BioMedNet HomeLibraryDatabasesCollaborationsJob ExchangeShopping MallYour Room


FULL TEXT (TEXT + FULL FIGURES)

Current Opinion in Biotechnology
Vol. 6, No. 5, October 1995
Advances in the use of Pichia pastoris for high-level gene expression
[Review article]
Mike Romanos
Current Opinion in Biotechnology 1995, 6:527-533.
 
Viewing options [Help]

text only | + thumbnails | + full figures
 
 
Associated links
 
 
-> Publications by
Mike Romanos
 
 
-> Related fulltext articles on BioMedNet
 
 
-> Fulltext articles on BioMedNet that cite this article
 


Outline


Abstract
The Pichia pastoris system has the potential for very high level production of foreign proteins. This, together with the recent availability of this system in kit form, has changed Pichiafrom being an expression system with only specialized biotechnological applications to one that is widely used for rapid expression in the laboratory. The development of G418 selection protocols has simplified the rapid isolation of multicopy transformants efficiently expressing intracellular proteins. In addition, several strategies are now also available for optimizing secretion, such as the generation of clones with progressively increasing vector copy number, expression screening in microtitre plates, and minimizing proteolysis by a number of techniques.

Abbreviations
AOX1—alcohol oxidase 1;
EGF—epidermal growth factor;
HSA—human serum albumin;
IGF-1—insulin-like growth factor 1;
Muts—methanol utilization slow;
PHO-1—acid phosphatase 1.


Introduction

In the past few years, the Pichia pastoris expression system has rapidly achieved much wider use thanks to increasing awareness of the early successes and to its sale in kit form by Invitrogen (San Diego, USA). From being a specialist system, used primarily by yeast groups in biotechnology companies, it is now a mainstream expression host used alongside Escherichia coli, Saccharomyces cerevisiae, and baculovirus. This was clear from the diverse interests of delegates at a recent Pichia meeting (San Diego, USA, 1994) organized by Invitrogen.

Pichia is an industrial methylotrophic yeast initially chosen for production of single-cell protein because of its ability to grow to very high cell density in simple defined media. From this basis, a highly efficient expression system was designed using the methanol-inducible alcohol oxidase 1 (AOX1) promoter and vectors that integrate into the Pichiagenome [1]. Several examples show that Pichia can routinely achieve percentage yields (5–40% of cell protein) much higher than baker's yeast, and often equivalent to E. coli or baculovirus [1][2]. Additionally, scale-up of Pichia culture to high cell density is simple and has resulted in enormous yields on a volumetric basis (e.g. >12 gl-1 for tetanus toxin fragment C [3] and >3 gl-1 secreted human serum albumin [HSA] [4]).

Although most Pichia expression vectors are very similar, different strategies can be employed because a selection of host phenotypes and chromosomal sites of integration is available. This gives greater scope for optimizing expression, but has caused some confusion, particularly among users unfamiliar with yeast genetics. In this review, I attempt to clarify the choices that are available and give suggestions as to when they should be used. I also discuss some of the most recent developments and results with the Pichia system, and provide an update on some of the many Pichia-derived products in commercial development or about to reach the market.



Pichia: advantages and disadvantages

Pichia has the following main advantages: first, extremely high yields of intracellular proteins; second, very high levels of secretion into an almost protein-free medium; third, ease of fermentation to high cell density; and fourth, genetic stability and scale-up without loss of yield. It is proving valuable in producing large amounts of protein for analytical studies; one interesting recent application is in efficient in vivo isotopic labelling of proteins for NMR [5••]. The system is almost certainly the simplest of any to scale up, a feature that makes it very attractive for rapid production of biological products for clinical trials.

Fig. 1.Two typical Pichia expression vectors, pPIC3 and pPIC3K.(a) Vector pPIC3 comprises a 5' AOX1 region including promoter, cloning site polylinker, AOX1 terminator (AOX1t), HIS4marker, 3' AOX1 region and ampicillin-resistance marker (Ap). (b) Vector pPIC3K, in addition to the above, contains the kanamycin-resistance (kanr) marker for G418 selection of multicopy transformants.

Return to text reference [1]

Like any other expression system, however, Pichia is no panacea, and examples of low yields or failure of expression are also accumulating, though many remain unpublished. Probably the commonest problem encountered is proteolysis of secreted polypeptides [1], though a number of ways of overcoming this have become available. Another common problem is inefficient secretion of complex foreign proteins (e.g. HIV-1 gp120 [6]). Despite many examples of yields in the grammes per litre range, heterologous secretion is more demanding than intracellular expression and not guaranteed to work. Finally, some genes do not give any detectable protein, often because yeast transcriptional terminators result in truncated mRNA. The problem was first described for the highly AT-rich tetanus toxin gene in S. cerevisiae and solved by gene synthesis to increase the GC content [2]. The same problem has been seen in Pichia and solved in the same way (e.g. with the Bacillus sphaericus Bsp2 insecticidal toxin [P1] and with HIV-1 Env [6]). It is noteworthy that the Env DNA, which is not AT-rich, is efficiently transcribed in S. cerevisiae.



Pichiavectors and strains

As Pichia has no stable episomal vectors, integrating vectors are employed. All use HIS4 as a selectable marker and have the same general organization, as exemplified by pPIC3 (Fig. 1). Some of the most useful vectors and their properties are listed in Table 1. The most commonly used host strain is GS115 (his4), but the more recently available protease-deficient strains (e.g. SMD1168;his4, pep4) are finding increasing use in reducing proteolytic cleavage of secreted proteins. KM71 (his4, aox1) and other strains that have an inactive AOX1 gene, are fundamentally different in that they grow very slowly on methanol as a carbon source, for example, during induction (i.e. they have a Muts phenotype, rather than the Mut+ phenotype of GS115).

Table 1. Pichia expression vectors.
Vector name Unique cloning sites Selection markers Comments References
Intracellular production
pPIC3 BamHI, NcoI* SnaBI, EcoRI, AvrII and NotI HIS4 Polylinker vector [12••]
pPIC3K BamHI, NcoI* SnaBI, EcoRI AvrII and NotI HIS4 and kanr Polylinker vector with G418-selection for multicopy clones [12••]
pHIL-D1 EcoRI HIS4 One of the first basic vectors [9]
pHIL-D2 EcoRI HIS4 Contains f1 ori and NotI sites for transplacement [a]
pHIL-D3 AsuII and EcoRI HIS4 Contains f1 ori and native AsuII site for unaltered AOX1 5' untranslated region [a]
pHIL-D4 EcoRI HIS4 and kanr G418-selection of multicopy clones [a]
pHIL-D5 EcoRI HIS4 and kanr Contains f1 ori, NotI sites for transplacement and G418-selection for multicopy clones [a]
pHIL-D7 AsuII and EcoRI HIS4 and kanr Contains fi ori, a native AsuII cloning site, NotI sites for transplacement, and uses G418-selection for multicopy clones [a]
pAO815 EcoRI HIS4 Contains f1 ori and a BamHI site for generation of multicopy expression units in vitro [1][16]
pHIL-S1 XhoI, EcoRI, SmaI and BamHI HIS4 Contains Pichia PHO1 signal and f1 ori [a]
pPIC9 XhoI, SnaBI, EcoRI, AvrII and NotI HIS4 Contains a S. cerevisiae alpha-factor leader [12••]
pPIC9K XhoI*, SnaBI, EcoRI, AvrII and NotI HIS4 and kanr Contains a S. cerevisiae alpha-factor leader, with G418-selection for multicopy clones [12••]
pYAM7SP6 StuI, EcoRI, BglII NotI, XhoI, SpeI and BamHI HIS4 Contains a hybrid Pichia Pho1 signal with a Kex2 endopeptidase cleavage site [5••]
*The cloning site is not unique, thus three-way ligations are used. [a] K Sreekrishna, K Kropps, personal communication. (For further details contact K Sreekrishna, Marion Merrell Dow Inc, 2110 East Galbraith Road, PO Box 156300, Cincinnati, Ohio 45215, USA.)
Return to table reference [1]

Expression vectors are directed to integrate into the Pichiagenome in one of two ways, depending on where the DNA is cut before transformation (see Table 2). Digestion to give a DNA fragment with homology to AOX1 at both ends leads to replacement of genomic AOX1 by the fragment (i.e. transplacement) and generates a Muts recombinant strain. Linearization of the vector, by cutting either 5' to the AOX1 promoter or within the HIS4 marker, directs integration of the plasmid at the homologous sites in the genome. The Mut phenotype of the latter type of transformant is dictated by the host strain used.

Table 2. Types of Pichia transformant.
Host strain Vector digest to direct integration* Resulting His+ transformants Comments
GS115 (Mut+) SacI (AOX1integration) Vector is integrated 5' to the genomic AOX1 gene, leaving the AOX1 gene undisrupted (i.e. Mut+ phenotype) High-frequency transformation using either sphaeroplasting or electroporation. Ideal for routine use. ~ 100% of transformants express protein. Multi-copy integrants (up to 10 copies) arise at low frequencies
SalI and StuI (HIS4 integration) Vector is integrated within genomic his4 locus. AOX1 gene undisrupted (i.e. Mut+ phenotype) High-frequency transformation using either sphaeroplasting or electroporation. ~ 100% of transformants express protein. Note potential to generate His+ 'pop-outs' lacking foreign gene‡ . Multi-copy integrants (up to 10 copies) arise at low frequency
BglII† (AOX1 transplacement) BglII fragment replaces genomic AOX1 gene, generating Muts phenotype. (Only 5–25% of transformants are of this type, the remainder are mainly AOX1 or HIS4 integrants [i.e. Mut+ phenotype]) Low-frequency transformation, preferably using the sphaeroplast method. This is best method for multi-copy clones, yielding a 1–10% frequency and up to 30 copies. Generates a heterogeneous pool of Muts and Mut+ transformants, including some non-expressers
KM71 (Muts) SacI or SalI/StuI Same transformants, as GS115, except all are MutS because host AOX1 gene is already disrupted. Higher transformation frequency than GS115, especially with electroporation
*These sites are common to all vectors and can be used generally, unless present in the foreign gene. † The vector pHIL-D2 has NotI sites in place of BglII and can be used when the foreign gene contains BglII sites. ‡ This problem does not often occur in small-scale cultures.
Return to table reference [1]

Some unexpected features of Pichia transformations increase the number of types of transformant and thus the potential for confusion. With integration at AOX1 or HIS4, multicopy transformants (up to 10) can arise from repeated recombination events. On the other hand, transplacement would be expected to yield only single-copy Muts transformants; however, a detailed analysis has revealed three sources of heterogeneity among the transformants [3]. First, a high proportion of 'transformants' contain no vector, express no foreign protein and probably represent his4gene conversions. Second, only 5–30% are true transplacements (Muts), the remainder have integrations at AOX1 or HIS4 and are Mut+. Finally, 1–10% of Mutstransformants have up to 30 integrated copies of the transplacing fragment as tandem head to tail repeats that probably arise by a mechanism involving in vivo ligation [3]. The Mut+ population also contains multicopy clones. Although transplacement usually requires the laborious sphaeroplast transformation technique because of low frequencies, it yields a highly divergent population of transformants, which is useful for detailed optimization studies. Also, it seems to be the only way of obtaining very high copy number transformants.



Gene dosage is critical for maximal expression

In some of the earliest studies, expression of beta-galactosidase and hepatitis B surface antigen was not improved by increasing vector copy number [7][8]. In numerous subsequent examples, however, the isolation of multicopy integrants has resulted in dramatically higher yields [3][9][10][11]. In a detailed study of tetanus toxin fragment C, our group [3] showed that expression was correlated with copy number (1–14 copies), whereas site of integration and Mut phenotype had, at most, only a minor effect on yield. This is perhaps surprising because the Mut phenotype affects both the growth rate during induction and the accumulation of large amounts of endogenous alcohol oxidase. Muts strains may yield a higher proportion of correctly folded product in situations where folding is rate-limiting (e.g. in the case of hepatitis B surface antigen [8]).

The extremely high level of alcohol oxidase (5–30%) expressed from AOX1in induced wild-type Pichia had suggested that one copy of an AOX1 expression vector would produce maximal mRNA levels. In a recent paper, mRNA levels were analyzed from a series of transformants containing increasing copy numbers (1–12) of an HIV-1 Env expression vector [12••]. The Env mRNA level increased progressively with copy number up to the maximum number tested; at a single copy of the vector, it was two-thinspace to threefold less than AOX1 mRNA, but with greater than three copies it exceeded AOX1 mRNA. A progressive increase was also seen with fragment C, suggesting that transcription is, in general, limiting in Pichiacontaining only a single copy of the vector and that it is sensible to routinely maximize copy number. Despite this, other factors, such as protein stability, must have a major effect because final yields of different proteins vary greatly, even with multicopy clones. For example, 12 copies of Env vector yielded 2.5% of total cell protein, whereas 14 copies of fragment C vector yielded 27%.

Aside from gene dosage, a critical factor that has long been recognized to affect induction efficiency is aeration ofPichia cultures. This results from the tendency of cultures, especially Mut+ strains, to become oxygen-limited in shake-flask inductions, and it probably explains the consistently large increase (e.g. 5–10-fold) in yield that is observed when switching to fermenters.



Secretion in Pichia

Many users have been attracted to Pichia by the very high reported levels of protein secretion in high-density cultures, such that the product can comprise >80% of the protein in the medium. Yet, secretion is complex and is dependent not only on factors such as gene dosage and Mut phenotype, but also on other factors that affect the yield and quality of product (e.g. signal sequence, processing, proteolysis and glycosylation).

Perhaps the most contentious issue, as demonstrated by the lively debate at the recent meeting on Pichia in San Diego, is Mut phenotype. Because secretory vesicles in S. cerevisiae localize to the bud, it had been widely believed that secretion could only occur in dividing cells. Secretion of mouse alpha-amylase fromS. cerevisiae has, however, been shown to be as efficient in non-dividing as dividing cells [13]. The group at SIBIA Inc (La Jolla, California) had favoured Mut+ strains and have used these in many examples of high-level secretion (e.g. epidermal growth factor [EGF], insulin-like growth factor-1 [IGF-1], aprotinin, etc. [14] [P2] [P3••]). Even so, results with HSA [4] and murine EGF [15] demonstrate that Muts strains can also yield high levels. Many more recent successful examples utilize either Mut+ or Muts strains.

In the case of gene dosage, agreement is absolute that an optimal, rather than maximal, copy number is usually required. In many cases, secretion efficiency has been improved with several vector copies [P2][P3••]. Examples also exist where the maximum copy number tested was optimal (e.g. with murine EGF [15]). With bovine lysozyme, however, increasing the copy number from one to three reduced the level of secreted product [16], and for HIV gp120, a copy number of greater than one reduced secretion and increased accumulation of intracellular products [6]. It would appear that less efficiently secreted proteins are likely to block the secretory pathway at higher expression levels.

Several foreign proteins have been efficiently secreted using their native signal peptide (e.g. HSA, where a strain containing three copies of the gene, expressed from a modifiedAOX2 promoter, gave a staggering yield of 10 gl-1 [4][P4]). With bacterial alpha-amylase, however, the yield using a yeast signal was two-thinspaceto threefold higher [17•], and in general, yeast signals are more likely to be successful [2]. Secretion of EGF and murine EGF using the S. cerevisiae alpha-factor leader was shown to be highly efficient, and analysis of the product showed authentic processing at the Kex2 endopeptidase cleavage site [15][P2]. The vectors pPIC9 and pPIC9K, which contain the alpha-factor leader sequence with convenient cloning sites [12••], have recently become available from Invitrogen and are now widely used. Recent successful examples using the alpha-factor leader include the following: single-chain Fv antibody fragments [18•]; a 9 kDa thrombomodulin fragment [19]; blood factor XII [19]; a fragment of amyloid beta-protein [20]; oncostatin M (SJ McAndrew et al., abstract, Current Topics in Gene Expression Systems: Pichia pastoris, San Diego, USA, October 1994) coffee-bean alpha-galactosidase (A Zhu, LF Kimball, Current Topics in Gene Expression Systems: Pichia pastoris, San Diego, USA, October 1994), and cathepsin B (JS Mort et al., abstract, Current Topics in Gene Expression Systems: Pichia pastoris, San Diego, October 1994). An alternative signal sequence, that of the Pichiaacid phosphatase 1 (PHO1) gene, is used in the vector pHIL-S1, which is also available from Invitrogen.

Studies using S. cerevisiae have shown that the particular yeast signal peptide used can affect efficiency of secretion [2]. Recently, a synthetic hybrid signal based on Pho1, with an additional 19 residues, including a Kex2 cleavage site, has been found to improve secretion of tick anticoagulant protein and some other proteins two-thinspace to threefold in Pichia [5••]. Even so, it is not clear that general rules concerning the signal peptide can be applied to any protein.

The problem of proteolytic instability in the medium has been encountered with several proteins secreted from Pichia. It has been seen in shake flasks, but usually appears to be far worse in fermenters, because of the higher concentration of proteases or the different medium used. Three different approaches have been used successfully to overcome proteolysis [1]: adding amino acid or peptide supplements to the growth medium, buffering the pH of the medium to a value where degradation is reduced (e.g. pH 3), or using protease-deficient host strains. In the case of IGF-1, no protein was produced unless the medium was buffered to pH 3, but a 50% increase was then achieved using a pep4 strain grown in a medium buffered to pH 3 [P3••].

S. cerevisiae has been avoided as a host for the development of human therapeutic glycoproteins because yeast-derived glycoproteins are antigenic and frequently hyperglycosylated (i.e. they contain extensive outer chain mannose units [50 to 150 residues] that can mask function). Pichia-derived invertase is, however, not hyperglycosylated and has an outer-chain length of 8–14 units, compared with >50 in S. cerevisiae[21]. BulkPichia glycoprotein was found to be less frequently hyperglycosylated, to have a shorter outer-chain length (<30 residues), and to lack the highly antigenic terminal alpha1,3-mannose linkages present in S. cerevisiae [22][23]. Nevertheless, it should be emphasized that glycosylation in Pichia and that in mammals is not identical, and it is not known how antigenicPichia glycoproteins are or how the pharmacological properties of heterologous products might be affected.

For the development of vaccines, totally authentic glycosylation may be unnecessary, provided the proteins elicit a protective immune response. Even so, results with two viral glycoproteins, HIV1 gp120 and Epstein–Barr virus gp350, were not promising. HIV-1 gp120 was hyperglycosylated [6], whereas gp350 was less highly glycosylated than the S. cerevisiae-derived material (CA Scorer, personal communication), but neither of these proteins was recognized by antibodies raised against the native protein. In contrast, the Bm86 cattle tick membrane glycoprotein, secreted using the invertase signal peptide, was not hyperglycosylated and formed immunogenic particles which partially protected cattle against challenge [24•].



Strategies for expression

For intracellular expression, it would seem reasonable to isolate transformants with maximum vector copy number. A G418-selection protocol using vectors containing the Tn903 kanr gene (e.g. pPIC3K) has recently been described [12••]. Using this procedure, multicopy clones (5–10 copies) can be isolated, even following electroporation, which had been thought to yield only single-copy transformants. This greatly simplifies the rapid isolation of multicopy clones and may be considered the method of choice for general laboratory use. In practice, this method works best using the KM71 strain and SacI-digested vector because of the higher transformation frequencies that are attainable. Where very high copy numbers (10–30) are required, however, it then appears that transplacement using the sphaeroplast transformation method must be used. Transformants can subsequently be screened for high copy number 'jackpot clones', either Muts or Mut+, using a rapid DNA dot-blot method [11]. Precise copy numbers should be determined using quantitative DNA dot blots [3].

For secretion, initial studies could be carried out with single-copy transformants, but it would be preferable to start with a series with different copy numbers. Such a series could be created in several ways. A range of multicopy transformants, isolated by G418-selection [12••] or DNA dot blot [11], could be analyzed for copy number [3]. An alternative method developed by SIBIA is to utilize a plasmid that can be used to generate multi-cassette vectors with up to eight copies in vitro [1][16]. This method has the disadvantage that several DNA cloning steps are required, but the advantage that the copy number is determined before transformation. Any of these approaches can be carried out in either Mut+ or Muts strains for comparison.

An alternative empirical approach to optimization has been used for tick anticoagulant protein [5••]. Transplacement was used to generate a heterogeneous pool of transformants and the level of secreted product from 91 Muts and Mut+ clones was tested in inductions in microtitre plates. Eleven transformants were further evaluated in shake-flask inductions and one chosen for fermenter optimization, giving a a final product level of 1.7 gl-1 of product. This method could be used in other cases where a simple method exists for quantitating the product.

Pichia transformants are generally tested in fermenters as soon as possible because shake-flask inductions are sub-optimal. Mut+and Muts strains each have their advantages: Mut+ strains are less likely to become poisoned by methanol, whereas Muts strains are less likely to become oxygen-limited. Although a matter of debate, reports exist of induction using Muts that is equally rapid (48 h) as that using Mut+ strains [3][10][11][15]. Because the increase in yield in going from shake-flask to fermenter inductions is not always predictable [11], it may be prudent to select several transformants before proceeding to detailed fermenter optimization.



Conclusions

The number of different proteins being expressed in Pichia is expanding rapidly, and results from ongoing studies will increase our knowledge of the capabilities and limitations of this expression system. The more wide adoption of Pichia, however, will require additional refinements (e.g. more auxotrophic and mutant strains, alternative selection markers, a drug-selection system that shows greater dose-dependence than G418, vectors for simultaneous expression of two proteins and alternative promoters to AOX1). These refinements, many of which should be available shortly, will enable the Pichia system to gain some of the flexibility of S. cerevisiae.

Pichia is already widely accepted as an important biotechnological host organism, and we are now at the exciting stage of observing products moving through to clinical trials and beyond. IGF-1 and HSA should soon be marketed products for the treatment of amylotrophic lateral sclerosis and as a serum replacement, respectively. Numerous cytokines, vaccines and other biological products are under development, and a Cuban group has developed a hepatitis B vaccine that is currently being sold in South America.



Acknowledgements

I would like to thank my colleagues in the field, especially Jeff Clare, Koti Sreekrishna, Jim Cregg, Rich Buckholz, David Higgins, Mick Hunter and Bennet Cohen, for continually sharing information with me.



References and recommended reading
Papers of particular interest, published within the annual period of review, have been highlighted as:
• of special interest.
•• of outstanding interest.
  1. Cregg JM, Vedvick TS, Raschke WC:
    Recent advances in the expression of foreign genes in Pichia pastoris.
    Biotechnology 1993, 11: 905–910. [Cited by]
    Return to citation reference [1] [2] [3] [4] [5] [6]

  2. Romanos MA, Scorer CA, Clare JJ:
    Foreign gene expression in yeast: a review.
    Yeast 1992, 8: 423–488. [MEDLINE] [Cited by]
    Return to citation reference [1] [2] [3] [4]

  3. Clare JJ, Rayment FB, Ballantine SP, Sreekrishna K, Romanos MA:
    High-level expression of tetanus toxin fragment C inPichia pastoris strains containing multiple tandem integrations of the gene.
    Biotechnology 1991, 9: 455–460. [Cited by]
    Return to citation reference [1] [2] [3] [4] [5] [6] [7] [8]

  4. Barr KA, Hopkins SA, Sreekrishna K:
    Protocol for efficient secretion of HSA developed from Pichia pastoris.
    Pharm Eng 1992, 12: 48–51. [Cited by]
    Return to citation reference [1] [2] [3]

  5. •• Laroche Y, Storme V, De Meutter J, Messens J, Lauwereys M:
    High-level secretion and very efficient isotopic labelling of tick anticoagulant peptide (TAP) expressed in the methylotrophic yeast. Pichia pastoris.
    Biotechnology 1994, 12: 1119–1124. [Cited by]
    A rapid microtitre plate expression screen is employed to obtain several efficient TAP-secreting transformants from a pool for further optimization. The authors achieved very high level secretion using a hybrid Pho1 signal peptide containing a Kex2 cleavage site. Also reports an efficient method for 15N and 13C isotopic labelling of a protein that can then be used to determine the solution structure by NMR.
    Return to citation reference [1] [2] [3] [4]

  6. Scorer CA, Buckholz RG, Clare JJ, Romanos MA:
    The intracellular production and secretion of HIV-1 envelope protein in the methylotrophic yeast Pichia pastoris.
    Gene 1993, 136: 111–119. [MEDLINE] [Cited by]
    Return to citation reference [1] [2] [3] [4]

  7. Cregg JM, Madden KN: Development of transformation systems and construction of methanol-utilisation-defective mutants of Pichia pastoris by gene disruption. In Biological Research on Industrial Yeast. Edited by Stewart GG, Russell I, Klein RD, Hiebsch RR. Boca Raton: CRC Press, 1987, 2: 1–18.
    Return to citation reference [1]

  8. Cregg JM, Tschopp JF, Stillman C, Siegel R, Akong M, Craig WS, Buckholz RG, Madden KR, Kellaris PA, Davies GR et al:
    High-level expression and efficient assembly of hepatitis B surface antigen in the methylotrophic yeast Pichia pastoris.
    Biotechnology 1987, 5: 479–485. [Cited by]
    Return to citation reference [1] [2]

  9. Sreekrishna K, Nelles L, Potenz R, Cruze J, Mazzaferro P, Fish W, Motohiro F, Holden K, Phelps D, Wood P, Parker K:
    High-level expression, purification, and characterisation of recombinant human tumour necrosis factor synthesised in the methylotrophic yeast Pichia pastoris.
    Biochemistry 28: 4117–4125. [MEDLINE] [Cited by]
    Return to citation reference [1] [2]

  10. Sreekrishna K, Potenz RB, Cruze JA, McCombie WR, Parker KA, Nelles L, Mazzaferro PK, Holden KA, Harrison RG, Wood PJ et al:
    High level expression of heterologous proteins in methylotrophic yeast Pichia pastoris.
    J Basic Microbiol 1988, 28: 265–278. [MEDLINE] [Cited by]
    Return to citation reference [1] [2]

  11. Romanos MA, Clare JJ, Beesley KM, Rayment FB, Ballantine SP, Makoff AJ, Dougan G, Fairweather NF, Charles IG:
    Recombinant Bordetella pertussis pertactin (P69) from the yeastPichia pastoris: high-level production and immunological properties.
    Vaccine 1991, 9: 901–906. [MEDLINE] [Cited by]
    Return to citation reference [1] [2] [3] [4] [5]

  12. •• Scorer CA, Clare JJ, McCombie WR, Romanos MA, Sreekrishna K:
    Rapid selection using G418 of high copy number transformants of Pichia pastoris for high-level foreign gene expression.
    Biotechnology 1994, 12: 181–184. [Cited by]
    Polylinker vectors for intracellular expression (pPIC3 and pPIC3K) and alpha-factor secretion vectors (pPIC9 and pPIC9K) are constructed with and without the kanamycin-resistance marker for G418-selection. The authors describe a rapid method for selecting multicopy transformants using G418-selection and electroporation. They analyze HIV Env mRNA levels from strains with 1–12 copies of the expression vector and show a progressive increase with copy number.
    Return to citation reference [1] [2] [3] [4] [5] [6] [7] [8]

  13. Hovland P, Flick J, Johnston M, Sclafani RA:
    Galactose as a gratuitous inducer of GAL gene expression in yeasts growing on glucose.
    Gene 1989, 83: 57–64. [MEDLINE] [Cited by]
    Return to citation reference [1]

  14. Vedvick T, Buckholz RG, Engel M, Urcan M, Kinney J, Provow S, Siegel RS, Thill GP:
    High-level secretion of biologically active aprotinin from the yeast Pichia pastoris.
    J Ind Microbiol 1991, 7: 197–201. [MEDLINE] [Cited by]
    Return to citation reference [1]

  15. Clare JJ, Romanos MA, Rayment FB, Rowedder JE, Smith MA, Payne MM, Sreekrishna K, Henwood CA:
    Production of mouse epidermal growth factor in yeast: high-level secretion usingPichia pastoris strains containing multiple gene copies.
    Gene 1991, 105: 205–212. [MEDLINE] [Cited by]
    Return to citation reference [1] [2] [3] [4]

  16. Thill GP, Davis GR, Stillman C, Holtz G, Brierley R, Engel M, Buckholtz R, Kinney J, Provow S, Vedvick T, Seigel RS: Positive and negative effects of multicopy integrated expression vectors on protein expression in Pichia pastoris. In Proceedings of the 6th International Symposium on Genetics of Microorganisms. Edited by Heslot H, Davies J, Florent J, Bobichon L, Durand G, Penasse L. Paris: Societe Francaise de Microbiologie, 1990, 2: 477–490.
    Return to citation reference [1] [2] [3]

  17. • Paifer E, Margolles E, Cremata J, Montesino R, Herrera L, Delgado J-M:
    Efficient expression and secretion of recombinant alpha amylase in Pichia pastoris using two different signal sequences.
    Yeast 1994, 10: 1415–1459. [MEDLINE] [Cited by]
    Improved secretion efficiency and product yield (from 0.9 gl-1 to 2.5 gl-1) is achieved by replacing the bacterial signal peptide sequence with that from S. cerevisiae SUC2.
    Return to citation reference [1]

  18. • Ridder R, Schmitz R, Legay F, Gram H:
    Generation of rabbit monoclonal antibody fragments from a combinatorial phage display library and their production in the yeast Pichia pastoris.
    Biotechnology 1995, 13: 255–260. [Cited by]
    The alpha-factor leader vector, pPIC9, is used to direct expression, allowing secretion of functional antibody single-chain Fv fragment at >100 mgl-1 in P. pastoris.
    Return to citation reference [1]

  19. White CE, Kempi NM, Komives EA:
    Expression of highly disulfide bonded proteins in Pichia pastoris.
    Structure 1994, 2: 1003–1005. [Full text] [MEDLINE] [Cited by]
    Return to citation reference [1] [2]

  20. Van Nostrand WE, Schmaier AH, Neiditch BR, Siegel RS, Raschke WC, Sisodia SS, Wagner SL:
    Expression, purification and characterisation of the Kunit-type proteinase inhibitor domain of the amyloid-protein precursor-like protein-2.
    Biochim Biophys Acta 1994, 1209: 165–170. [MEDLINE] [Cited by]
    Return to citation reference [1]

  21. Tschopp JF, Sverlow G, Kosson R, Craig W, Grinna L:
    High-level secretion of glycosylated invertase in the methylotrophic yeast Pichia pastoris.
    Biotechnology 1987, 5: 1305–1308. [Cited by]
    Return to citation reference [1]

  22. Grinna LS, Tschopp JF:
    Size distribution and general structural features of N-linked oligosaccharides from the methylotrophic yeast, Pichia pastoris.
    Yeast 1989, 5: 107–115. [MEDLINE] [Cited by]
    Return to citation reference [1]

  23. Trimble RB, Atkinson PH, Tschopp JF, Townsend RR, Maley F:
    Structure of oligosaccharides on Saccharomyce SUC2invertase secreted by the methylotrophic yeast Pichia pastoris.
    J Biol Chem 1991, 266: 22807–22817. [MEDLINE] [Cited by]
    Return to citation reference [1]

  24. • Rodriguez M, Rubiera R, Penichet M, Montesinos R, Cremata J, De La Fuente J:
    High level expression of the B. microplus Bm86 antigen in the yeast Pichia pastoris forming highly immunogenic particles for cattle.
    J Biotechnol 1994, 33: 135–146. [MEDLINE] [Cited by]
    The Bm86 membrane glycoprotein from gut epithelial cells of the cattle tick is expressed in Pichia using the invertase signal peptide. The product is purified as 17–45 nm particles that are able to partially protect cattle in immunization experiments.
    Return to citation reference [1]



Patents
• of special interest.
•• of outstanding interest.
  1. Sreekrishna K, Prevatt WD, Thill GP, Davis GR, Koutz P, Barr KA, Hopkins SA:
    Production of Bacillus entomotoxins in methylotrophic yeast. .
    1993, EP0586892A1.
    Return to citation reference [1]

  2. Siegel RS, Buckholz RG, Thill GP, Wondrack LM:
    Production of epidermal growth factor in methylotrophic yeast cells. .
    1990, WO90/10697.
    Return to citation reference [1] [2] [3]

  3. •• Brierley RA, Davis GR, Holtz GC:
    Production of insulin-like growth factor-1 in methylotrophic yeast cells. .
    1994, US5,324,639.
    These authors use in vitro ligation to generate a multicopy alpha-factor expression vector for IGF-1 secretion. Secretion levels increase progressively with copy number, up to six copies. Because of proteolysis, no product is detected, unless the induction medium is buffered to pH 3. A further 50% increase is observed when using a pep4 protease-deficient strain.
    Return to citation reference [1] [2] [3]

  4. Miura M, Ishida Y, Oi H, Murakami K, Nakagawa Y, Kawabe H:
    Mutant AOX2 promoter, microorganism carrying same, method of preparation thereof and production of heterologous protein using such microorganism.
    1992, EP92105201.5.
    Return to citation reference [1]



Author Contacts
M Romanos, Glaxo Wellcome, Langley Court, Beckenham, Kent BR3 3BS, UK.
Return to author list


Copyright

Copyright © 1995 Current-Opinion.com
Help   Feedback      Search   Map
Black Line
© 1998-1999 BioMedNet. All rights reserved.
email: info@biomednet.com.