Yeast surface display of antibodies via the heterodimeric interaction of two coiled-coil adapters

Journal of Immunological Methods 354 (2010) 11–19

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Journal of Immunological Methods j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j i m

Research paper

Yeast surface display of antibodies via the heterodimeric interaction of two coiled-coil adapters Kevin Caili Wang ⁎, Chirag A. Patel, Jian Wang, Jinqing Wang, Xinwei Wang, Peter Peizhi Luo, Pingyu Zhong ⁎ Abmaxis Inc., USA 1 Department of Biologics Research, Merck Research Laboratories, Merck & Co., Inc. 770 Sumneytown Pike, West Point, PA 19486, USA

a r t i c l e

i n f o

Article history: Received 28 August 2009 Received in revised form 12 January 2010 Accepted 14 January 2010 Available online 25 January 2010 Keywords: Yeast display Adapter-directed display Coiled-coil adapters Heterodimeric interaction scFv antibodies Outer wall protein

a b s t r a c t A novel adapter-directed yeast display system with modular features was developed. This display system consists of two modules, a display vector and a helper vector, and is capable of displaying proteins of interest on the surface of Saccharomyces cerevisiae through the interaction of two small adapters that are expressed from the display and helper vectors. In this report, an anti-VEGF scFv antibody gene was cloned into the display vector and introduced alone into yeast S. cerevisiae cells. This led to the expression and secretion of a scFv antibody that was fused in-frame with the coiled-coil adapter GR1. For display purposes, a helper vector was constructed to express the second coiled-coil adapter GR2 that was fused with the outer wall protein Cwp2, and this was genetically integrated into the yeast genome. Co-expression of the scFv–GR1 and GR2–Cwp2 fusions in the yeast cells resulted in the functional display of anti-VEGF scFv antibodies on the yeast cell surfaces through pairwise interaction between the GR1 and GR2 adapters. Visualization of the co-localization of GR1 and GR2 on the cell surfaces confirmed the adapter-directed display mechanism. When the adapter-directed phage and yeast display modules are combined, it is possible to expand the adapter-directed display to a novel cross-species display that can shuttle between phage and yeast systems. © 2010 Elsevier B.V. All rights reserved.

1. Introduction The display of heterologous proteins on the surface of genetic packages is a powerful method for molecular evolution in the laboratory. The ability to construct libraries of enormous molecular diversity and to select for molecules with desired properties has made this technology suitable for a wide range of applications, from protein engineering to the discovery of new

Abbreviations: scFv, single chain Fv fragment; VEGF, vascular endothelial growth factor; PBS, phosphate-buffered saline; FACS, fluorescence-activated cell sorting; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis. ⁎ Corresponding authors. Department of Biologics Research, WP26-413A, Merck Research Laboratories, Merck & Co., Inc. 770 Sumneytown Pike, West Point, PA 19486, USA. E-mail addresses: [email protected] (K.C. Wang), [email protected] (P. Zhong). 1 A wholly-owned subsidiary of Merck & Co., Inc. 0022-1759/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.jim.2010.01.006

biological agents. Among the various display systems that have been developed thus far, yeast surface display has some unique advantages: a eukaryotic expression apparatus that is suitable for eukaryotic protein folding and post-translational modification, the ability to express and display large and complex proteins, and in particular, the quantitative and visualizable selection of discrete populations through fluorescence-activated cell sorting (FACS). With these benefits, yeast display has become increasingly attractive, and the number of publications reporting the use of yeast display for a variety of applications is growing exponentially (Gai and Wittrup, 2007). The display of a heterologous protein on the cell surface of the eukaryotic host S. cerevisiae has been achieved by fusing the protein to an outer cell wall protein. Thus far, more than a dozen yeast cell wall proteins have proven capable of displaying peptides and proteins through fusion. These include a-agglutinin (Agal and Aga2), Cwp1p, Cwp2p, Gas1p, Yap3p, Flo1p, Crh2p,

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Pir1, Pir2, Pir4, and Icwp in S. cerevisiae (Schreuder et al., 1993; Shibasaki et al., 2009; Pepper et al., 2008; Kondo and Ueda, 2004; Breinig and Schmitt, 2002; Abe et al., 2003), HpSed1p, HpGas1p, HpTip1p, and HpCwp1p in Hansenula polymorpha (Kim et al., 2002), and Hwp1p, Als3p, and Rbt5p in Candida albicans (Mao et al., 2003). Of all the display systems based on cell wall protein fusions, the system using Aga2p fusion (Boder and Wittrup, 1997) has been applied successfully to display and engineer various proteins including human growth factors, cytokines, cell surface receptors, the human MHC complex, and antibodies. Moreover, such display platforms are currently being used to discover novel antibodies from large non-immune human naïve antibody libraries (Feldhaus et al., 2003; WeaverFeldhaus et al., 2004). Through direct fusion with an outer wall protein that has a GPI attachment signal, the protein of interest is expressed as part of the cell wall protein. An additional molecular cloning step is required, in which the gene of interest is transferred to an expression vector, before soluble protein can be obtained for downstream biochemical characterization (Feldhaus et al., 2003). In contrast to the system described above, our newlydeveloped adapter-directed yeast display is designed with two modules: a display vector and a helper vector. In the display vector, the sequence that codes for a cell wall protein is avoided. Instead, the protein of interest (here, an scFv antibody) is fused to a small and interactive coiled-coil adapter GR1. This display vector was used directly for the expression and secretion of soluble scFv protein without the need for further molecular cloning. For display purposes, a new S. cerevisiae strain was engineered in which a helper vector is integrated to express the outer wall protein Cwp2 fused with a second coiled-coil adapter GR2, which is able to form a pairwise heterodimer with the first adapter GR1. The scFv antibody was displayed on the cell surface via heterodimeric interaction between the GR1 and GR2 adapters. In combination with the adapter-directed phage modules that were constructed previously, it becomes possible to expand the adapter-directed display into an entirely new display platform, i.e., a cross-species display system that can shuttle between phage and yeast. 2. Materials and methods 2.1. Yeast strains and culture Saccharomyces cerevisiae strain YPH499 [MATa ura3-52 lys2801 (amber) ade2-101(ocher) trp1-63 his3-200 leu2-1] (Stratagene, La Jolla, CA) was grown in YPD medium (10 g/L yeast extract, 20 g/L peptone, 20 g/L dextrose) at 30 °C with continuous shaking at 250 rpm. S. cerevisiae strain KQP600 was grown in SD-CAA minus Ura medium [20 g/L dextrose, 5 g/L casamino acids (-Ura, -Trp, -Ade), 1.7 g/L yeast nitrogen base, 5.3 g/L ammonium sulfate, 40 mg/L adenine, 100 mg/L tryptophan, 10.19 g/L Na2HPO4·7H2O, 8.56 g/L NaH2PO4·H2O]. Yeast cells harboring both display and helper vectors were grown in SDCAA-minus TRP and URA medium.

surface GPI anchoring protein Cwp2. This vector was derived from the commercial vector pUC19 by inserting a synthetic DNA fragment between the AatII and PciI restriction sites. This synthetic DNA comprised sequences for three expression cassettes: (1) a yeast URA3 auxotrophic marker for selection; (2) zeocin, for selection; (3) the outer wall protein Cwp2 (amino acids 25–92), fused at its N-terminus with the GR2 adapter (TSRLEGLQSENHRLRMKITELDKDLEEVTMQLQDVGGC), a Myctag, an SG linker (S2G3SG3SGSG4SG3SGS3) and the S/T-rich region from Flo1 (amino acids 1088–1432). The Cwp2 fusion was controlled by the galactose-inducible GAL1 promoter and the secretory signal sequence of Flo1. The final vector sequence was confirmed by sequencing. We constructed the display vector pMAT12 for the expression and secretion of soluble scFv in yeast cells. As shown in Fig. 2B, pMAT12 was created by inserting a synthetic DNA fragment between the Aatll and Pcil restriction sites of the commercial vector pUC19. This synthetic DNA fragment was comprised of three parts. The first part was an expression cassette for the scFv-adapter GR1 (EEKSRLLEKENRELEKIIAEKEERVSELRHQLQSVGGC) fusion, which was driven by the yeast GAL1 promoter. The sequence of an anti-VEGF scFv antibody was constructed downstream of a yeast signal sequence (for the yeast endo-B-1,3-glucanase protein Bgl2p). The HA and His6 tag sequences were included upstream of the GR1 sequence to allow for protein detection and Ni-NTA purification. The second part was the yeast CEN/ARS element, to allow for replication, and the third part was an expression cassette that coded for the yeast TRP1 auxotrophic marker. The synthetic DNA sequence was confirmed by sequencing. 2.3. Construction of the S. cerevisiae strain KQP600 The vector pMAT8 was linearized using the restriction enzyme AatII and then transformed into S. cerevisiae strain YPH499 using the Frozen-EZ Yeast Transformation II Kit according to the manufacturer's instructions (Zymo Research, Orange, CA). Clones in which pMAT8 was integrated were selected and grown for 3 days on a SD-CAA minus Ura plate (20 g/L dextrose, 5 g/L casamino acids (-Ura, -Trp, -Ade), 1.7 g/L yeast nitrogen base, 5.3 g/L ammonium sulfate, 40 mg/L adenine, 100 mg/L tryptophan, 10.19 g/L Na2HPO4·7H2O, 8.56 g/L NaH2PO4·H2O, and 15 g/L agar). To confirm the surface expression of the adapter 2 (GR2) fusions on the yeast cells in which the pMAT8 vector was integrated into the chromosome, we performed a galactose induction experiment. Yeast cells derived from a single colony were grown in 50 mL of YDP medium at 30 °C overnight (OD600 = 15–20). Then, 3–5 mL of the overnight culture was transferred to 50 mL of SG-CAA-minus Ura medium (20 g/L galactose, 1 g/L dextrose, 5 g/L casamino acids (-Ura, -Trp, -Ade), 1.7 g/L yeast nitrogen base, 5.3 g/L ammonium sulfate, 40 mg/L adenine, 100 mg/L tryptophan, 10.19 g/L Na2HPO4·7H2O, 8.56 g/L NaH2PO4·H2O) and incubated for 20 h at 22 °C. The induced cells were then harvested for fluorescent labeling. 2.4. Surface display of the scFv antibody on yeast

2.2. Construction of the vectors The yeast helper vector pMAT8 shown in Fig. 2A expresses a fusion protein comprising the adapter GR2 with the yeast

The yeast strain KQP600 in which the pMAT8 vector had been chromosomally integrated was used for surface display of the scFv antibody. The display vector pMAT12 was transformed

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into the KQP600 strain using the Frozen-EZ Yeast Transformation II Kit (Zymo Research). Cells from a single colony on the SD minus TRP and URA plate (Teknova) were grown in 50 mL of SD-CAA-minus TRP and URA medium overnight at 30 °C with continuous shaking at 250 rpm. The cells were then transferred to 50 mL of SG-CAA-minus TRP and URA medium with a starting OD600 of 1–3, and incubated for 20 h in the presence of galactose at 18–22 °C with continuous shaking. The cells were harvested, washed with wash buffer (1% BSA–PBS), and incubated with anti-tag antibodies or biotinylated VEGF for fluorescence labeling. 2.5. Fluorescent labeling of S. cerevisiae cells The galactose-induced KQP600 cells were harvested and fluorescently labeled. Approximately 107 yeast cells in 250 µL wash buffer were incubated with 4 µg/mL of an anti-Myc chicken antibody (Invitrogen) at room temperature for 1 h. The washed cells were then probed with the Alexa-647labeled goat anti-chicken secondary antibody (Invitrogen) diluted 1/200 in 500 µL of wash buffer for 1 h at room temperature. After washing, we analyzed the fluorescently labeled GR2–Myc–Cwp2 fusions on the yeast cell surface using flow cytometry (the DB FACSAria system). Wild type YPH499 cells were also labeled using the same procedure to serve as a control. The KQP600-T12 cells were fluorescently labeled with anti-tag antibodies as follows. The galactose-induced cells (∼107 yeast cells in 250 µL wash buffer) were incubated for 1 h at room temperature with 0.8 µg/mL anti-HA rat antibody (Roche) and/or 4 µg/mL of anti-Myc chicken antibody and then probed with the secondary antibodies during a 1-h incubation: Alexa-488-labeled donkey anti-rat antibody (1:200, from Invitrogen) and/or Alexa-594-labeled goat anti-chicken antibody (1:200). After the cells were washed with wash buffer, the fluorescently labeled scFv–DH–GR1 and/or GR2–Myc–Cwp2 fusion proteins on the yeast cell surface were visualized under a Zeiss Axiovert 135 fluorescent microscope and analyzed using flow cytometry. The surface-displayed scFv was detected with biotinylated human VEGF (R&D Systems, Minneapolis, MN) and Streptavidin that was labeled with the Alexa fluor 633 dye (Invitrogen). The cells from the 20-h induction with galactose were incubated for 1 h at room temperature with 10 µg/mL of recombinant human VEGF that had been biotinylated using the Biotinxx Microscale Protein Labeling kit (Invitrogen). They were then incubated with 40 µg/mL Streptavidin-Alexa fluor 633 in 500 µL wash buffer for 1 h at room temperature. The cells were washed twice with ice-cold wash buffer and resuspended in wash buffer. The stained yeast cells were kept on ice until analyzed using FACS. 2.6. FACS and fluorescence microscopy analysis The fluorescence of the cells was measured using a FACSAria instrument (BD Biosciences, San Jose, CA). The gate was set to encompass the singlet events on the FSC vs. SSC plot. Alexa647-labeled GR2–Myc–Cwp2 fusions on the yeast surface were detected using a photo multiplier tube (PMT) with a 660/ 20-nm bandpass filter. The Alexa-594-labeled GR2–Myc–Cwp2 fusions on the yeast surface were detected using a PMT with a

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610/20-nm bandpass filter. Alexa-488-labeled scFv that was displayed on the yeast surface was detected using a PMT with a 530/30-nm bandpass filter. Alexa-633-labeled VEGF, which checks the functionality of the surface-displayed scFv, was detected using a PMT with a 660/20-nm bandpass filter. The data was acquired and analyzed using BD FACSDiva 6.1 software (BD Biosciences, San Jose, CA) and WinMDI2.9 software. Unstained cells were used to define background levels. The fluorescently labeled yeast cells were visualized under a Zeiss Axiovert 135 fluorescent microscope. Photographs were taken using a Plan-Neofluar 100×/1.30 oil objective lens. 2.7. Expression and secretion of the scFv antibody The YPH499 strain was transformed with the expression vector pMAT12 according to the protocol included in the Frozen-EZ Yeast Transformation II Kit (Zymo Research). Yeast cells from a single colony on the SD minus TRP plate were grown in 50 mL of SD-CAA-minus TRP medium overnight at 30 °C with continuous shaking at 250 rpm, and then transferred to 1 L of SD-CAA-minus TRP for 2 days of growth. The cells were then grown in SG-CAA-minus TRP medium with a starting OD600 of ∼10 for 2 days of galactose induction at 20 °C with continuous shaking. After centrifugation at 5000 ×g for 20 min, the culture supernatant was neutralized by adding 10% 1 M Tris–HCl, pH 8, followed by precipitation with 65% ammonium sulfate overnight at 4 °C. The precipitated proteins were pelleted by centrifuging at 17,000 ×g and resuspended in 100 mL PBS. After overnight dialysis against PBS with 3 buffer changes, the concentrated culture supernatant was purified using an anti-HA affinity column. The HA-tagged anti-VEGF scFv–GR1 fusion was also expressed from a bacterial expression vector driven by the bacterial lac promoter in TG1 cells. An overnight culture of TG1 cells (harboring the bacterial expression vector) in 50 mL of 2YT/2% glucose/100 µg/mL carbenicillin was grown in a 37 °C shaker incubator. On the second day, 1 L of 2YT/0.1% glucose/ 100 µg/mL carbenicillin was inoculated with 10 mL of the overnight culture. The culture was grown at 30 °C with shaking for approximately 3–4 h until OD600 ∼1. IPTG was added to the culture to a final concentration of 0.5 mM. After induction, the cells were incubated overnight at 22 °C. Following this, the cells were harvested by centrifugation at 10,000 rpm for 10– 15 min, and the soluble scFv was extracted from the cell periplasm in two steps. First, the TG1 cell pellet was resuspended in 20 mL of pre-chilled PPB buffer (20% sucrose, 2 mM EDTA, and 30 mM Tris–HCl, pH 8) and incubated on ice for 1 h. The supernatant that contained the soluble scFv was collected by centrifugation. Subsequently, the cell pellet was resuspended in 20 mL of pre-chilled 5 mM magnesium sulfate and incubated for 1 h on ice. The two supernatants were combined and purified using an anti-HA affinity column. 2.8. Affinity purification of the scFv antibody Briefly, the scFv sample was loaded into a column with a 1 mL bed volume of anti-HA agarose (Sigma) and washed with 10 mL of PBS to remove unbound proteins. The HA-tagged scFv protein was then eluted with 0.1 M glycine–HCl buffer (pH 2.5). The eluted fractions were neutralized by adding 10% 1 M

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Tris–HCl buffer (pH 8) and dialyzed against PBS. Finally, the samples were analyzed using 4–12% SDS-PAGE. 2.9. Protein concentration determination The protein concentration was determined using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific) according to the manufacturer's instructions. Diluted BSA standards were prepared, ranging from 2000 µg/mL to 25 µg/mL. The standard or scFv samples were mixed with the working reagent (50:1, Reagent A: B) in microplate wells and incubated at 37 °C for 30 min. The absorbance was measured at 562 nm using a plate reader. A standard curve was prepared by plotting the absorbance vs. the concentration of the standards, and the protein concentrations of scFv samples were determined from the standard curve. 2.10. Affinity measurements using surface plasmon resonance technology The binding constants (Kd) of the scFvs that were purified from S. cerevisiae and E. coli were measured using a Biacore 2000 instrument with either the antigen or the scFv antibody immobilized on a CM5 chip. To immobilize the antigen, we diluted the recombinant human VEGF (R&D Systems, 293VE/CF) to 20 µg/mL in 10 mM sodium acetate pH 6.5, and injected it over a CM5 chip, using the Amine Coupling kit (GE, BR-1000-50) according to the manufacturer's instructions. The final amount of VEGF immobilized was ∼300 RU. We prepared 5 scFv samples at different concentrations in HBS-P running buffer using 1/1 serial dilution (GE, BR-1003-68). The samples were injected at a flow rate of 20 µL/min for 2 min, and this was followed by a 10-minute dissociation period at the same flow rate with HBS-P buffer. The surface was regenerated using a 30-second injection of 10 mM Glycine–HCl, pH 1.5 at a flow rate of 50 µL/min. The response from a reference flowcell was subtracted from the sample sensorgram during data analysis. The Kds were determined by fitting the five concentration sensorgrams globally to a 1:1 Langmuir model. To immobilize the antibody, we diluted the purified scFvs to 10 µg/mL in 10 mM sodium acetate, pH 5.0, and injected the dilutions manually over a CM5 chip at 5 µL/min. The final immobilization levels were ∼450 RU for both the S. cerevisiae and E. coli scFvs. Four different concentrations of the recombinant human VEGF (20 nM, 40 nM, 80 nM, and 160 nM in PBS) were injected at a flow rate of 30 µL/min for 5 min, and this was followed by a 10-minute dissociation period using the same flow rate. The surface was regenerated using the same conditions as noted above. 3. Results 3.1. The design of the adapter-directed yeast display system The adaptor-directed yeast system is comprised of two modules: a display vector that expresses and secretes the protein of interest and a yeast helper vector that expresses a recombinant yeast outer wall protein. In the display vector, the target gene is fused in-frame with an adapter sequence (adapter 1) instead of the sequence that codes for a yeast cell wall protein. Upon induction with galactose, the protein of

interest is expressed as a secretory protein fused with a small adapter, as illustrated in the left panel of Fig. 1A. Diverse DNA sequences can be inserted into this vector to construct an expression library. In the yeast helper vector, adapter 2 (which is able to form a pairwise heterodimer with adapter 1) is fused in-frame with a yeast outer wall protein, Cwp2. This helper vector is integrated genetically into the yeast genome. Introducing the display and helper vectors into yeast (on induction by galactose) leads to the expression and transport of both adapter fusions to the endoplasmic reticulum (ER), where the two adapters associate and the resulting heterodimer is stabilized by the formation of a disulfide bond. The heterodimers are processed further through the Golgi apparatus and are subsequently anchored in the outer cell wall, resulting in the display of the target protein on the surface of the yeast cells (as illustrated in the right panel of Fig. 1A). We chose two coiled-coil sequences as the adapters in this study. These were derived from the intracellular C-termini of the gamma-aminobutyric acid (B) receptors, GABAB-R1 and GABAB-R2. The adapter sequences, termed GR1 and GR2, are shown in Fig. 1B. A previous study has proven that these sequences preferentially form parallel coiled-coil heterodimers (Kammerer et al, 1999). To stabilize the heterodimer on the yeast surface further, we added an extension sequence (ValGlyGlyCys) after each coiled-coil sequence to lock the heterodimeric coiled-coil pair together via a disulfide bond. 3.2. Yeast strain with chromosomal integration of yeast helper vector To construct the yeast helper vector pMAT8, we fused the S. cerevisiae outer wall protein Cwp2 at its N-terminus with the adapter GR2 sequence. This yeast helper vector supports the display of the GR1 fusion on the yeast cell surface. In the pMAT8 vector, the GR2–Cwp2 fusion was driven by the yeast galactoseinducible promoter GAL1 and directed by the secretory signal derived from the S. cerevisiae outer wall protein Flo1, as illustrated in Fig. 2A. To increase the accessibility of the cell surfaceanchored GR2–Cwp2 fusions further, an SG linker of 25 amino acids and the S/T-rich spacer of 345 amino acids derived from Flo1 were inserted between the GR2 adapter and Cwp2. The Myc-tag sequence was added after the adapter GR2 sequence for detection of the surface-attached adapter GR2 fusion. A S. cerevisiae strain with chromosomal integration of pMAT8 vector was generated by transformation of the linearized vector into S. cerevisiae strain YPH499, as described in the Materials and methods. Yeast clones in which pMAT8 was integrated were selected and grown on a SD-CAA minus Ura plate for 3 days, resulting in the new S. cerevisiae strain, KQP600. To investigate whether the newly created S. cerevisiae strain KQP600 was able to produce the surface GR2 fusions, cells from a single colony were grown in SG-CAA-minus Ura medium at 22 °C for 20 h to induce the production of GR2–Cwp2 fusions. The cell surface-anchored GR2–Cwp2 fusions were then detected using the anti-Myc-tag chicken antibody and the fluorescence Alexa-647-labeled goat anti-chicken secondary antibody. FACS analysis (Fig. 3) indicated that up to 94% of the individual KQP600 yeast cells in the tested population expressed a detectable level of the adapter GR2 fusions on the

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Fig. 1. (A) A schematic representation of the experimental design of the adapter-directed yeast display system. The system has two components: a display vector and a yeast helper vector. Introduction of the display vector alone into S. cerevisiae leads to the expression and secretion of the protein of interest that is fused with a small adapter GR1 on induction with galactose (left panel). In the yeast helper vector, the adapter GR2, which is able to form a pairwise heterodimer with the adapter GR1, is fused in-frame with the yeast outer cell wall protein Cwp2. Introducing the display and helper vectors into a yeast cell leads to expression and transportation of both adapter fusions into the endoplasmic reticulum (ER), where the association of the two adapters occurs. This is followed by stabilization of the heterodimer by the formation of a disulfide bond. The heterodimers are processed further through the Golgi apparatus and subsequently anchored on the outer cell wall, resulting in the display of the target protein on the surface of the yeast cells (right panel). (B) The amino acid sequences of adapter 1 (GR1) and adapter 2 (GR2), with the VGGC extensions.

Fig. 2. Maps of the yeast helper and display vectors. (A) A schematic representation of the yeast helper vector pMAT8. The helper vector expresses the yeast outer cell wall protein Cwp2 fused in-frame with the adapter GR2 and a myc-tag (for detection). (B) A schematic representation of the yeast display vector pMAT12 that expresses an scFv protein fused in-frame with a HA-tag, His6-tag and the adapter GR1.

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Fig. 3. Flow cytometry of S. cerevisiae KQP600 cells expressing the Myc-tagged adapter GR2 on the cell surface. The cells were induced with galactose for 20 h and probed with the anti-Myc-tag chicken antibody and the fluorescent Alexa647-labeled goat anti-chicken secondary antibody. Of the individual KQP600 yeast cells, 94% expressed a detectable level of adapter GR2 fusions on the cell surface. As a control, galactose-induced YPH499 cells were not stained fluorescently.

cell surfaces that were accessible to the anti-Myc antibody. As a control, the cells of the parental S. cerevisiae strain (YPH499) did not show detectable surface fluorescence. This data therefore suggests that the yeast helper vector that was genetically integrated into S. cerevisiae strain KQP600 provided enough adapter GR2 molecules on the cell surface after induction using galactose. This is essential for display of the GR1 fusion in this new display system. 3.3. Yeast surface display of an scFv antibody via the interaction of two adapters The display vector pMAT12 was constructed to express and secrete an anti-VEGF scFv antibody that was fused with the adapter GR1 sequence. As illustrated in Fig. 2B, the scFv–GR1 fusion was expressed under the control of the galactoseinducible GAL1 promoter and directed to the secretion pathway by the signal sequence that was derived from the S. cerevisiae endo-B-1,3-glucanase protein Bgl2p. HA and His6 tags were included at the C-terminus of scFv for detection and purification purposes. Next, we transformed the pMAT12 vector into the newly created S. cerevisiae strain KQP600 that expresses the GR2– Cwp2 fusions. On simultaneous induction with galactose in the S. cerevisiae cells harboring both the display and helper vectors, we expected the scFv protein to be displayed on the cell surface through the interaction of the two adapters. To test for the surface display of the HA-tagged scFv proteins, we labeled the induced cells with anti-HA rat antibody and an Alexa-488-labeled donkey anti-rat antibody. As shown in Fig. 4, FACS analysis indicated that a majority (up to 81%) of the individual KQP600-T12 cells within the tested population had high, detectable levels of the scFv proteins on the cell surface. In contrast, the KQP600 cells (control) showed no detectable fluorescence on the cell surface. To confirm that the display of scFv was dependent on the GR2 fusion generated by the pMAT8 helper vector, we also transformed the display vector pMAT12 into the parental YPH499 strain. After galactose induction, we stained the cells with anti-HA rat antibody and the Alexa-488-labeled donkey anti-rat antibody. No fluorescence was detectable on the

Fig. 4. Flow cytometry of S. cerevisiae KQP600-T12 cells expressing the HAtagged scFv antibody on the cell surface. After 20 h of galactose induction, both the KQP600 and KQP600-T12 cells (KQP600 cells harboring the pMAT12 display vector) were probed with anti-HA rat antibody and the Alexa-488labeled donkey anti-rat antibody. Up to 81% of individual KQP600-T12 cells had detectable levels of scFv on the cell surface, whereas the control KQP600 cells showed no detectable surface fluorescence.

surface of the YPH499-T12 cells using FACS (data not shown). These data suggested that the scFv was displayed through the interaction of the two adapters. 3.4. Co-localization of adapter 1 and adapter 2 on the surface of yeast To verify that both the adapter GR1 and GR2 fusions were present on the surface further, we fluorescently labeled the KOP600-T12 cells using both anti-Myc and anti-HA antibodies. The fluorescently stained cells were observed under a fluorescence microscope. As illustrated in Fig. 5A, the green fluorescence (which probed for the HA-tagged GR1 fusions, using the anti-HA rat antibody and the Alexa-488-labeled donkey antirat antibody) was localized over the entire wall of the cells. The red fluorescence (which probed for the Myc-tagged GR2 fusions, using the anti-Myc chicken antibody and the Alexa594-labeled goat anti-chicken antibody) was distributed over the cell walls in a nearly identical manner, as confirmed by the yellow color that results from the overlay of the green and red fluorescence. Furthermore, the same doubly-stained cells were also analyzed using flow cytometry to assess the fluorescence staining in relation to the cell populations. The FACS data in Fig. 5B indicated that a majority (72%) of individual cells had both green and red fluorescent staining on their surfaces. Taken together, the results of the fluorescence microscopy and the flow cytometry analysis confirm that the scFv antibody is displayed on the surface of the KQP600-T12 cells through the mechanism that we designed for that purpose, i.e., through the heterodimeric interaction of the two adaptors. 3.5. Antigen-binding activity of the scFv antibody on the surface of the yeast cells To examine the functionality of the surface-displayed scFv antibody, we incubated the galactose-induced KQP600-T12 cells with biotinylated VEGF antigen for 1 h, and then probed the cells with fluorescent Alexa-633-labeled Streptavidin. The cells were subsequently analyzed using flow cytometry. The FACS data that are shown in Fig. 6 indicate that up to 88% of the individual cells within the tested population bound specifically to the VEGF antigen. In contrast, the same cell

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Fig. 6. VEGF antigen binding to scFv on the cell surfaces. KQP600-T12 cells that were induced with galactose were probed with Streptavidin-Alexa 633 in the presence of biotinylated VEGF antigen. FACS data indicated that up to 88% of the individual cells bound specifically to the VEGF antigen. In contrast, the same cell population stained with Streptavidin-Alexa 633 in the absence of the VEGF antigen did not show cell surface fluorescence.

Fig. 5. Surface co-localization of adapters GR1 and GR2 for scFv display. The KQP600-T12 cells were fluorescently labeled doubly using the anti-Myc and anti-HA antibodies to detect the HA-tagged adapter GR1 and Myc-tagged adapter GR2 on the cell surface. (A) The fluorescently stained cells were observed under a fluorescence microscope. The green fluorescence (which probed for HA-tagged GR1) and the red fluorescence (which probed for Myc-tagged GR2) were co-localized over the entire cell wall. The yellow color results from the overlay of the red and green fluorescence; (B) FACS analysis indicated 72% of individual cells had both green and red fluorescence staining on their surfaces.

population, when stained with fluorescent Alexa-633-labeled Streptavidin without the VEGF antigen, did not show any cell surface fluorescence. This result shows that the scFv antibody retains its ability to bind VEGF when displayed on the cell surface. 3.6. Secretion of the scFv antibody from the display vector We reasoned that the display vector could be used to produce soluble protein directly in yeast, as the yeast cell wall sequence is absent in the display vector. To test this, we induced the YPH499 cells harboring the display vector pMAT12 only (i.e., without the helper vector pMAT8) in SG medium containing galactose for two days. The culture supernatant was harvested and purified using an anti-HA affinity column. From

1 L of culture, we obtained 0.3 mg soluble scFv. This yield of purified scFv is in the range that has been reported previously using a centromere (CEN)-based low-copy vector in the S. cerevisiae system (Hackel et al., 2006; Shusta et al., 1998). SDSPAGE analysis showed a major 36 kD band that corresponded to the scFv-HA-GR1 fusion and a weaker 31 kD band. Mass spectrometry analysis suggested that the 31 kD band corresponds to the scFv protein without the adapter GR1, generated by protease cleavage after the HA-tag sequence. We also expressed the same anti-VEGF scFv in E. coli and purified it using periplasm extraction as a control. The purification yield was approximately 0.5 mg/L. The binding affinities of both yeast and E. coli scFvs were measured on a Biacore instrument, immobilizing either antigen or the scFv antibody on a CM5 chip. The kinetic sensograms of both yeast and E. coli scFvs were shown in Fig. 7. The data in Table 1 indicate that the scFv proteins that were generated from S. cerevisiae and E. coli had very similar antigen binding kinetics and affinities. In conclusion, these data confirmed that the display vector can be used directly for the expression and secretion of soluble, functional scFv protein, without the need for further molecular cloning.

4. Discussion By directly fusing heterologous proteins to outer cell wall proteins, they can be incorporated into the outer cell wall of yeast and therefore be displayed on the yeast surface. Such outer wall protein fusion-based display systems have been successfully utilized to display over twenty heterologous proteins (Pepper et al., 2008; Kondo et al., 2004). In this study, we report a non-outer wall protein fusionbased yeast display system. In this system, the heterologous scFv antibody is fused with a small coiled-coil adapter protein, GR1, in the display vector (instead of an outer cell wall protein). With a second coiled-coil adapter (GR2) fused to the cell wall protein Cwp2 that is produced from the yeast helper vector, we were able to display the scFv protein functionally on the cell surface through interaction between the GR1 and GR2 adapters. The display mechanism was confirmed by the co-localization of these two adapters on the yeast cell surface.

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Fig. 7. Binding affinity measurements by Biacore 2000 of the scFvs expressed in S. cerevisiae (A) and E. coli (B) against recombinant human VEGF. The purified scFvs were diluted to 10 µg/mL and immobilized on a CM5 chip. 4 concentrations of VEGF were injected to the CM5 chip to determine the kinetic and binding constants of each scFv antibody.

To produce a soluble protein that is selected from a display library in the outer wall protein fusion-based display system, the gene of interest needs to be amplified and then cloned into a secretion vector (Feldhaus et al., 2003). In contrast, using the adapter-directed display system, the protein of interest is not fused to any outer wall protein and will not be expressed as a part of the cell wall proteins. Therefore, display vectors that have been isolated from a library of yeast cells can be used directly for the soluble expression of proteins without further molecular cloning. This feature will benefit downstream biochemical characterization of the lead proteins. As a proof of concept experiment, we have demonstrated that the anti-VEGF scFv protein fused with the small coiled-coil adapter is secreted into the yeast culture medium. The purified yield of the scFv protein is consistent with previous studies that used a CENbased low-copy vector in S. cerevisiae. The expression yield can be improved further by using S. cerevisiae strains that overexpress yeast chaperones such as protein disulfide isomerase

Table 1 Affinities of the scFv antibodies from S. cerevisiae and E. coli. scFvs

ka a (1/Ms)×104 Kd a (1/s)×10−5 Kd (nM) Immobilization

S. cerevisiae E. coli S. cerevisiae E. coli

1.19 ± 0.07 1.78 ± 0.08 3.09 ± 0.09 2.60 ± 0.09

a

Mean ± SE.

1.84 ± 0.17 2.24 ± 0.18 3.65 ± 0.23 2.47 ± 0.24

1.54 1.26 1.18 0.95

Antigen on CM5 Antigen on CM5 Antibody on CM5 Antibody on CM5

(PDI) and the heavy chain binding protein (BiP) (Hackel et al., 2006). Furthermore, we demonstrated that the antigenbinding affinity of yeast scFv is the same as that of scFv generated from E. coli cells. So far, we have also succeeded in using this display system for multiple chain Fab display and affinity maturation (data not shown). The adapter-directed display is a modular system that has two separated modules (display vector and helper vector) connected by a pair of heterodimeric adapters. The concept of adapter-directed display has been used successfully for the phage surface display of antibodies (Wang et al., 2007, 2010). The common features between the adapter-directed phage and yeast displays are that the protein to be displayed is independent from the surface-anchoring proteins and that they utilize the same set of interactive adapters. These modular features provide the adapter-directed system with a unique flexibility through the combination of different modules. With a cross-species display vector and two speciesspecific helper vectors (such as the yeast helper vector described in this study and the helper phage vectors reported previously), it becomes possible to create a cross-species display system for shuttling the display of the protein of interest between the prokaryotic phage and eukaryotic yeast systems (data not shown). This unique feature cannot be realized with the conventional fusion-based yeast display systems, in which the protein of interest is fused with a yeast-specific anchoring sequence that is not able to function in the second species, such as phage.

K.C. Wang et al. / Journal of Immunological Methods 354 (2010) 11–19

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