleic Acids Res. 10, 459 (1982). 9 M. A. Innis, M. J. Holland, P. E. McCabe, G. E. Cole, V. P. Wittman, R. Tai, K. W. Watt, D. H. Gelfard, J. P. Holland, and J. H. Mead, Science 228, 21 (1985). ~0j. L. Harley and J. E. Donelson, Nature (London) 286, 860 (1980). l~ A. B. Rose and J. R. Broach, this volume [22]. 12G. Tsumper and J. Carbon, Gene 10, 157 (1980). ~3F. Fasiolo, J. Bonnet, and F. Lacroute, J. Biol. Chem. 56, 2324 (1981). 14 D. A. Barnes and J. Thorner, Mol. Cell. Biol. 6, 2828 (1986). ~5F. Bolivar, R. L. Rodriguez, P. Y. Green, M. C. Betlach, H. L. Heyneker, H. W. Boyer, Y. H. Crosa, and S. Falkow, Gene 2, 95 (1977).

424

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~.Q.I~ MatureProteinCodingSequence Xba ' , ~ / R 5 , 2~ ori ginS~n " I~ L ~ I ' - ~ ~i / R ~n . ~ R. ~

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Carrying Expression Unit on Low-Copy Plasmid or Integrated into Genomic DNA If high expression is not required or is deleterious to the cell, plasmids containing a centromere with a chromosomal origin of replication ~6 may be used to carry the expression system. These plasmids maintain low copy number, from 1 - 4 copies/cell. Plasmids which disrupt centromere function by an inducible promoter may also be used to raise and lower plasmid copy number at will. 17 Integrative plasmids may also be used if one or more copies of the expression system is desired in the chromosome. 18 Linear DNA integrations can be used to substitute an expressionsecretion system for a particular region of DNA in the yeast genome using a method described by Rothstein. ~9 A similar strategy has been used to incorporate several copies of an expression system at a targeted region of the yeast genome. 2°

Promoters Used for Heterologous Gene Expression Many 5'-flanking sequences from various yeast genes have been used for expression and are discussed in various articles in this volume. However, the experiments discussed here have mainly used the yeast 3-phosphoglycerate kinase (PGK) gene promoter, 2~,22 the yeast chelatin prorooter, 23,24 and the yeast a-factor promoter. 25,26 In each case, sufficient 5'-flanking sequence is present to provide for optimal promoter function. Generally, one kilobase or more is necessary for optimal function since ~6L. Clarke and J. Carbon, Nature (London) 287, 504 (1980). ~7L. Panzeri, I. Groth-Clausen, J. Shepard, A. Stotz, and P. Philippsen, Chromosomes Today 8, 46 (1984). ~8R. A. Hitzeman, C. Y. Chen, F. E. Hagie, J. M. Lugovoy, and A. Singh, "Recombinant DNA Products: Insulin, Interferon, and Growth Hormone," p. 47. CRC Press, Boca Raton, Florida, 1984. ~9 R. J. Rothstein, this series, Vol. 101, p. 202. 20 A. Singh, C. N. Chang, J. M. Lugovoy, M. D. Matteucci, and R. A. Hitzeman, "Genetics: New Frontiers," p. 169. Oxford and IBH Publishing Co., New Delhi, Bombay, Calcutta, 1983. 2t R. A. Hitzeman, F. E. Hagie, J. S. Hayflick, C. Y. Chen, P. H. Seeburg, and R. Derynck, Nucleic Acids Res. 10, 7791 (1982). 22 S. M. Kingsman, D. Cousens, C. A. Stanway, A. Chambers, M. Wilson, and A. J. Kingsman, this volume [27]. 23 M. Karin, R. Najarian, A. Haslinger, P. Valenzuela, J. Welch, and S. Fogel, Proc. Natl. Acad. Sci. U.S.A. 81, 337 (1984). 24 T. Etcheverry, this volume [26]. :~ J. Kurjan and I. Herskowitz, Cell 30, 933 (1982). 26 A. Singh, E. Y. Chen, J. M. Lugovoy, C. N. Chang, R. A. Hitzeman, and P. H. Seeburg, Nucleic Acids Res. 11, 4049 (1983).

426

EXPRESSION IN YEAST

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some promoter activation sequences27 are quite far upstream of the ATG. The chelatin promoter results in 10 times more mRNA (when induced) than the PGK or a-factor promoters using the same test genes (data not shown). The chelatin promoter (chelp) is about 5× inducible on a 2-/tm plasmid using 0.15-0.3 m M Cu 2+ ion. 2s If more control is desired, one should use the GALI-IO promoter, which is essentially turned off during growth on glucose and turned on during growth o n g a l a c t o s e . 29

Transcription Termination or Processing Signals for Heterologous Genes Many different 3'-flanking sequences have also been used. In the general plasmid shown in Fig. 1, the FLIP gene from 2-/tm circle plasmid is used for termination and the transcript polyadenylation occurs near the XbaI site close to the 2-/tm origin.~°,~ The PGK transcription termination region can also be used and sometimes results in higher steady states of mRNA than the FLIP terminator (data not shown). It is recommended that enough Y-flanking region is used (usually -> 300 bp) and at least part of the normal yeast structural gene (to make sure junctional information has not been lost); however, a few exact constructions containing homologous 3'-flanking sequences immediately after the stop codon of the heterologous structural gene do function well. 3°,3~These data suggest that the end of the normal yeast structural gene is not necessary for proper transcription termination and polyadenylation. Two mammalian cDNAs have 3'-flanking regions which are known to function in yeast [e.g., y-IFNa2 and IFN-a2~s]. Whether this is a universal phenomenon has not been explored, but, if an extensive 3'-region is not removed, a Northern analysis of the mRNA 33 made from a Fig. 1-type ptasmid can easily determine whether the yeast or heterologous 3'-flanking sequence is being used. We generally remove most of the 3'-flanking sequence on the heterologous gene (-< 30 bp remain). It has been previously published that the absence of a yeast 3'-flanking sequence on homologous a4 or heterologous ts genes results in a 10-fold or more reduction in mRNA and protein. Transcripts become very long, ending at various regions downstream from the normal stop regions. 27 K. Struhl, Cell49, 295 (1987). 2s T. Etcheverry, W. Forrester, and R. Hitzeman, Bio Technology 4, 726 (1986). 29 M. Johnston and R. W. Davis, Mot. Cell. Biol. 4, 1440 (1984). 3o C. Y. Chen, H. Opperrnan, and R. A. Hitzeman, Nucleic Acids Res. 12, 8951 (1984). 3~ C. Y. Chen and R. A. Hitzeman, Nucleic Acids Res. 15, 643 (1987). 32 R. Dcrynck, A. Singh, and D. V. Goeddel, Nucleic Acids Res. 11, 1819 0983). 33 p. R. Dobner, E. S. Kawasaki, L. Y. Yu, and F. C. Bancroft, Proc. Natl. Acad. Sci. U.S.A. 78, 2230 (1981). K. S. Zaret and F. Sherman, Cell 28, 563 (1982).

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Promoter- Heterologous Gene Junctions Between the homologous promoter and the ATG of the secretion signal, we normally use the DNA sequence TCTAGAATTC, which contains XbaI and EcoRI restriction sites and is present in the transcript. This sequence is substituted for the first 10 bases at the junction between the homologous promoter and the ATG initiation coding sequence of its natural gene. We have determined that this sequence does not affect mRNA levels or protein production for the homologous promoter and its natural gene. 3~ We have also used other linker sequences such as BamHI and XhoI without effects on expression. Of course, ATGs in the linker prior to the correct ATG must be avoided since the linker sequence is included in the mRNA. Such linkers are placed at the 5' end of the heterologous structural gene using M 13 mutagenesis a5 or by reconstruction with synthetic DNA from a restriction site within the structural gene.

Secretion Signal Sequences Figure 1 also shows a general schematic representation ofa presequence (secretion signal) used to secrete the desired mature protein. Two types of secretion signals have been used in such expression systems: those from heterologous genes and those from homologous genes. Heterologous signals have generally been used to secrete their natural gene products from yeast) 6 This is discussed in more detail in the following sections. Homologous signals have been more often chosen for hybrid expression units containing a natural yeast secretion signal and a heterologous gene (as shown in Fig. 1) to allow the production of the mature protein product in the fermentation media. In most cases, the signals have been obtained from the yeast invertase gene, 37 the c~-factor genes,25,26an acid phosphatase gene, 38 and the killer toxin gene. 39 The nature of the processing of these proteins during the secretion process has been determined. The desired heterologous protein product is usually the one with the same amino terminus as that of the protein from its natural environment. This is accomplished by attaching the DNA junction of the signal peptidase 3 cleavage site directly to the DNA of the first encoded amino acid of the 35 M. J. Zoller and M. Smith, Nucleic Acids Res. 10, 6487 (1982). 36 R. A. Hitzeman, D. W. Leung, L. J. Perry, W. J. Kohr, H. L. Levine, and D. V. Goeddel, Science 219, 620 (1983). 37 M. Carlson, R. Taussig, S. Kustu, and D. Botstein, Mol. Cell. Biol. 3, 439 (1983). 3s B. Meyhack, W. Bajwa, H. Rudolph, and A. Hinnen, EMBO J. 1, 675 (1982). 39 K. A. Bostian, V. E. Burn, S. Jayachandran, and D. J. Tipper, Nucleic Acids Res. 11, 1077 (1983).

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EXPRESSION IN YEAST

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mature peptide-encoding sequence (as shown in Fig. 1B). The fight side of the signal cleavage site seems to be very tolerant of different amino acids, and such a construct does give desired processing in many cases. 4° Processing can also be obtained at the prosequence level (as shown in Fig. I A). The best example of this is the use of one of the or-factor genes of yeast. Processing occurs for the natural system after a Lys-Arg site at the end of the prosequence due to the action of the KEX2 gene endopeptidase. 4~ Various groups have determined that the attachment of the mature heterologous gene DNA after this Lys-Arg-encoding sequence results in correct processing of the mature protein even with many changes on the right side of the junction. 42-~

Secretion Signal and Prosequence Junctions with Heterologous Genes The exact connection of the signal sequence or the prosequence to the mature heterologous gene is done by means of convenient restriction sites shown in Fig. 1 as RS1, RS2, and RS3. Reconstruction is accomplished in this vector or in simpler vectors using synthetic DNA. Junctions can also be engineered using oligonucleotide mutagenesis 35 for loop-out of undesired sequences or insertion of useful restriction sites. A stretch of synthetic DNA linkers between the presequence or prosequence and the transcription termination region may be useful for the insertion of the heterologous genes. However, since exact in-frame (translational) constructions are desired without extraneously encoded amino acids, unwanted DNA sequences must then be removed by oligonucleotide mutagenesis.

Examples of Use of Heterologous and Homologous Secretion Signals in Yeast

Heterologous Signals The first heterologous gene products were secreted from yeast using their natural secretion signal sequences) 6 Thus, DNA modifications within the structural gene were not necessary. These were cDNAs of human genes, 4o C. N. Chang, M. Matteucci, L. J. Perry, J. J. Wulf, C. Y. Chen, and R. A. Hitzeman, Mol. Cell. Biol. 6, 1812 (1986). 4~ D. Julius, A. Brake, L. Blair, R. Kunisawa, and J. Thorner, Cell37, 1075 (1984). 4~ A. J. Brake, J. P. Merryweather, D. G. Coit, U. A. Heberkin, F. R. Masiarz, G. T. Mullenbaeh, M. S. Urdea, P. Valenzuela, and P. J. Barr, Proc. Natl. Acad. Sci. U.S.A. 81, 4642 (1984). 43 A. Singh, J. M. Lugovoy, W. J. Kohr, and L. J. Perry, Nucleic Acids Res. 12, 8927 (1984). 44 G. A. Bitter, K. K. Chen, A. R. Banks, and P. H. Lai, Proc. Natl. Acad. Sci. U.S.A. 81, 5330 (1984).

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including interferon a (IFN-a), as well as leukocyte interferons I F N ~ I and IFN-t~2. A hybrid signal encoded by DNA sequences from pre-IFN-al and pre-IFN-a2 was also used productively for the secretion of IFN-a2. Figure 2 shows two of these amino-terminal sequences (structures 1 and 2) that result in the secretion of IFN-al and IFN-a2. Although 45-64% of the interferon in the media was properly processed between amino acids - l and + 1 (Gly-Cys) by the signal peptidase, 3 significant proportions were misprocessed between amino acids - 4 and - 3. A small amount (8%) of a different misprocessing also occurred in structure 2 (Fig. 2). These misprocessed forms suggest a difference in signal peptidase recognition for these signals in yeast versus human cells. The 50-70% of the interferon that remained cell-associated show similar levels of correct and incorrect processing and misprocessing. Expression of the human growth hormone (hGH) cDNA 45 results in properly processed hGH in yeast media ~s (see Fig. 2, 3). This suggests that signal peptidase recognition is not flawed for this signal. However, only 10% of the expressed material is secreted while 90% of the hGH remains cell-associated and retains the entire signal sequence, suggesting that differences exist in the efficiency of signal recognition between yeast and human secretion pathways. Another class of heterologous signals is represented by structure 4 (Fig. 2). The expression of the HSA cDNA t has been previously reported using the yeast chelatin promoter. 2s This human secretion signal works well in yeast, producing about 50% of the HSA free of the cell in yeast fermentation media. All of the HSA in the media is correctly processed at the prosequence cleavage site. These data are consistent with the data of Bathurst et al., 46 who showed that human proalbumin obtained from human cells is cleaved in K E X 2 yeast extract but not in a k e x 2 - yeast mutant extract. Thus, this processing is most likely done by the K E X 2 - e n coded endoprotease and is exactly like the processing seen after the amino acid pair Lys-Arg in the prosequence of tx-factor. 4~ However, in proHSA the amide bond is cleaved after an Arg-Arg amino acid pair. In the general yeast plasmid shown in Fig. 1, this pre- and prosequence of HSA results not only in HSA secretion with proper processing but also in the secretion and desired processing of other heterologous genes. Structure 5 in Fig. 2 is an example of this for human immunodeficiency virus

45 D. V. Goeddel, H. L. Heyneker, T. Hozumi, R. Arentzcn, K. Itakura, D. G. Yansura, M. J. Ross, G. Miozzari, R. Crea, and P. H. Sccburg, Nature (London) 281, 544 (1979). I. C. Bathurst, S. O. Brennan, R. W. Carreli, L. S. Cousens, A. J. Brake, and P. J. Barr, Science 235, 348 (1987).

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(HIV) gpl20. 47 HIV gpl20, is secreted from wild-type yeast as a hyperglycosylated form containing the processing after the dibasic Arg-Arg residues in the prosequence. Therefore, HSA DNA sequences may be as useful as the homologous a-factor pre- and prosequences which have been used for secretion and processing of heterologous gene products (see next section). Furthermore, these HSA sequences are shorter (24 encoded amino acids versus 85 for a-factor) than the homologous signal from a-factor (prosequence contains three N-linked glycosylation sites). In fact, in a side-byside comparison of a-factor and HSA pre/prosequences used for the secretion and processing of gpl20, secretion levels produced were essentially identical. Figure 3 shows the great potential of the expression-secretion unit encoding preHSA-proHSA-HSA (structure 4 Fig. 2) under the control of the chelatin promoter. A 10-liter fermentation run produces levels of HSA in the crude media as seen in lanes 3-5. Greater than 90% of the Coomassie blue-staining material is correctly processed HSA of 65,000 molecular weight. Interestingly, these high media levels seen in high-density fermentations are not seen in shake flasks. In shake flasks, most of the HSA remains cell-associated in the periplasm. Thus, if such yeast systems are used to obtain secreted proteins in media, vigorous fermentation conditions may be required for some proteins. It should also be noted that the percentage of material secreted varies from one heterologous gene production to another. It is also possible to use the signal sequences of HSA to produce fusion proteins in yeast media. Structures 6 and 7 in Fig. 2 are examples of such fusions. These constructions are just like structure 4; however, in this case, the carboxyl end encoding DNA of mature HSA has been modified to eliminate the translation stop and replace this DNA with a linker sequence for the amino acids Thr, Leu, Glu, and Phe. This linker DNA contains the sequence TCTAGAATTC, which contains the overlapping restriction sites XbaI and EcoRI. The EcoRI site has been used to connect the mature invertase-encoding DNA to HSA-encoding DNA in the proper reading frame. We modified the 5' end of yeast invertase gene 48 to contain an EcoRI site immediately before the ATG of mature invertase. This particular construction uses the 3'-flanking sequence of the yeast invertase gene for transcription termination. Extremely large quantities of the fusion protein are secreted (comparable to structure 4 in Fig. 2) and much of this is found in the media and is glycosylated. Structure 7 was made in a similar 47 L. A. Lasky, J. E. Groopman, C. W. Fennie, P. M. Benz, D. J. Capon, D. J. Dowbenko, G. R. Nakamura, W. M. Nunes, M. E. Renz, and P. W. Berman, Science 233, 209 (1986). 48 R. Taussig and M. Carlson, Nucleic Acids Res. 11, 1943 (1983).

432

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FIG. 3. Coomassie blue-stained SDS-polyacrylamidc gel of HSA. One-microliter samples of crude media from yeast fermentation runs were applied to the SDS-polyacrylamide 00%) gel in lanes 3-5. Lanes 3-5 contain HSA (strain 30-4) produced by construct 4 (Fig. 2). Lane 1 has protein standards with the second down being bovine serum albumin, which migrates essentially identically to HSA produced and secreted by yeast.

fashion and contains the small coding region of somatostatin 49 at the 3' end of mature HSA. Again, large amounts of the fusion protein are found in the media (comparable to structure 4 in Fig. 2 produced levels). The use of such fusions as carriers of small peptides or as antigens shows great promise. Such constructions may be of general research use for obtaining portions of proteins or entire proteins in extremely pure form for antibody production and for functional studies. These fusions may also be advantageous when direct secretion signal constructions do not secrete the protein of interest. Many other heterologous signal sequences have been used for secretion of their normally mature protein products (for review, see Ref. 50); however, there are few examples of processing determination or of the use of 49 K. ltakura, T. Hirose, R. Crea, A. D. Riggs, H. L. Heyneker, F. Bolivar, and H. W. Boyer, Science 198, 1056 (1977).

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SECRETION OF HETEROLOGOUS PROTEINS FROM YEAST

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heterologous signals for the secretion of other heterologous gene products (hybrid secretion proteins). Almost every hybrid secretion gene has been made using a homologous signal and a heterologous mature protein encoding sequence, as is discussed in the next section. However, the authors wish to point out the usefulness of a highly characterized heterologous signal attached to another heterologous gene.

Homologous Signals Figure 4 shows examples of how homologous (yeast) secretion signals can be used for the secretion and proper processing of heterologous gene products. Structure 8 demonstrates how the yeast invertase protein is processed during its secretion, driven by its amino-terminal peptide secretion signal sequence. 37 Knowing the natural junction sequence, we used this signal in structure 9 to secrete human interferon, IFN-a2, from yeast into the growth media. *° Several other human gene products have also been secreted from yeast using this signal.*° All these constructions resulted in properly processed protein in the media due to protein cleavage at the level of the signal peptidase. These results strongly suggest that the amino acid to the right of the signal peptidase cleavage site can be varied without affecting cleavage. A variant of the invertase presequence (Fig. 4, structure 10) results in secreted and properly processed IFN-a2.*O This construction had an alanine missing in the signal as well as a methionine at the right-hand side of the signal peptidase junction. The significance of the missing alanine will not be discussed. However, the junctional change of methionine to the right of the cleavage site further shows a flexibility in signal peptidase recognition of hybrid junctions. Another demonstration of a junctional change is demonstrated by structure 11. When this structure is expressed in yeast, protein secretion results and 100% properly processed HSA is found in the growth media. For this construction and the next, the yeast PGK promoter was used in the general yeast expression-secretion vector (Fig. 1). Structure 12 (Fig. 4) is similar to structure 11 but has an insert of 6 amino acids (Arg Gly Val Phe Arg Arg) at the signal peptidase cleavage site of structure 11. These additional amino acids are the prosequence encoded by the cDNA of HSA. Production and secretion for structures l l and 12, as well as 4, are essentially identical when all other parts of the expression plasmid are kept the same. Structure 12 also gives correctly processed HSA in the yeast media. However, unlike previously discussed structures, this one contains a homologous secretion signal and a heterologous proseso R. C. Das and J. L. Shultz, BiotechnoL Prog. 3, 43 (1987).

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SECRETION OF HETEROLOGOUS PROTEINS FROM YEAST

435

quence. Correct processing is at the level of the yeast KEX2 gene endoprotease as described in the previous section. Thus, another option is available for the secretion and processing of a heterologous gene product. Structure 15 reveals still another option for secretion and processing. This structure is similar to 8 (Fig. 4); however, at the carboxyl end encoding DNA of mature invertase, the translation stop codon has been replaced by an EcoRI restriction site using M 13 oligonucleotide mutagenesis. An EcoRI site was also placed at the beginning of the prosequence of structure 4 (Fig. 2). Then modified structures 8 and 4 were placed together using these EcoRI sites to give structure 15. Structure 15 encodes a procleavage site between mature invertase and mature HSA. It is interesting that, on expression of this gene in yeast, one obtains active invertase secreted as well as normal-size secreted HSA as determined by Western analysis. These results suggest that the HSA prosexluence can be used at the carboxyl end of a normally secreted protein in yeast to get processing of a heterologous gene protein, which is being carded along through the secretion pathway. It will be interesting to see if such an expression- secretion system may be more effective than the attachment of pre- and prosequences alone in the secretion of some proteins (especially those that are normally not secreted in their natural environment). Finally, we have saved the most frequently used secretion system for the last part of this discussion. This system is based on the yeast a-factor pre- and prosequences. Two a-factor genes have been cloned from yeast. 25,26 These genes produce the a mating pheromone, a small peptide which prepares the opposite mating type (a) for cell fusion and zygote formation3 ~The processing of the pheromone precursor from one of these gene products has been extensively characterized.25.41,~2 The signal sequence is attached to a large prosequence to give a combined pre/prosequence of 85 amino acids. This prosequence contains three N-linked glycosylation sites (unlike that of HSA). The prosequence, as well as intervening amino acid sequences between four tandem copies of the mature pheromone, are clipped by the K E X 2 gene product after Lys-Arg residues. The cleaved a-factor pheromones retain Glu-Ala-Glu/Asp-Ala at their amino-terminal ends and Lys-Arg at the carboxyl end. The amino-terminal sequences are then removed by a dipeptidylaminopeptidase which removes two amino acids at a time. 52,53The Lys-Arg residues are removed by another protease encoded by KEXI, "~2 however, this protease is irrelevant to the processing of heterologous gene products. 5~ I. Herskowitz and Y. Oshima, "The Molecular Biology of the Yeast Saccharomyces: Life Cycle and Inheritance," p. 181. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1981. s2 D. Julius, L. Blair, A. Brake, G. Sprague, and J. Thorner, Cell32, 839 (1983). ~3 R. B. Wickner, Genetics 76, 423 (1974).

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Singh et al. 4a have described the use of structure 13 in Fig. 4 to obtain Met IFN-al. The a-factor promoter was used in a Fig. l-type plasmid. The pre- and prosequences of the a-factor gene were attached by means of an XbaI and EcoRI restriction site linker [encodes Leu-Glu-Phe, which allows for convenient in-frame (translational) connection of other genes as well]. It was not expected that this amino acid sequence would be removed during secretion of the protein. However, the K E X 2 protease cleavage at the procleavage site (after Lys-Arg) was expected, as well as the removal of Glu-Ala-Glu-Ala. All the yeast media interferon retained the Glu-Ala-GluAla. Further studies suggest that, at high expression levels, the protease that performs this function may be limiting. In order to overcome this problem, structure 14 (Fig. 4) was made which places the Lys-Arg next to the Cys of mature IFN-al. Correctly processed IFN-al was obtained at levels of about 50% of the media protein. Since this time, many constructions like this have been made with different amino acid sequences to the fight of Lys-Arg and all have been fairly well processed. An accompanying article about a-factor discusses this in greater detail. 54

Glycosylation of Heterologous (3ene Products Problem

When proteins travel through the secretion pathway of yeast, core glycosylation occurs at the endoplasmic reticulum and outer-chain glycosylation occurs in the Golgi bodies. One type of glycosylation happens at Asn residues within the amino acid sequence Asn-X-Ser/Thr (X is any amino acid) (see Fig. 5). s5'56 An accompanying article by B a i l o u 57 describes this glycosylation process in great detail. However, when heterologous gene products are glycosylated during secretion from yeast, they are glycosylated somewhat differently than what normally occurs in the cell from which the gene was obtained. For example, most of the genes described in this article are from human cells where the glycosylation process is similar. However, the sugar content at Asn residues is somewhat different. Human cells have a very similar inner core but lack the extensive "flowering" of the large outer chain which can be repeated several times. This characteristic results in a gross difference 54 A. J. Brake, this volume [34]. " C . E. Ballou, "This Molecular Biology of the Yeast Saccharomyces: Metabolism and Gene Expression," p. 335. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1982. 56 p. K. Tsai, J. Frevert, and C. E. Ballou, J. Biol. Chem. 259, 3805 (1984). 37 C. E. Ballou, this volume [36].

[35]

SECRETION OF HETEROLOGOUS PROTEINS FROM YEAST

437 I I

~r/ Thr

x [ M ~---~sM ~

T: M

M~

M .~-~ M

T: M T: M

T: M T: M T: M

I

Asn

I

x

M

t:

T: T: M

M

[ Outer Chain I

[ Inner Core

I

FIG. 5. Asparagine-linked glycosylation in yeast. The primary protein sequence is at the fight. M refers to mannose residues, P is phosphate, and GNAc is N-acetylglucosamine. Only one type of mutant that affects outer-chain glycosylation is shown, mnn9. 56 Note that this structure has been modified since the submission of this chapter. 57

between the sugar content of a heterologous human protein produced in yeast and mammalian cells. Other differences also exist between yeast and human glycosylation. The mannose content relative to other sugars in human glycosylated proteins is not as great as in yeast. Several other sugars are added within the inner core by a series of reactions that vary from one human cell type to another. The extent of the difference between human and yeast glycosylation can be easily seen with expression of the gpl20 gene of HIV. This retrovirus envelope glycoprotein gene has been previously truncated by placing a premature translation stop within the hydrophobic region between gpl20 and gp41. This truncated protein has been shown to be secreted by CHO cells. 4v We have further modified the gpl20 gene as described earlier in this article (Fig. 2, structure 5). The pre- and prosequences of human HSA were attached to the first 489 amino acids ofgpl20. The general expression plasmid in Fig. 1 was used with the yeast chelatin promoter and the yeast PGK transcription terminator. This portion of the gpl20 gene should produce a secreted protein monomer of about 54,000 Da if it is not glycosylated. However, there are 24 possible N-linked glycosylation sites. In mammalian cells, this results in a protein monomer of 120,000 Da due to extensive sugar addition. In yeast, this gene expression results in a hyperglycosylated form ofgp 120 of greater than 600,000 Da. This is shown by the Western blot 5a in Fig. 6. A reducing SDS-polyacrylamide gel was run with media from yeast expressing the gpI20 gene (lane 1) and from the same yeast strain containing a plasmid without this gene (lane 2). When the yeast medium material was treated with endo H (which removes the 58 W. N. Burnette, Anal. Biochem. 112, 195 (1981).

1

2

3

" 200

497

"68

"44 FIG. 6. HIV gpl20 secreted by wild-type yeast and a mnn9 mutant yeast. This is a Western blot ss ofa SDS-polyacrylamide (7%) gel. Lane 1 contains culture mexiium from yeast 20B-12 containing construction 5 (Fig. 2). Lane 2 is medium from yeast 20B-12 containing construction 4 (Fig. 2) (no gpl20). Lane 3 is medium from yeast tx mnn9 trpl- containing construction 5. Antiserum against HIV gpl20 was incubated with blot, after gel proteins were blotted onto nitrocellulose. Antibody binding to gpl20 was visualized using t25I-labeled protein A and autoradiography. Standard protein migration is as indicated (MW × 10-3).

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sugars), the gpl20 migrates at about 60,000 Da (data not shown). This hyperglycosylated form of gp 120 does not bind to the CD4 receptor 59 and binds poorly to polyvalent and monovalent antibody columns (antibodies to the mammalian cell-produced material47). Partial Solution A mnn9 mutant 57 yeast characterized by Tsai et al. 56 (kindly provided by Clinton Ballou) behaves as shown in Fig. 5. The mutant lacks an active a-l---,6-mannosyltransferase that initiates outer-chain glycosylation. Therefore, the mnn9 mutant produces an N-glycosylated protein which totally lacks the outer chain. We crossed this mnn9 mutant with strain S1916° to obtain an ct mnn9 trpl spore. This strain was transformed with the gp120 gene expression plasmid described above and the production of gp 120 protein in the yeast medium was examined. In Fig. 6, lane 3, the medium from this strain is shown by Western analysis to produce a smear at around 120,000 Da. This is identical to the mammalian-produced material. When compared by SDS-gel electrophoresis, it is difficult to notice any difference between the two. In fact, with storage, some of the same-size degradation products appear in both (data not shown). Therefore, relative sugar addition to this protein containing an extremely high number of glycosylated sites is essentially identical with respect to size in this yeast mutant and in mammalian cells. Unfortunately, this drop in size does not significantly improve binding to CD4 (the receptor protein), although binding to polyvalent antibody columns is greatly improved. The lack of binding to CD4 suggests that there may still be folding differences between yeast and mammalian cell-produced gp 120. Whether this is related to inner-core glycosylation differences is unknown. Recent experiments by Moir and Dumais 61 which describe the expression and secretion of human a-1-antitrypsin suggest similar conclusions. They found that ix-1-antitrypsin secreted by the wild-type yeast was hyperglycosylated (3-N-glycosylation sites) in comparison to human cell-derived material. However, when it was produced in the mnn9 yeast, it was slightly smaller than the natural protein. Recently, we have cloned the wild-type M N N 9 gene using the extremely sensitive nature of mnn9 yeast to the antibiotic hygromyein B. We have verified the identity of the gene by several criteria and have sequenced the structural gene and its flanking regions. This gene can now be conve~9 D. H. Smith, R. A. Byrn, S. A. Marsters, T. Gregory, J. E. Groopman, and D. J. Capon, Science 238, 1704 (1987). 6o Yeast strain S191, a trpl met15 from Arjun Sing,h, unpublished results. 61 D. T. Moir and D. R. Dumais, Gene 56, 209 (1987).

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niently deleted in the yeast genome (e.g., in a ura3-- yeast strain using a linear DNA containing URA3 flanked by MNN9 sequences). Such deletions are useful since we have previously had some trouble with reversion of the original mnn9 mutant. Concluding C o m m e n t s If one intends to produce and secrete a heterologous gene product using yeast, several options are available to accomplish this. If it is a secreted protein in its normal cell environment, one can first try its own amino-terminal secretion signal. Many have been functional; however, the amount of protein produced, the amount secreted, and the processing which occurs are all variable from one gene product to another. If the heterologous signal does not function in any of the desired aspects mentioned above, homologous or heterologous signals that are known to function in a somewhat predictable fashion in yeast may be tried. This article has described how such hybrid constructions can be made and the many options available.

[36] Isolation, Characterization, and Properties of Saccharornyces cerevisiae mnn Mutants with Nonconditional Protein Glycosylation Defects B y CLINTON E. BALLOU

Introduction Three classes of mutant have been obtained that are defective in protein glycosylation in yeast, one based on selection by [3H]mannose-killing and called alg (asparagine-linked giycosylation defective) mutants, l one based on changes in cell density owing to accumulation of membrane material and called sec (secretion defective) mutants, 2 and one based on changes in cell surface carbohydrate structure and called mnn (mannan defective) mutants) The first two selections are designed to detect conditional mutants, whereas the last selects nonconditional mutants because the cells must grow in order to express the change in phenotype. The alg mutants are affected mostly in early steps involving synthesis of the dolichol-linked precursor oligosaccharide and often lead to underglycosylation of proteins. The sec mutants are defective in the processing of glycoproteins through I T. C. Huffaker and P. W. Robbins, Proc. Natl. Acad. Sci. U.S.A. 80, 7466 (1983). 2 p. Novick, C. Field, and R. Schekman, Cell 21, 205 (1980). 3 W. C. Raschke, K. A, Kern, C. Antalis, and C. E. Ballou, J. BioL Chem. 248, 4660 (1973).

METHODS IN ENZYMOLOGY, VOL. 185

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