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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
  • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
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  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
  • C07K14/33—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Clostridium (G)
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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
  • C12N15/62—DNA sequences coding for fusion proteins
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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
  • C12N15/62—DNA sequences coding for fusion proteins
  • C12N15/625—DNA sequences coding for fusion proteins containing a sequence coding for a signal sequence
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
  • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
  • C07K2319/74—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor

Definitions

• the invention relates to a yeast cell surface display system, to a method of displaying a polypeptide on a yeast cell surface, and to vectors and proteins for use therein.
• Microbial cell surface display of proteins is a widely used approach for industrial applications including vaccine development, gene therapy, cell-based diagnostics, high-throughput polypeptide library screening, whole-cell biocatalysis, bioremediation, biosensors and even biofuels production (Chen and Georgiou 2002; Wu et al. 2008).
• Microbial cell surface display systems are typically based on the expression of translational fusions of a carrier protein and a desired passenger protein (such as one demonstrating desirable enzymatic activity) (Lee et al. 2003).
• Systems have been developed for displaying desired proteins on the outer surface of both prokaryotic and eukaryotic cells, including Gram-positive (Gunneriusson et al. 1996; Samuelson et al.
• the cell wall proteins used include ⁇ -agglutinin, Aga1, Cwp1 , Cwp2, Tipi p, Srp1 (Van der Vaart et al. 1995, 1997), FIoIp (Theunissen et al. 1993; Tanino et al. 2007), Sedip (Hardwick et al. 1992), TiMp (Marguet et al. 1988) and YCR89W (Oliver et al. 1992).
• These cell wall proteins contain the glycosyl phosphatidylinositol (GPI) signal motif and are covalently cross-linked to /?-1 ,6-glucan in the cell wall.
• GPI glycosyl phosphatidylinositol
• the tethering of a lipase to the yeast cell wall using the mannose-oligosacharide binding property of a FIoIp lacking the GPI domain has also been described (Matsumoto et al. 2002).
• Further yeast strains have been reported that are equipped with a variety of functional displayed proteins including antibodies, enzymes and combinatorial protein libraries (Breinig et al. 2006; Furukawa et al. 2006; Lee et al. 2006; Parthasarathy et al. 2006; D ⁇ rauer et al. 2008).
• a yeast cell surface display system comprising a) at least one scaffoldin protein having at least one functional cohesin domain, and b) at least one anchoring protein, wherein the anchoring protein is capable of tethering the scaffoldin protein to a surface of a yeast cell.
• the anchoring protein to be cell wall protein 1 (Cwp1 ) or a protein having at least 80% homology to SEQ ID NO 1 ; and for the N-terminus thereof to be fused to the C-terminus of the scaffoldin protein; for the yeast cell surface display system to further include a secretion signal, such as Trichoderma reesei XYN2 or a secretion signal having at least 80% homology to SEQ ID NO 2, fused to the N-terminus of the scaffoldin protein; for the scaffoldin to be a chimeric protein comprising at least one C. cellulolyticum cohesin domain and at least one C.
• Cwp1 cell wall protein 1
• the N-terminus thereof to be fused to the C-terminus of the scaffoldin protein
• the yeast cell surface display system to further include a secretion signal, such as Trichoderma reesei XYN2 or a secretion signal having at least 80% homology to SEQ ID NO 2, fused to the N
• thermocellum cohesin domain for the scaffoldin protein to be from Clostridium; for the scaffoldin to be SEQ ID NO 3 or a protein having at least 80% homology to SEQ ID NO 3; and for the yeast to be S. cerevisiae.
• the invention also provides a vector having nucleic acid encoding a scaffoldin protein comprising at least one cohesin domain operably linked to expression elements for expressing and displaying the scaffoldin protein on the yeast cell surface.
• nucleic acid encoding cell wall protein 1 (Cwp1), or nucleic acid having at least 80% homology to SEQ ID NO 4, ligated in frame with, and 3' to, the nucleic acid encoding the scaffoldin protein
• the vector to further include a nucleic acid encoding a secretion signal, such as Trichoderma reesei XYN2 or nucleic acid having at least 80% homology to SEQ ID NO 5, ligated in frame with, and 5' to, the nucleic acid encoding the scaffoldin protein
• for the scaffoldin protein to be a chimeric protein comprising at least one C. cellulolyticum cohesin domain and at least one C. thermocellum cohesin domain
• for the scaffoldin protein to be from
• the invention still further provides a fusion protein comprising a) at least one scaffoldin protein having at least one functional cohesin domain, and b) at least one anchoring protein, wherein the anchoring protein is capable of tethering the scaffoldin protein to a surface of a yeast cell, and wherein the N-terminus of the anchoring protein is fused to the C-terminus of the scaffoldin protein.
• thermocellum cohesin domain for scaffoldin protein to be from Clostridium; for the scaffoldin to be SEQ ID NO 3 or a protein having at least 80% homology to SEQ ID NO 3; and for the yeast to be S. cerevisiae.
• the invention yet further provides a yeast cell transformed with a vector having nucleic acid encoding a scaffoldin protein comprising at least one cohesin domain operably linked to expression elements for expressing and displaying the scaffoldin protein on the yeast cell surface.
• the invention also provides a method of displaying a polypeptide on a yeast cell surface, the method including the steps of a) transforming the yeast cell with a vector having nucleic acid encoding a scaffoldin protein comprising at least one cohesin domain operably linked to expression elements for expressing and displaying the expressed scaffoldin protein on the yeast cell surface, and b) contacting the yeast cell with a fusion protein comprising a dockerin domain and a polypeptide to be displayed.
• nucleic acid encoding cell wall protein 1 (Cwp1), or nucleic acid having at least 80% homology to SEQ ID NO 4, ligated in frame with, and 3' to, the nucleic acid encoding the scaffoldin protein
• the vector to further include a nucleic acid encoding a secretion signal, such as Thchoderma reesei XYN2 or nucleic acid having at least 80% homology to SEQ ID NO 5, ligated in frame with, and 5' to, the nucleic acid encoding the scaffoldin protein
• the scaffoldin to be a chimeric protein comprising at least one C. cellulolyticum cohesin domain and at least one C.
• thermocellum cohesin domain for the scaffoldin protein to be from Clostridium; for the nucleic acid encoding the scaffoldin to be SEQ ID NO 6 or nucleic acid having at least 80% homology to SEQ ID NO 6; for the expression elements to include at least one element selected from the group including the phosphoglycerate kinase I gene promoter (PGK1 P ), nucleic acid at least 80% homologous to SEQ ID NO 7, the phosphoglycerate kinase I gene terminator (PGK1 ⁇ ), and nucleic acid at least 80% homologous to SEQ ID NO 8; for the yeast to be S.
• PGK1 P phosphoglycerate kinase I gene promoter
• PGK1 ⁇ phosphoglycerate kinase I gene terminator
• the dockerin domain of the fusion protein for the dockerin domain of the fusion protein to be SEQ ID NO 10, or a protein having at least 80% homology to SEQ ID NO 10; for the dockerin domain to be fused to a random linker having the sequence of SEQ ID NO 11 or poly-alanine linker having the sequence of SEQ ID NO 12; and for the C-terminus of the linker to be fused to the N-terminus of the dockerin domain.
• the term "functional cohesin domain” as used in this application may be defined as a cohesin domain capable of binding to a dockerin domain.
• Figure 1 shows the plasmid maps of (A) pMBRE2 ⁇ PGK1 p-XYNSEC- Scaf3-CWP2-PGK1 T expression cassette) and (B)
• Figure 3 is a micrograph of the immunofluorescent detection of cell surface displayed scaf3p at 1000 x magnification, (A) Parent strain S. cerevisiae NI-C-D4 without the Scaf3 gene, (B) S. cerevisiae Nl-C-D4( pMBRE21) expressing the Cwp2-scaf3 fusion protein with the primary antibody against the CBM- module of the scaf3p (rabbit anti-CBM) and goat anti-rabbit
• IgG-AlexaFluor 488 as secondary antibody
• C S. cerevisiae NI-C-D4(pMBRE2) expressing the scaf3-Cwp2 fusion protein with the primary antibody against the CBM- module of the scaf3p (rabbit anti-CBM) and goat anti-rabbit IgG-AlexaFluor 488 as secondary antibody
• the first column represents the fluorescent image
• the second column the light microscopy image
• the third column shows the combination of the two
• Figure 4 is a Western-like blot showing the detection of membrane and cell wall associated scaf3p using the dockerin containing protein Cel ⁇ A-Dockcf as a probe: Lane 1 : Biotinylated SDS molecular weight marker B2787 (Sigma
• Lane 2 Purified scaf3p from Clostridium
• Lane 3 Membrane-bound protein fraction isolated from S. cerevisiae NI-C-D4 parent strain
• Lane 4 Membrane-bound protein fraction isolated from S. cerevisiae Nl-C- D4(pMBRE21) strain
• Lane 5 Membrane-bound protein fraction isolated from S. cerevisiae NI-C-D4(pMBRE2) strain
• Lane 6 Cell wall associated protein fraction isolated from S. cerevisiae NI-C-D4 parent strain
• Lane 7 Cell wall associated protein fraction isolated from S. cerevisiae Nl-C- D4(pMBRE2) strain
• Lane 8 Cell wall associated protein fraction isolated from S. cerevisiae NI-C-D4(pMBRE21) strain
• Lane 3 Membrane-bound protein fraction isolated from S. cerevisiae NI-C-D4 parent strain
• Lane 4 Membrane-bound protein fraction isolated from S. cerevisiae Nl-C
• Figure 5 is a graph of the comparative endoglucanase activity
• FIG. 6 is a micrograph of the comparative attachment of S. cerevisiae NI-C-D4 parent strain (A) and S. cerevisiae Nl-C-
• Figure 7 is a Western-like blot showing the detection of ⁇ the
• SEQ ID NO 2 is the amino acid sequence of the Trichoderma reesei XYN2 secretion signal
• SEQ ID NO 3 is the amino acid sequence of the scaffoldin protein
• SEQ ID NO 4 is the DNA sequence of the Cwp1 module
• SEQ ID NO 5 is the DNA sequence of the Trichoderma reesei XYN2 secretion signal
• SEQ ID NO 6 is the DNA sequence of the scaffoldin protein
• SEQ ID NO 7 is the DNA sequence of the PGK1 promoter
• SEQ ID NO 12 is the amino acid sequence of the poly-alanine linker.
• an expression vector encoding a chimeric scaffoldin protein was cloned in order to express the scaffoldin as a fusion protein with cell wall protein 1 (Cwp1 ), together with the T. reeseiXYN2 secretion signal.
• the terminal C. thermocellum cohesin domain was fused to the yeast Cwp2p. Since the presence of the Cwp2 anchoring module at the C-terminal extremity of the fusion protein might hamper the folding of the adjacent cohesin(s) and/or interfere with the binding to the corresponding dockerin domain, another genetic construct was prepared in which the Cwp2p module preceded the N-terminus of scaf3.
• an expression vector was constructed containing the green fluorescent protein (GFPmut2) fused in frame with the T. reesei XYN2 secretion signal upstream of a C. thermocellum dockerin domain and placed under the constitutive control of the PGK1 regulatory sequences. Included in the latter design was the separation of the GFP and dockerin domains by two different linkers to ensure proper folding of the GFP and dockerin domains.
• GFPmut2 green fluorescent protein fused in frame with the T. reesei XYN2 secretion signal upstream of a C. thermocellum dockerin domain and placed under the constitutive control of the PGK1 regulatory sequences.
• the multicopy episomal S. cerevisiae-E. coli shuttle vector pDLG59 (La Grange 1999), containing the promoter (PGK1 P ) and terminator (PGK1 T ) sequences of the yeast phosphoglycerate kinase I gene (PGK1), as well as the cell wall binding module of the cell wall protein 2 (CWP2) was used.
• the 121-bp XYNSEC Trichoderma reesei XYN2 secretion signal
• the 717-bp PCR-generated GFPmut2 fragment (with native start and stop codons excluded) was digested using the built-in SgIII and Xho ⁇ sites and sub- cloned into pHVXII (Volschenk et al. 1997), creating pMBRE3 (plasmid map not shown).
• the 267-bp PCR-generated dockerincf (C.
• thermocellum dockerin fragment (with its native stop codon) containing the two different linker sequences was digested with Xho ⁇ , and cloned into the pMBRE3 vector, thereby generating pMBRE4-A (containing the Ala-io-Linker) and pMBRE4-B (containing the random linker sequence), respectively (plasmid maps not shown).
• NI-C-D4 yeast strain and transformants NI-C-D4(pMBRE2) and Nl- CD4(pMBRE21), were grown overnight at 30°C in sc complete and S ⁇ ura media, respectively. After culturing, 2 ml cells were centrifuged at 6000 rpm for 3 minutes and washed with 1 mi l x phosphate buffered saline solution (PBS) pH 7.0.
• PBS mi l x phosphate buffered saline solution
• the cells were resuspended in 200 ⁇ l 1 x PBS containing 1% bovine serum albumin (BSA) and 1 :500 primary antibody raised against the CBM-module of the scaf3p (rabbit anti-CBM) and incubated for 1 hour at room temperature whilst slowly shaking (antibody supplied by H-P Fierobe).
• the cells were washed with 1 x PBS and resuspended in 200 ⁇ l 1 x PBS containing 1% BSA and goat anti-rabbit IgG-AlexaFlour 488 (1:250, 2 mg/ml, Invitrogen) and incubated for 1 hour at room temperature whilst slowly shaking.
• the cells were washed three times with 1 x PBS and resuspended in 200 ⁇ l 1 x PBS and viewed using a fluorescent microscope at 1000 x magnification (Nikon Eclipse E400, Nikon).
• the YeastBuster protein extraction reagent (MERCK, Germany) was used according to the manufacturer's specifications. The isolated proteins were stored at -20 ° C. Similarly, 15 ⁇ l of each sample containing the intracellular protein fraction was loaded onto SDS-PAGE gels.
• Proteins were isolated based on the procedures of Beki et al. (2003) and Del Carratore et al. (2000).
• a 10 ml SC "ura yeast preculture was grown overnight at 30 ° C and inoculated into 100 ml SC 'ura medium and grown overnight at 30 ° C. After culturing, 60 ml cells were centrifuged at 5000 rpm for 5 minutes at 4 ° C ( ⁇ 1 g yeast cells). The cells were resuspended in 10 ml HCS Buffer (50 mM HEPES, 10 mM CaCI 2 pH 7, 2 M Sorbitol) and 1 mg Zymolyase T20 per gram cells were added and incubated for 1 hour at 37 ° C.
• 10 ml HCS Buffer 50 mM HEPES, 10 mM CaCI 2 pH 7, 2 M Sorbitol
• the cells were centrifuged at 5000 rpm for 5 minutes and the supernatant decanted.
• the cells were washed twice with 10 ml HCS Buffer at 5000 rpm for 5 minutes and the spheroplasts resuspended in 10 ml HCS Buffer.
• the spheroplasts were sonicated (Omni- Ruptor 400, Omni International Inc.) on ice 3 times for 90 seconds at a 50% power setting followed by centrifugation at 13 000 rpm for 30 minutes.
• the supernatant which contained the cell-membrane bound protein fractions was transferred to a sterile McCartney Bottle and kept at -2O 0 C.
• the pellets obtained, containing the cell wall-associated protein fraction, were resuspended in 2 ml HCS Buffer and kept at - 20 ° C. As with the intracellular and extracellular protein fraction, 15 ⁇ l of each sample containing either the cell-membrane bound or cell wall-associated protein fraction was loaded onto SDS-PAGE gel.
• the Cel5A-Dockcf protein (purified enzyme supplied H-P Fierobe) was used as a probe.
• This protein is a C. cellulolyticum endoglucanase appended with a C. thermocellum dockerin (Fierobe et al. 2001 ).
• the Cel ⁇ ADockcf by protein was biotinylated on the lysine groups with a biotinyl ⁇ /-hydroxysuccinimide ester as described by the manufacturer (Biotin protein labeling kit, Roche).
• the scaf3 protein (purified protein supplied by H-P Fierobe) was used as a probe and was biotinylated as described above. All four protein fractions isolated from NI-C-D4, NI-C-D4(pMBRE5-A) and NI-CD4(pMBRE5-B) were separated by 12% (v/v) SDS-PAGE according to the method of Laemmli (1970) and transferred onto a polyvinylidene fluoride (PVDF) membrane (Millipore, USA). Nonspecific binding was blocked by incubating (1 hour at room temperature) the membrane with 3% blocking solution (same composition as described above).
• PVDF polyvinylidene fluoride
• NI-C-D4 and NI-C-D4(pMBRE2) cells were grown for 72 hrs at 3O 0 C in 25ml 2 x gQ ⁇ mpiete and 2 x SC "ura medium, respectively, buffered with succinic acid (20g/L) and pH 6.0 (adjusted with sodium hydroxide). Cells (25 ml) were centrifuged at 3000 rpm for 3 minutes and washed with 1 ml 5OmM Tris-Maleate buffer (pH 6.0) containing 5mM Calcium Chloride (CaCI 2 ).
• Washed cell cultures were divided in half and to one half of the cells 1 ml of NI-CD4 supernatant (negative control) was added and to the other half 1 ml of 1:100 diluted Cel ⁇ A- Docket protein (5.3 mg protein/ml). Cells were incubated for 1 hour at room temperature, thereafter they were washed 3 times with an excess volume of 5OmM Tris-Maleate buffer pH 6.0 containing 5 mM CaCl 2 .
• the cells were resuspended in 1 ml Tris-Maleate buffer (50 mM, pH 6.0) containing 5 mM CaCb as well as 1% (w/v) low viscosity carboxymethylcellulose sodium salt (Sigma, Germany) as substrate and incubated for 3.5 hours at 37°C while shaking.
• 1 ml Cel5A-Dockcf protein final dilution of 1 :100
• Tris-Maleate buffer 50 mM, pH 6.0
• 5 mM CaCI 2 was also directly incubated for 3.5 hours at 37 0 C while shaking in the presence of 1% (w/v) low viscosity carboxymethylcellulose sodium salt.
• NI-C-D4, NI-C-D4(pMBRE2) and NI-C-D4(pMBRE21) yeast cells were treated with rabbit anti-CBM and goat anti-rabbit IgG-Alexa Fluor 488 and subjected to fluorescent microscopy (Figure 3).
• Scaf3 that is expressed and targeted to the yeast cell surface is capable of binding to Cel5A-dockerincf
• thermocellum cohesin domain Binding of purified biotinylated Cel ⁇ A-dockerincf to the C. thermocellum cohesin domain was detected in the cell membrane ( Figure 4: lane 5) and cell wall debris protein fractions ( Figure 4: lane 7) of the yeast containing pMBRE2. No chemiluminescent signal could be detected for the intracellular and extracellular protein fractions, even for the large scale extracellular protein fraction isolation (results not shown). Purified scaf3 protein was used as positive control ( Figure 4: lane 2).
• the Far Western blot results confirmed both the functional expression and targeting of scaf3 to the cell wall of S. cerevisiae, and that the yeast produced cohesin domain (as part of the scaf3 protein) is able to bind to a purified dockerin domain (as part of the Cel ⁇ A-dockerinrt protein).
• This data confirmed the importance of the orientation of the Cwp2 module relative to scaf3p in the fusion protein and the influence of this orientation on scaf3 functionality.
• the functional expression and targeting of the scaf3p to the yeast cell surface was further assessed by determining the acquired endoglucanase enzyme activity in the yeast NI-C-D4 and NI-C-D4(pMBRE2) (PGK1 P -XYNSEC-Scaf3- CWP2-PGK1 T ) incubated in the presence of purified Cel5A-Dockcf fusion protein and carboxymethylcellulose (Figure 5). Endoglucanase activity (hydrolytic activity towards cellulose) of the Cel5A-Dockrt fusion protein on carboxymethylcellulose in the absence of any yeast cells was taken as the 100% level (Figure 5).
• NI-C-D4(pMBRE2) showed a 2 fold increase in endoglucanase activity (28% of maximum activity) over the parent strain Nl-C- D4 (14% of maximum activity).
• the difference in endoglucanase activity between cells of NI-CD4(pMBRE2) and NI-C-D4 was calculated as significant (P ⁇ 0.05, two-tailed test) using the Mann-Whitney U statistical test.
• the correct targeting and functioning of the scaf3 protein on the cell surface of S. cerevisiae was assessed for the capacity of S. cerevisiae cells expressing the cell wall-targeted scaf3 protein to bind to cellulose as mediated by the C. cellulolyticum CBM.
• the rationale of this qualitative approach was based on the fact that the native Clostridium scaffoldin promotes the binding of the cellulosomes to cellulose via the CBM, thus functional display of the scaf3p on the yeast cell surface should enable yeast cells to attach to cellulose. Upon microscopic inspection of cells of S.
• the green fluorescent protein (GFPmut2) was fused in frame with the C. thermocellum dockerin domain and separated by two different linkers to ensure proper folding.
• the scaffoldin protein from the cellulosome of Clostridium species which is responsible for the degradation of crystalline cellulose is herein proposed as a candidate carrier protein for cell surface displaying proteins in yeast.
• the protein is relatively large, containing a scaffoldin subunit (160-189 kDa), a non-catalytic modular polypeptide containing an internal carbohydrate binding module (CBM) and multiple copies (8 and 9 in the case of C. cellulolyticum and C. thermocellum, respectively) of cohesin domains inherently flexibly linked to one another.
• CBM carbohydrate binding module
• the relatively large size of the scaffoldin protein is proposed to increase the distance of the displayed protein from the cell wall, thereby decreasing steric effects of the yeast cell wall on the functionality of the displayed protein.
• the nature of the inherent flexible linking of the cohesin domains to one another is proposed to confer advantageous conformational flexibility in the display of desired proteins.
• a further advantage of the invention relates to the use of a bacterial scaffoldin protein in a yeast cell surface display system. This facilitates the expression and flexible display in S. cerevisiae of proteins of eukaryotic origin that require complex post-translation modification and that are therefore not easily produced in their native form in recombinant bacterial systems.
• scaffoldin proteins for presenting proteins on a yeast cell surface is herein proposed to serve as a highly flexible platform for numerous applications including high throughput screening of peptide and enzyme libraries, whole cell sorbents, recombinant biocatalysts, and cell-based diagnostics, including high throughput drug discovery/screening, directed protein evolution and protein engineering applications.

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