Abstract
Yeast surface display (YSD) has been shown to represent a powerful tool in the field of antibody discovery and engineering as well as for selection of high producer clones. However, YSD is predominantly applied in Saccharomyces cerevisiae, whereas expression of heterologous proteins is generally favored in the non-canonical yeast Pichia pastoris (Komagataella phaffii). Establishment of surface display in P. pastoris would therefore enable antibody selection and expression in a single host. Here we describe the generation of a Pichia surface display (PSD) system based on antibody expression from episomal plasmids. By screening a diverse set of expression vectors using Design of Experiments (DoE), the effect of different genetic elements on the surface expression of antibody fragments was analyzed. Among the tested genetic elements, we found that the combination of P. pastoris formaldehyde dehydrogenase (FLD1) promoter, S. cerevisiae invertase 2 signal peptide (SUC2), and α-agglutinin cell wall protein (SAG1) including an autonomously replicating sequence of Kluyveromyces lactis (panARS) were contributing most strongly to higher display levels of three tested antibody fragments. Employing this combination resulted in the display of antibody fragments for up to 25% of cells. Despite significantly reduced expression levels in PSD compared to well-established YSD in S. cerevisiae, similar fractions of antigen binding single-chain variable fragments (scFvs) were observed (80% vs. 84%). In addition, plasmid stability assays and flow cytometric analysis demonstrated the efficient plasmid clearance of cells and associated loss of antibody fragment display after removal of selective pressure.
Key points
• First report of antibody display in P. pastoris using episomal plasmids.
• Identification of genetic elements conferring highest levels of antibody display.
• Comparable antigen binding capacity of displayed scFvs for PSD compared to YSD.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
Data availability
The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.
References
Andreu C, del Olmo ML (2018) Yeast arming systems: pros and cons of different protein anchors and other elements required for display. Appl Microbiol Biotechnol 102:2543–2561. https://doi.org/10.1007/s00253-018-8827-6
Aw R, Barton GR, Leak DJ (2017) Insights into the prevalence and underlying causes of clonal variation through transcriptomic analysis in Pichia pastoris. Appl Microbiol Biotechnol 101:5045–5058. https://doi.org/10.1007/s00253-017-8317-2
Aw R, McKay PF, Shattock RJ, Polizzi KM (2018) A systematic analysis of the expression of the anti-HIV VRC01 antibody in Pichia pastoris through signal peptide optimization. Protein Expr Purif 149:43–50. https://doi.org/10.1016/j.pep.2018.03.013
Barrero JJ, Casler JC, Valero F, Ferrer P, Glick BS (2018) An improved secretion signal enhances the secretion of model proteins from Pichia pastoris. Microb Cell Fact 17:1–13. https://doi.org/10.1186/s12934-018-1009-5
Boder ET, Wittrup KD (1997) Yeast surface display for screening combinatorial polypeptide libraries. Nat Biotechnol 15:553–557. https://doi.org/10.1038/nbt0697-553
Brown SR, Staff M, Lee R, Love J, Parker DA, Aves SJ, Howard TP (2018) Design of experiments methodology to build a multifactorial statistical model describing the metabolic interactions of alcohol dehydrogenase isozymes in the ethanol biosynthetic pathway of the yeast Saccharomyces cerevisiae. ACS Synth Biol 7:1676–1684. https://doi.org/10.1021/acssynbio.8b00112
Camattari A, Goh A, Yip LY, Tan AHM, Ng SW, Tran A, Liu G, Liachko I, Dunham MJ, Rancati G (2016) Characterization of a panARS-based episomal vector in the methylotrophic yeast Pichia pastoris for recombinant protein production and synthetic biology applications. Microb Cell Fact 15:1–11. https://doi.org/10.1186/s12934-016-0540-5
Chao G, Lau WL, Hackel BJ, Sazinsky SL, Lippow SM, Wittrup KD (2006) Isolating and engineering human antibodies using yeast surface display. Nat Protoc 1:755–768. https://doi.org/10.1038/nprot.2006.94
Cherf GM, Cochran JR (2015) Applications of yeast surface display for protein engineering. Methods Mol Biol 1319:155–175. https://doi.org/10.1007/978-1-4939-2748-7_8
Cregg JM, Barringer KJ, Hessler AY, Madden K (1985) Pichia pastoris as a host system for transformations. Mol Cell Biol 5:3376–3385. https://doi.org/10.1128/mcb.5.12.3376-3385.1985
Cregg JM, Cereghino JL, Shi J, Higgins DR (2000) Recombinant protein expression in Pichia pastoris. Mol Biotechnol 16:23–52. https://doi.org/10.1385/MB:16:1:23
Engler C, Kandzia R, Marillonnet S (2008) A one pot, one step, precision cloning method with high throughput capability. PLoS ONE 3:e3647. https://doi.org/10.1371/journal.pone.0003647
Gai SA, Wittrup KD (2007) Yeast surface display for protein engineering and characterization. Curr Opin Struct Biol 17:467–473. https://doi.org/10.1016/j.sbi.2007.08.012
Gu Y, Gao J, Cao M, Dong C, Lian J, Huang L, Cai J, Xu Z (2019) Construction of a series of episomal plasmids and their application in the development of an efficient CRISPR/Cas9 system in Pichia pastoris. World J Microbiol Biotechnol 35:1–10. https://doi.org/10.1007/s11274-019-2654-5
Hegemann JH, Klein S, Heck S, Güldener U, Niedenthal RK, Fleig U (1999) A fast method to diagnose chromosome and plasmid loss in Saccharomyces cerevisiae strains. Yeast 15:1009–1019. https://doi.org/10.1002/(SICI)1097-0061(199907)15:10B%3c1009::AID-YEA396%3e3.0.CO;2-I
Hoggard T, Liachko I, Burt C, Meikle T, Jiang K, Craciun G, Dunham MJ, Fox CA (2016) High throughput analyses of budding yeast ARSs reveal new DNA elements capable of conferring centromere-independent plasmid propagation. G3 (Bethesda) 6:993–1012. https://doi.org/10.1534/g3.116.027904
Inokuma K, Kurono H, den Haan R, van Zyl WH, Hasunuma T, Kondo A (2020) Novel strategy for anchorage position control of GPI-attached proteins in the yeast cell wall using different GPI-anchoring domains. Metab Eng 57:110–117. https://doi.org/10.1016/j.ymben.2019.11.004
Jin Z, Zhang K, Zhang L, Zheng SP, Han SY, Lin Y (2014) Quantification analysis of yeast-displayed lipase. Anal Biochem 450:46–48. https://doi.org/10.1016/j.ab.2013.12.035
Karbalaei M, Rezaee SA, Farsiani H (2020) Pichia pastoris: a highly successful expression system for optimal synthesis of heterologous proteins. J Cell Physiol 235:5867–5881. https://doi.org/10.1002/jcp.29583
Karim AS, Curran KA, Alper HS (2013) Characterization of plasmid burden and copy number in Saccharomyces cerevisiae for optimization of metabolic engineering applications. FEMS Yeast Res 13:107–116. https://doi.org/10.1111/1567-1364.12016
Kato M, Fuchimoto J, Tanino T, Kondo A, Fukuda H, Ueda M (2007) Preparation of a whole-cell biocatalyst of mutated Candida antarctica lipase B (mCALB) by a yeast molecular display system and its practical properties. Appl Microbiol Biotechnol 75:549–555. https://doi.org/10.1007/s00253-006-0835-2
Khasa Y, Conrad S, Sengul M, Plautz S, Meagher MM, Inan M (2011) Isolation of Pichia pastoris PIR genes and their utilization for cell surface display and recombinant protein secretion. Yeast 28:213–226. https://doi.org/10.1002/yea.1832
Kim H, Yoo SJ, Kang HA (2015) Yeast synthetic biology for the production of recombinant therapeutic proteins. FEMS Yeast Res 15:1–16. https://doi.org/10.1111/1567-1364.12195
Klis FM, Mol P, Hellingwerf K, Brul S (2002) Dynamics of cell wall structure in Saccharomyces cerevisiae. FEMS Microbiol Rev 26:239–256. https://doi.org/10.1111/j.1574-6976.2002.tb00613.x
Kuroda K, Matsui K, Higuchi S, Kotaka A, Sahara H, Hata Y, Ueda M (2009) Enhancement of display efficiency in yeast display system by vector engineering and gene disruption. Appl Microbiol Biotechnol 82:713-719. https://doi.org/10.1007/s00253-008-1808-4
Lee CC, Williams TG, Wong DW, Robertson GH (2005) An episomal expression vector for screening mutant gene libraries in Pichia pastoris. Plasmid 54:80–85. https://doi.org/10.1016/j.plasmid.2004.12.001
Li H, Sethuraman N, Stadheim TA, Zha D, Prinz B, Ballew N, Bobrowicz P, Choi BK, Cook WJ, Cukan M, Houston-Cummings NR, Davidson R, Gong B, Hamilton SR, Hoopes JP, Jiang Y, Kim N, Mansfield R, Nett JH, Rios S, Strawbridge R, Wildt S, Gerngross TU (2006) Optimization of humanized IgGs in glycoengineered Pichia pastoris. Nat Biotechnol 24:210–215. https://doi.org/10.1038/nbt1178
Liachko I, Youngblood RA, Keich U, Dunham MJ (2013) High-resolution mapping, characterization, and optimization of autonomously replicating sequences in yeast. Genome Res 23:698–704. https://doi.org/10.1101/gr.144659.112
Liachko I, Dunham MJ (2014) An autonomously replicating sequence for use in a wide range of budding yeasts. FEMS Yeast Res 14:364–367. https://doi.org/10.1111/1567-1364.12123
Liachko I, Youngblood RA, Tsui K, Bubb KL, Queitsch C, Raghuraman MK, Nislow C, Brewer BJ, Dunham MJ (2014) GC-rich DNA elements enable replication origin activity in the methylotrophic yeast Pichia pastoris. PLoS Genet 10:e1004169. https://doi.org/10.1371/journal.pgen.1004169
Lin S, Houston-Cummings NR, Prinz B, Moore R, Bobrowicz B, Davidson RC, Wildt S, Stadheim TA, Zha D (2012) A novel fragment of antigen binding (Fab) surface display platform using glycoengineered Pichia pastoris. Journal I Methods 375:159–165. https://doi.org/10.1016/j.jim.2011.10.003
Liu L, Gomathinayagam S, Hamuro L, Prueksaritanont T, Wang W, Stadheim TA, Hamilton SR (2013) The impact of glycosylation on the pharmacokinetics of a TNFR2: Fc fusion protein expressed in glycoengineered Pichia pastoris. Pharm Res 30:803–812. https://doi.org/10.1007/s11095-012-0921-3
Love KR, Panagiotou V, Jiang B, Stadheim TA, Love JC (2010) Integrated single-cell analysis shows Pichia pastoris secretes protein stochastically. Biotechnol Bioeng 106:319–325. https://doi.org/10.1002/bit.22688
McCafferty J, Griffiths AD, Winter G, Chiswell DJ (1990) Phage antibodies: filamentous phage displaying antibody variable domains. Nature 348:552–554. https://doi.org/10.1038/348552a0
Mead DJ, Gardner DC, Oliver SG (1986) The yeast 2 μ plasmid: strategies for the survival of a selfish DNA. Mol Gen Genet 205:417–421. https://doi.org/10.1007/BF00338076
Mergler M, Wolf K, Zimmermann M (2004) Development of a bisphenol A-adsorbing yeast by surface display of the Kluyveromyces yellow enzyme on Pichia pastoris. Appl Microbiol Biotechnol 63:418–421. https://doi.org/10.1007/s00253-003-1361-0
Mogelsvang S, Gomez-Ospina N, Soderholm J, Glick BS, Staehelin LA (2003) Tomographic evidence for continuous turnover of Golgi cisternae in Pichia pastoris. Mol Biol Cell 14:2277–2291. https://doi.org/10.1091/mbc.e02-10-0697
Montgomery DC (2013) Design and analysis of experiments. John Wiley & Sons Inc, Hoboken, NJ
Moura MVH, da Silva GP, Machado ACDO, Torres FAG, Freire DMG, Almeida RV (2015) Displaying lipase B from Candida antarctica in Pichia pastoris using the yeast surface display approach: prospection of a new anchor and characterization of the whole cell biocatalyst. PLoS ONE 10:e0141454. https://doi.org/10.1371/journal.pone.0141454
Näätsaari L, Mistlberger B, Ruth C, Hajek T, Hartner FS, Glieder A (2012) Deletion of the Pichia pastoris KU70 homologue facilitates platform strain generation for gene expression and synthetic biology. PLoS ONE 7:e39720. https://doi.org/10.1371/journal.pone.0039720
Nakamura Y, Nishi T, Noguchi R, Ito Y, Watanabe T, Nishiyama T, Aikawa S, Hasunuma T, Ishii J, Okubo Y, Kondo A (2018) A stable, autonomously replicating plasmid vector containing Pichia pastoris centromeric DNA. Appl Environ Microbiol 84:e02882-e2917. https://doi.org/10.1128/AEM.02882-17
Obst U, Lu TK, Sieber V (2017) A modular toolkit for generating Pichia pastoris secretion libraries. ACS Synth Biol 6:1016–1025. https://doi.org/10.1021/acssynbio.6b00337
Peña DA, Gasser B, Zanghellini J, Steiger MG, Mattanovich D (2018) Metabolic engineering of Pichia pastoris. Metab Eng 50:2–15. https://doi.org/10.1016/j.ymben.2018.04.017
Puxbaum V, Mattanovich D, Gasser B (2015) Quo vadis? The challenges of recombinant protein folding and secretion in Pichia pastoris. Appl Microbiol Biotechnol 99:2925–2938. https://doi.org/10.1007/s00253-015-6470-z
Rakestraw JA, Aird D, Aha PM, Baynes BM, Lipovšek D (2011) Secretion-and-capture cell-surface display for selection of target-binding proteins. Protein Eng Des Sel 24:525–530. https://doi.org/10.1093/protein/gzr008
Rhiel L, Krah S, Günther R, Becker S, Kolmar H, Hock B (2014) REAL-Select: full-length antibody display and library screening by surface capture on yeast cells. PLoS ONE 9:e114887. https://doi.org/10.1371/journal.pone.0114887
Rossanese OW, Soderholm J, Bevis BJ, Sears IB, O’Connor J, Williamson EK, Glick BS (1999) Golgi structure correlates with transitional endoplasmic reticulum organization in Pichia pastoris and Saccharomyces cerevisiae. J Cell Biol 145:69–81. https://doi.org/10.1083/jcb.145.1.69
Ryckaert S, Martens V, De Vusser K, Contreras R (2005) Development of a S. cerevisiae whole cell biocatalyst for in vitro sialylation of oligosaccharides. J Biotechnol 119:379–388. https://doi.org/10.1016/j.jbiotec.2005.04.010
Ryckaert S, Pardon E, Steyaert J, Callewaert N (2010) Isolation of antigen-binding camelid heavy chain antibody fragments (nanobodies) from an immune library displayed on the surface of Pichia pastoris. J Biotechnol 145:93–98. https://doi.org/10.1016/j.jbiotec.2009.10.010
Sasagawa T, Matsui M, Kobayashi Y, Otagiri M, Moriya S, Sakamoto Y, Ito Y, Lee CC, Kitamoto K, Arioka M (2011) High-throughput recombinant gene expression systems in Pichia pastoris using newly developed plasmid vectors. Plasmid 65:65–69. https://doi.org/10.1016/j.plasmid.2010.08.004
Schwarzhans JP, Luttermann T, Wibberg D, Winkler A, Hübner W, Huser T, Kalinowski J, Friehs K (2017) A mitochondrial autonomously replicating sequence from Pichia pastoris for uniform high level recombinant protein production. Front Microbiol 8:780. https://doi.org/10.3389/fmicb.2017.00780
Scott JK, Smith GP (1990) Searching for peptide ligands with an epitope library. Science 249:386–390. https://doi.org/10.1126/science.1696028
Shaheen HH, Prinz B, Chen MT, Pavoor T, Lin S, Houston-Cummings NR, Moore R, Stadheim TA, Zha D (2013) A dual-mode surface display system for the maturation and production of monoclonal antibodies in glyco-engineered Pichia pastoris. PLoS ONE 8:e70190. https://doi.org/10.1371/journal.pone.0070190
Shibasaki S, Ueda M, Iizuka T, Hirayama M, Ikeda Y, Kamasawa N, Osumi M, Tanaka A (2001) Quantitative evaluation of the enhanced green fluorescent protein displayed on the cell surface of Saccharomyces cerevisiae by fluorometric and confocal laser scanning microscopic analyses. Appl Microbiol Biotechnol 55:471–475. https://doi.org/10.1007/s002530000539
Ståhl S, Uhlén M (1997) Bacterial surface display: trends and progress. Trends Biotechnol 15:185–192. https://doi.org/10.1016/S0167-7799(97)01034-2
Su GD, Zhang X, Lin Y (2010) Surface display of active lipase in Pichia pastoris using Sed1 as an anchor protein. Biotechnol Lett 32:1131–1136. https://doi.org/10.1007/s10529-010-0270-4
Tanino T, Ohno T, Aoki T, Fukuda H, Kondo A (2007) Development of yeast cells displaying Candida antarctica lipase B and their application to ester synthesis reaction. Appl Microbiol Biotechnol 75:1319–1325. https://doi.org/10.1007/s00253-007-0959-z
Tang H, Wang S, Wang J, Song M, Xu M, Zhang M, Shen Y, Hou J, Bao, X (2016) N-hypermannose glycosylation disruption enhances recombinant protein production by regulating secretory pathway and cell wall integrity in Saccharomyces cerevisiae. Sci Rep 6:1-13. https://doi.org/10.1038/srep25654
Tuller T, Waldman YY, Kupiec M, Ruppin E (2010) Translation efficiency is determined by both codon bias and folding energy. Proc Natl Acad Sci U S A 107:3645–3650. https://doi.org/10.1073/pnas.0909910107
Vervecken W, Kaigorodov V, Callewaert N, Geysens S, De Vusser K, Contreras R (2004) In vivo synthesis of mammalian-like, hybrid-type N-glycans in Pichia pastoris. Appl Environ Microbiol 70:2639–2646. https://doi.org/10.1128/AEM.70.5.2639-2646.2004
Vogl T, Glieder A (2013) Regulation of Pichia pastoris promoters and its consequences for protein production. N Biotechnol 30:385–404. https://doi.org/10.1016/j.nbt.2012.11.010
Vogl T (2015) Synthetic biology to improve protein expression in Pichia pastoris. Dissertation, Graz University of Technology. https://diglib.tugraz.at/synthetic-biology-to-improve-protein-expression-in-pichia-pastoris-2015. Accessed 29 June 2022
Weis R, Luiten R, Skranc W, Schwab H, Wubbolts M, Glieder A (2004) Reliable high-throughput screening with Pichia pastoris by limiting yeast cell death phenomena. FEMS Yeast Res 5:179–189. https://doi.org/10.1016/j.femsyr.2004.06.016
Weninger A, Hatzl AM, Schmid C, Vogl T, Glieder A (2016) Combinatorial optimization of CRISPR/Cas9 expression enables precision genome engineering in the methylotrophic yeast Pichia pastoris. J Biotechnol 235:139–149. https://doi.org/10.1016/j.jbiotec.2016.03.027
Westermann S, Drubin DG, Barnes G (2007) Structures and functions of yeast kinetochore complexes. Annu Rev Biochem 76:563–591. https://doi.org/10.1146/annurev.biochem.76.052705.160607
Xu Y, Liu K, Han Y, Xing Y, Zhang Y, Yang Q, Zhou M (2021) Codon usage bias regulates gene expression and protein conformation in yeast expression system P. pastoris. Microb Cell Fact 20:1–15. https://doi.org/10.1186/s12934-021-01580-9
Yang S, Lv X, Wang X, Wang J, Wang R, Wang T (2017) Cell-surface displayed expression of trehalose synthase from Pseudomonas putida ATCC 47054 in Pichia pastoris using Pir1p as an anchor protein. Front Microbiol 8:2583. https://doi.org/10.3389/fmicb.2017.02583
Yang X, Tang H, Song M, Shen Y, Hou J, Bao X (2019) Development of novel surface display platforms for anchoring heterologous proteins in Saccharomyces cerevisiae. Microb Cell Fact 18:1–10. https://doi.org/10.1186/s12934-019-1133-xFigures
Zavec D, Gasser B, Mattanovich D (2020) Characterization of methanol utilization negative Pichia pastoris for secreted protein production: New cultivation strategies for current and future applications. Biotechnol Bioeng 117:1394–1405. https://doi.org/10.1002/bit.27303
Acknowledgements
We would like to thank Jan Drewes (Miltenyi Biotec) for establishing various laboratory automation protocols and providing useful input during troubleshooting. In addition, we are grateful to Marius Terfrüchte, Alexandra Schmitz, and Sven Karstaedt (Miltenyi Biotec) for the transfer of P. pastoris cells and for supporting the work in this study.
Author information
Authors and Affiliations
Contributions
Conceived and designed the experiments: DG, FT and JCD. Performed the experiments: DG. Analyzed the data: DG, FT, JCD, MD, and VN. Wrote the paper: DG and MD. Reviewed and edited the manuscript: VN and MW. All authors read and approved the manuscript.
Corresponding author
Ethics declarations
Ethics approval
This article does not contain any studies with human participants or animals performed by any of the authors.
Competing interests
DG, FT, JCD, MD, VN, and MW are employees of Miltenyi Biotec B.V. & Co. KG. DG has relevant IP to the findings disclosed. No relevant financial or non-financial interests exist for the remaining authors.
Additional information
Publisher's note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary Information
Below is the link to the electronic supplementary material.
Rights and permissions
Springer Nature or its licensor holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.
About this article
Cite this article
Gätjen, D., Tomszak, F., Dettmann, JC. et al. Design of a novel switchable antibody display system in Pichia pastoris. Appl Microbiol Biotechnol 106, 6209–6224 (2022). https://doi.org/10.1007/s00253-022-12108-5
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00253-022-12108-5