Chapter 5 Yeast Endoplasmic Reticulum Sequestration Screening for the Engineering of Proteases from Libraries Expressed in Yeast Li Yi, Joseph M. Taft, Qing Li, Mark C. Gebhard, George Georgiou, and Brent L. Iverson Abstract There is significant interest in engineering proteases with desired proteolytic properties. We describe a high-throughput fluorescence-activated cell sorting (FACS) assay for detecting altered proteolytic activity of protease in yeast, at the single cell level. This assay relies on coupling yeast endoplasmic reticulum (ER) retention, yeast surface display, and FACS analysis. The method described here allows facile screening of large libraries, and of either protease or substrate variants, including the screening of protease libraries against substrate libraries. We demonstrate the application of this technique in the screening of libraries of Tobacco Etch Virus protease (TEV-P) for altered proteolytic activities. In addition, the generality of this method is also validated by other proteases such as human granzyme K and the hepatitis C virus protease, and the human Abelson tyrosine kinase. Key words Protease engineering, FACS, ER retention, Yeast surface display
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Introduction Proteases constitute the largest enzyme family in humans, and they play important roles in almost all physiological functions [1–3]. Few protease-related drugs have been approved by the FDA [4], perhaps due to the lack of a powerful high-throughput screening method for the protease engineering. To solve this problem, we developed the yeast endoplasmic reticulum sequestration screening (YESS) system, which allows the simultaneous expression and co-localization of a protease and its substrate, followed by cell surface display of the substrate [5]. Co-expression is achieved by inserting the protease and substrate genes downstream of the GAL10 and GAL1 inducible promoters, which are arranged in a tail-to-tail fashion on the plasmid [6]. The N-termini of the proteins contain a signal sequence which directs the protease and
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substrate to the endoplasmic reticulum (ER) [5, 7]. At the C-termini of the proteins, the ER retention signal peptide (FEHDEL) retains the protein and its substrate within the lumen of the ER [8, 9]. By fusing the substrate with the Aga2 protein, as in preceding yeast display technologies, the substrate is trafficked to the outer membrane of the cell, where it is displayed via disulfide bonding to the lipid-anchored membrane protein Aga1 [7]. Co-expression of a protease and its substrate offers the possibility of screening both enzyme and substrate libraries. As with other display technologies, the phenotype-genotype linkage allows the researcher to recover variants that possess desired phenotypes by screening or selection followed by recovery and sequencing of the genes from the recovered clones. YESS allows each yeast cell to act as an independent vessel for a unique protease-substrate reaction, the progress of which is monitored by FACS (Fig. 1). The Aga2-substrate fusion protein contains epitope tags (e.g., FLAG, H6, or HA) on either side of the putative cleavage sequence. Reactions are probed by staining cells with fluorophore-labeled epitope tag-specific antibodies. Fluorescence from both N-terminal and C-terminal epitope tags indicates no cleavage has occurred;
Fig. 1 Overview of the Yeast Endoplasmic Reticulum Sequestration Screening (YESS) system. The cleaved or uncleaved substrates are transported to the yeast cell surface through the Aga2-Aga1 surface display system. After staining with anti-FLAG-PE and anti-6×His-FITC antibodies, cells exhibiting relatively high PE fluorescence, but little or no FITC fluorescence, are isolated through FACS, which indicates specific cleavage at only the desired substrate sequence
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N-terminal fluorescence indicates putative cleavage of the substrate; lack of fluorescence indicates promiscuous cleavage (Fig. 1). Repeated rounds of FACS are used to isolate clones with the desired catalytic properties. Through the use of selection and counter-selection, YESS can be utilized to increase proteolytic activity towards a target peptide while simultaneously reducing activity towards undesirable substrates [5]. Alternatively, substrate libraries can be screened to profile proteolytic specificity. Finally, libraries of enzymes can be screened against libraries of peptides in order to isolate novel combinations. Yi et al. performed “library-on-library” screening in order to isolate TEV protease (TEV-P) variants with novel substrate specificity [5]. The TEV-P gene was mutated with degenerate NNS codons in the S1 substrate-binding pocket. This library of TEV-P mutants was then co-transformed with variants of the TEV-P recognition sequence (ENLYFQ↓S) where the P1 position (Q) was randomized. Isolation of active protease-substrate pairs by FACS, followed by in vitro characterization, showed that two novel variants, TEV-PE10 and TEV-PH21, recognized new substrates ENLYFES and ENLYFHS with 5,000- and 1,100-fold specificity reversals, respectively, compared to TEV-P. The YESS system should be generally applicable to the directed evolution and analysis of proteases as well as other protein-modifying enzymes [5]. Hepatitis C virus protease and human Granzyme K protease displayed cleavage of their preferred substrates when expressed in the YESS system. Additionally, the catalytic domain of the human Abl1 tyrosine kinase was shown to phosphorylate a known substrate sequence fused to the C-terminus of Aga2.
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Materials All the solutions are prepared using deionized water. All reagents are stored at 4 °C unless indicated otherwise. Escherichia coli (E. coli) cells are grown at 37 °C (both plates and liquid media) unless indicated otherwise. Yeast cells are grown at 30 °C (both plates and liquid media) unless indicated otherwise.
2.1 Library Construction
1. The designed PAGE-purified primers were purchased from IDT DNA (IDT DNA, Coralville, IA, USA). 2. Phusion DNA polymerase (New England Biolabs). 3. Taq DNA polymerase (Life Technologies). 4. dNTPs (Fermentas). 5. E. coli JUDE-1(DH10B F′:: Tn10(Tetr)) competent cells, which have the Streptomycin resistance, were prepared by a previously published electrocompetent method [10].
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6. E. coli MC1061 [F− Δ(ara-leu)7697 [araD139]B/r Δ(codBlacI)3 galK16 galE15 λ− e14− mcrA0 relA1 rpsL150(strR) spoT1 mcrB1 hsdR2(r−m+)] competent cells, which have the Streptomycin resistance, were prepared by a previously published electrocompetent method [10]. 7. pTrc99A-MBP with an ampicillin marker was used as an expression plasmid for parental TEV-P and its S1 pocket saturation mutagenesis libraries [11]. 8. pMOPAC-12 with a chloramphenicol marker was used as an expression plasmid for parental TEV-P and its error-prone PCR mutagenesis libraries [12]. 9. pRK792 with an ampicillin marker was used as an expression plasmid for TEV-P and its variants. It was also used for protein expression of Maltose-binding protein (MBP)-glutathione S-transferase (GST) fusion complex [13]. 10. All restriction enzymes were purchased from NEB unless indicated otherwise. 11. Other than annotated, all chemicals were purchased from Sigma (Sigma-Aldrich, St. Louis, MO). 12. QIAprep Spin Miniprep kit (QIAGEN). 2.2 Yeast Transformation and Cell Sorting
1. The Saccharomyces cerevisiae strain, EBY100 (URA+, leu−, trp−), was a generous gift from Dr. Dane K. Wittrup (Massachusetts Institute of Technology, Cambridge, MA). 2. The pESD (Yeast Epitope tagging vector for Surface Display) plasmid (Fig. 2) was generated on the basis of the yeast surface display construct pCTCon2 (generously contributed by Dr. Dane K. Wittrup, Massachusetts Institute of Technology, Cambridge, MA) and the yeast epitope tagging vectors pESC-TRP vectors (generously contributed by Dr. Edward W. Marcotte, University of Texas at Austin). 3. YPD medium: 10 g/L Yeast extract, 20 g/L Peptone, and 20 g/L D-Glucose. 4. YNB-CAA-Glucose medium: 20 g/L glucose, 6.7 g/L yeast nitrogen base without amino acids (Thermo Fisher), 5.4 g/L Na2HPO4, 8.6 g/L NaH2PO4·H2O and 5 g/L casamino acids, pH 7.4. 5. YNB-CAA-Galactose medium: 20 g/L galactose, 6.7 g/L yeast nitrogen base without amino acids, 5.4 g/L Na2HPO4, 8.6 g/L NaH2PO4·H2O and 5 g/L casamino acids, pH 7.4. 6. Cell washing buffer A: 1× Phosphate buffered saline (PBS) buffer (Life Technologies, Carlsbad, CA, USA), containing 0.5 % bovine serum albumin (BSA) and 1 mM ethylenediaminetetraacetate (EDTA), pH 7.4.
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Fig. 2 Map of the vector pESD. (a) The pESD plasmid is constructed based on the pCTCon2 and the pESC-TRP vectors, which contains an ampicillin resistant gene, and a TRP1 gene responsible for the tryptophan biosynthesis. The pESD plasmid contains a GAL1/10 bi-directional promoter, leading to a simultaneous expression of protease and its substrates under the induction of galactose. (b) Salient features of the substrate and protease fusion proteins used in the YESS system. The protease gene is under the control of GAL1 promoter with an ER targeting signal sequence (MQLLRCFSIFSVIASVLA) and an ER retention sequence (FEHDEL) located at the N-terminal end and the C-terminal end, respectively. The ER targeting signal sequence leads the protease into the ER, and the ER retention sequence retains the protease in the ER. Under the control of GAL10 promoter, an Aga2 gene is fused with a five-part cassette which in order encodes the counter-selection substrate of protease, the FLAG tag sequence (DYKDDDDK), the designed substrate, the 6×His tag sequence (HHHHHH), and an ER retention sequence (FEHDEL). An ER targeting signal sequence is also anchored at the N-terminal end of the Aga2, facilitating the transportation of the Aga2-substrate cassette into the ER
7. Cell washing buffer B: 1× PBS, containing 0.5 % BSA, pH 7.4. 8. Cell washing buffer C: 1× PBS, pH 7.4. 9. Anti-FLAG-PE antibody (ProZyme). 10. Anti-6×His-FITC antibody (Genscript). 11. FACSAria II (BD Biosciences San Jose, CA, USA). 12. Zymoprep™ Yeast Plasmid Miniprep II kit (Zymo Research).
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2.3 TEV-P Purification and Characterization
1. The TEV-P and its variants were cloned into pRK792 vector for the protein purification. All the materials were prepared based on previously published protocol [13].
2.3.1 Purification of the TEV-P and Synthesis of Peptide Substrate
2. Substrate peptides, including TENLYFQSGTRRW, TENLYFE SGTRRW, and TENLYFHSGTRRW, are designed and purchased from Genscript. 3. KaleidaGraph software (Synergy Software).
2.3.2 Purification of the MBP-GST Fusion Complex
1. Luria Broth (LB) medium: 10 g/L Bacto-tryptone, 5 g/L yeast extract, 10 g/L NaCl, pH 7.5. 2. Cell lysis buffer: 50 mM Tris–HCl, 50 mM NaH2PO4, 200 mM NaCl, 25 mM imidazole, 10 % glycerol, pH 8.0, adding 2 mg/mL lysozyme, cOmplete EDTA-free protease inhibitor cocktail (1 tablet/50 mL, Roche, Basel, Switzerland), and 5 mM fresh DTT added immediately before use. 3. Wash buffer: 50 mM Tris–HCl, 50 mM NaH2PO4, 200 mM NaCl, 60 mM imidazole, pH 8.0, and 5 mM fresh DTT added immediately before use. 4. Elution buffer: 50 mM Tris–HCl, 50 mM NaH2PO4, 200 mM NaCl, 60 mM imidazole, pH 8.0, and 5 mM fresh DTT added immediately before use. 5. Storage buffer: 50 mM Tris–HCl, 1 mM EDTA, pH 7.5, and 5 mM fresh DTT added immediately before use. 6. Reaction buffer: 50 mM Tris–HCl, 1 mM EDTA, pH 7.5, and 2 mM fresh DTT added immediately before use. 7. Ni-NTA affinity resin (QIAGEN). 8. French Press (Thermo Scientific). 9. Thermomixer R (Eppendorf). 10. Phenomenex C18 reverse-phase column (Phenomenex). 11. Magic 2002 instrument (Micron Bioresources).
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Methods
3.1 Library Construction 3.1.1 Construction of the S1 Pocket Library of TEV-P
1. The designed primers, which contain a randomized NNS codon (N = A, T, G, or C; S = G or C) in place of the wild-type codon at T146, D148, H167, and S170, are used to amplify the TEV-P gene by splicing overlap extension PCR [14]. In addition, a KpnI restriction site and a PstI restriction site are introduced by the designed primers at the 5′ and 3′ ends of the whole PCR products, respectively. 2. The PCR products are purified and then digested with KpnI and PstI. The digested PCR products are purified through gel extraction (see Note 1).
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3. The pTrc99A-MBP vector is linearized with the same enzymes, KpnI and PstI, and also purified through gel extraction (see Note 1). 4. The purified PCR products are ligated into the linearized pTrc99A-MBP vector using T4 DNA ligase, followed by plasmid purification and desalting (see Note 2). 5. The ligation products are transformed into E. coli MC1061 competent cells, which are plated on ampicillin selective LB plates. 6. The library quality is determined, and the library DNA is extracted and saved for the following experiments (see Note 3). 3.1.2 Construction of the Error-Prone PCR Library of TEV-P
1. PAGE-purified primers are designed and purchased from IDT DNA. A SfiI restriction site is introduced by the designed primers at both the 5′ and 3′ ends of the whole PCR products. 2. The error-prone PCR is performed based on the previously published protocol using varied dNTPs concentrations [15]. 3. Both the PCR products and the pMOPAC12 plasmid are digested with SfiI at 50 °C for 6 h, and then purified through gel extraction. 4. The SfiI digested PCR products are ligated into the linearized pMOPAC12 vector using the T4 DNA ligase, followed by plasmid purification and desalting. 5. The ligation products are used to transform electrocompetent E. coli JUDE-1 cells, which are plated on chloramphenicol selective LB plates. 6. The library quality and error rate are determined, and the library DNA is extracted and saved for the following experiments (see Notes 3 and 4).
3.1.3 Incorporation of Mutagenesis Library into Yeast Construct
1. PCR amplify mutagenesis library with primers containing homology sequences of pESD plasmid for in vivo recombination. 2. PCR amplified mutagenesis library is gel purified, followed by additional product cleaning using Qiagen PCR purification kit (see Note 5). 3. The pESD plasmids are linearized with SalI and XhoI, followed by product cleaning using Qiagen PCR purification kit (see Note 5). 4. Purified mutagenesis library DNA and linearized pESD plasmids are mixed with different ratios for the following yeast cell transformation (see Note 6).
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3.2 Yeast Transformation and Cell Sorting 3.2.1 Yeast Transformation
3.2.2 Yeast Cell Sorting
1. The protease library is inserted downstream of the GAL1 promoter in pESD vector by homologous recombination following electroporation of Saccharomyces cerevisiae EBY100 (URA+, leu−, trp−) as previously described [16]. 2. If needed, the substrate library is inserted downstream of the GAL10 promoter in pESD vector using the similar strategy with the protease library incorporation (see Note 7). 1. Cells are grown to an OD600 of 2.0–3.0 in YNB-CAA-Glucose medium at 30 °C, and induced by changing to YNB-CAAGalactose medium with an initial OD600 of 0.5 (see Notes 8 and 9). 2. Following medium exchange, grow cells at 30 °C overnight, with shaking at a speed of 250 rpm. 3. For the first round of cell sorting, the total cells with the number equaling to ten times of the original library size are collected (see Note 10). 4. The collected cells are washed once with one volume of cell washing buffer A and twice with cell washing buffer B. During the washing steps, cells are kept at 4 °C. Cells are collected by centrifugation at 1,000 × g, 1 min. 5. The washed cells are then resuspended in cell washing buffer B with a density of 5 × 104 cells/μL, followed by incubation with fluorescently labeled anti-FLAG-PE antibody at a final concentration of 0.02 μg/μL (see Note 11). 6. Cells are then centrifuged at 4 °C at 1,000 × g for 1 min. The supernatant is discarded, followed by washing with cell washing buffer B (once) to completely remove the unbound antibodies. 7. The washed cells are labeled with the anti-6×His-FITC antibody at a final concentration of 0.02 μg/μL (see Note 11). 8. The antibody-labeled cells are washed twice with cell washing buffer C, and then resuspended in 1×PBS buffer for the cell sorting using FACSAria II (see Note 12). 9. The resuspended cells are analyzed using BD Biosciences FACSAria II flow cytometer. The gates are set on 575/30 nm emission filter for PE as well as 510/20 nm emission filter for FITC (see Note 13). 10. The sorting gate of the first round cell sorting is drawn to include as many target cells as possible (Fig. 3a) (see Note 14). 11. A smaller gate (Fig. 3b–e) is drawn to enrich the target cells in the following sorting rounds, which present high PE as well as low FITC signals (see Note 15).
3.2.3 Identification of Selected Mutants
1. After four rounds of cell sorting and resorting, the sorted cells are plated on the selective YNB-CAA-Glucose media plate, which lacks tryptophan.
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Fig. 3 The representative library sorting process. The cells containing the mutagenesis libraries are labeled with anti-FLAG-PE and anti-6×His-FITC antibodies. (a) Representative FACS data of the starting library; (b–e) Representative FACS data of the cells after the first, second, third, and fourth-round enrichment; (f) FACS data of the representative isolated single clone
2. After incubation at 30 °C for around 2 days, individual clones are picked up from the plates to grow in YNB-CAA-Glucose liquid medium. 3. The inoculated single clones are then induced with YNBCAA-Galactose medium, followed by staining with the antiFLAG-PE antibody and the anti-6×His-FITC antibody (see Note 16). 4. The labeled cells are re-analyzed using flow cytometry (Fig. 3f). 5. Based on the FACS results, the single yeast clones, which present the high PE signal accompanied with low FITC signals, are isolated. 6. The plasmid DNA of the isolated yeast clones is extracted and then transformed into E. coli for DNA enrichment (see Note 17). 7. The protease gene in the plasmid DNA is sequenced to gather the gene sequence information of the mutants.
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3.3 TEV-P Purification and Characterization
1. The pRK792 plasmid is used as the protein expression vector. TEV-P and selected variants were expressed and purified as previously described [13].
3.3.1 TEV-P Purification
2. All the purified enzymes were >95 % pure as determined by SDS-PAGE with Coomassie staining. 3. All the buffers contain 5 mM freshly prepared DTT to prevent TEV-P oxidation (see Note 18).
3.3.2 MBP-GST Fusion Protein Purification
To monitor the cleavage of fusion proteins by TEV-P or its variants, maltose-binding protein (MBP) and glutathione S-transferase (GST) protein are fused with an internal peptide linker containing ENLYFXS-H6, where X can be Q, H, or E. The H6 will facilitate the purification of the MBP-GST fusion complex using the Ni-NTA affinity resin. The respective fusions are designated MBP-ENLYFQSGST, MBP-ENLYFES-GST, and MBP-ENLYFHS-GST. 1. The MBP-GST fusion gene is cloned into the pRK792 vector and the protein is purified using Ni-NTA affinity resin (see Note 19). 2. The inoculated cells are grown in ampicillin selective LB media, and then induced with 1 mM IPTG when OD600 reaches 0.6. The induced cells are grown in a shaker at 20 °C for 20 h, 250 rpm. 3. The induced cells are collected by centrifugation at 8,000 × g for 15 min. Cell pellets are resuspended with cell lysis buffer (1:7 to 1: 10 ratio) in a glass beaker, adding 1 mg/mL lysozyme, 5 mM freshly prepared DTT, protease inhibitor cocktail (EDTA free, one tablet for 50 mL), 10 unit/mL DNAase. 4. The cell lysate is stirred with a stir bar for 1 h in the 4 °C cold room. The cells are further disrupted using a French Press. 5. The disrupted cell lysate is centrifuged at 30,000 × g for 1 h. The supernatant is then collected and filtered with 0.45 μm filter. 6. The supernatant is loaded into a column filled with Ni-NTA resin equilibrated with wash buffer. 7. The column is washed with at least 20-fold volume of wash buffer, and then followed by elution with elution buffer (see Note 20). 8. All the purified fusion protein should be >95 % pure as determined by SDS-PAGE with Coomassie staining.
3.3.3 TEV-P Characterization: Peptide Substrate Digestion
1. 5 μM to 6 mM of substrate peptide are incubated with 0.025–5 μM purified enzymes at 30 °C for 10–30 min. Reaction tubes were placed in a Thermomixer R (see Note 21). 2. The reactions are quenched with 0.5 % Trifluoroacetic acid (TFA) followed by freezing at −80 °C.
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3. All the enzymatic reactions are analyzed by HPLC on a Phenomenex C18 reverse-phase column using an acetonitrile gradient and a flow rate of 1 mL/min (see Notes 22 and 23). 4. The product-synthesizing rate is calculated upon the integration area at 280 nm (see Note 24). 5. The data is fitted to nonlinear regression of the MichaelisMenten equation using KaleidaGraph software. 3.3.4 TEV-P Characterization: Protein Substrate Digestion
1. The enzymatic reactions are performed the same way with the peptide substrate digestion. All the enzymatic assays were carried out in the reaction buffer using freshly purified enzyme. 2. 0.1 μg protease is mixed with 5 μg MBP-GST fusion protein substrate, which is reacted in microfuge tube on a Thermomixer R at 30 °C for 1 h. 3. The reactions are quenched with 0.5 % Trifluoroacetic acid (TFA) followed by freezing at −80 °C. 4. All the enzymatic reactions are analyzed by SDS-PAGE with Coomassie staining.
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Notes 1. To increase the efficiency of ligation and following transformation, the residual gel from the gel purification steps must be removed completely. A following DNA purification step using the Qiagen PCR purification kit can be used. 2. The ligation products need to be desalted to increase the efficiency of transformation through electroporation. 3. Library quality is evaluated by sequencing isolated plasmids. Twenty single clones are picked up from the pooled clones followed by plasmid DNA isolation using a QIAprep Spin Miniprep kit. 90 % correctness, which means 90 % of the sequenced plasmids contain the designed mutations, is required for a good library to be used for the following experiments. 4. The library error rate is calculated by the average number of mutations in 20 sequenced clones divided by the total number of residues of the native protease. 5. To enhance the transformation efficiency of the yeast cells, highly pure and concentrated DNA is preferred, with library DNA concentration above 0.5 μg/μL and the pESD plasmid concentration above 1 μg/μL. Generally, the mixed DNA volume should be less than 10 % of that of the competent cells in the electroporation cuvette.
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6. As for different mutagenesis libraries, the transformation DNA mix with varied ratios of the purified mutagenesis library against the linearized pESD plasmids should be tested to maximize the transformation efficiency. 7. The protease and the substrate genes in the pESD plasmid are under the control of the GAL1 and GAL10 promoters, respectively. The directed evolution of a protease against a substrate library, or a protease library against a substrate library, or a protease library against a specific substrate can be applied in the YESS system. The substrate library can be incorporated into the pESD vector using the similar strategy with the protease library. 8. The over-growth (OD600 higher than 3.0) of yeast cells before the induction will lower the protein expression and surface display efficiency. In addition, the initial OD600 of the induced cells should be kept lower than 1.0 but above 0.5. 9. Penicillin (final concentration of 100 units/mL) and streptomycin (final concentration of 100 μg/mL) can be added into the culture medium to avoid the bacterial contamination. 10. The total amount of cells that are collected for sorting in the first round should be ten times of the original library size to avoid the loss of the library diversity. 11. The anti-FLAG-PE antibody or the anti-6×His-FITC antibody is diluted in the cell washing buffer B for the antibody labeling. The antibody labeling process is performed at 4 °C for 15 min followed by room temp for 30 min. 12. A cell density concentration close to 5 × 106 cells/mL is preferred for loading into the FACSAria II for the cell sorting. 13. To avoid the interference of the PE signal to the FITC signal, a 510/20 nm emission filter for FITC is preferred. 14. The big sorting gate of the first round cell sorting can be drawn to be close to the major uncleaved cell population to lower the possibility of losing the target cells (Fig. 3a). 15. The total cells being analyzed and sorted for each round can be varied. It depends on the total number of the cells that are collected in the previous round. 16. 106 cells are enough for the FACS scanning for the single clones. 17. The plasmid DNA from the yeast cells is extracted using Zymoprep™ Yeast Plasmid Miniprep II kit. 18. The purified TEV-P is diluted in the storage buffer, with the concentration being kept lower than 0.5 mg/mL to avoid protein aggregation. 19. The MBP-ENLYFXS-H6-GST fusion gene is inserted into pRK792 construct, which contains an ampicillin resistant gene.
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20. The purified fusion protein is dialyzed with reaction buffer for the later enzymatic experiments. 21. All the enzymatic assays are performed using freshly purified enzyme. Kinetic assays are carried out in reaction buffer. 22. The reaction samples are centrifuged at 16,000 × g for 1 min. The supernatant is collected for the HPLC analysis. 23. The proteolysis products are confirmed using LC-MS (ESI), which was performed on a Magic 2002 instrument. 24. To minimize the product inhibitory effect, the substrate conversion percentage is controlled to be less than 5 % to approximate steady-state kinetics. References 1. Marnett AB, Craik CS (2005) Papa’s got a brand new tag: advances in identification of proteases and their substrates. Trends Biotechnol 23:59–64. doi:10.1016/j.tibtech.2004.12.010 2. Overall CM, Blobel CP (2007) In search of partners: linking extracellular proteases to substrates. Nat Rev Mol Cell Biol 8:245–257. doi:10.1038/nrm2120 3. Rawlings ND, Barrett AJ, Bateman A (2010) MEROPS: the peptidase database. Nucleic Acids Res 38:D227–D233. doi:10.1093/nar/ gkp971 4. Craik CS, Page MJ, Madison EL (2011) Proteases as therapeutics. Biochem J 435:1– 16. doi:10.1042/BJ20100965 5. Yi L, Gebhard MC, Li Q et al (2013) Engineering of TEV protease variants by yeast ER sequestration screening (YESS) of combinatorial libraries. Proc Natl Acad Sci U S A 110:7229–7234. doi:10.1073/pnas.1215994110 6. Johnston M, Davis RW (1984) Sequences that regulate the divergent GAL1-GAL10 promoter in Saccharomyces cerevisiae. Mol Cell Biol 4:1440–1448 7. Boder ET, Wittrup KD (1997) Yeast surface display for screening combinatorial polypeptide libraries. Nat Biotechnol 15:553–557. doi:10.1038/nbt0697-553 8. Hardwick KG, Lewis MJ, Semenza J et al (1990) ERD1, a yeast gene required for the retention of luminal endoplasmic reticulum proteins, affects glycoprotein processing in the Golgi apparatus. EMBO J 9:623–630 9. Pelham HR, Hardwick KG, Lewis MJ (1988) Sorting of soluble ER proteins in yeast. EMBO J 7:1757–1762
10. Makino T, Skretas G, Kang TH et al (2011) Comprehensive engineering of Escherichia coli for enhanced expression of IgG antibodies. Metab Eng 13:241–251. doi:10.1016/j. ymben.2010.11.002 11. Fisher AC, Haitjema CH, Guarino C et al (2011) Production of secretory and extracellular N-linked glycoproteins in Escherichia coli. Appl Environ Microbiol 77:871–881. doi:10.1128/AEM.01901-10 12. Jung ST, Reddy ST, Kang TH et al (2010) Aglycosylated IgG variants expressed in bacteria that selectively bind FcgammaRI potentiate tumor cell killing by monocyte-dendritic cells. Proc Natl Acad Sci U S A 107:604–609. doi:10.1073/pnas.0908590107 13. Tropea JE, Cherry S, Waugh DS (2009) Expression and purification of soluble His(6)tagged TEV protease. Methods Mol Biol 498:297–307.doi:10.1007/978-1-59745-1963_19 14. Varadarajan N, Cantor JR, Georgiou G et al (2009) Construction and flow cytometric screening of targeted enzyme libraries. Nat Protoc 4:893–901. doi:10.1038/nprot.2009.60 15. Drummond DA, Iverson BL, Georgiou G et al (2005) Why high-error-rate random mutagenesis libraries are enriched in functional and improved proteins. J Mol Biol 350:806–816. doi:10.1016/j.jmb.2005.05.023 16. Benatuil L, Perez JM, Belk J et al (2010) An improved yeast transformation method for the generation of very large human antibody libraries. Protein Eng Des Sel 23:155–159. doi:10.1093/protein/gzq002
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Introduction Proteases constitute the largest enzyme family in humans, and they play important roles in almost all physiological functions [1–3]. Few protease-related drugs have been approved by the FDA [4], perhaps due to the lack of a powerful high-throughput screening method for the protease engineering. To solve this problem, we developed the yeast endoplasmic reticulum sequestration screening (YESS) system, which allows the simultaneous expression and co-localization of a protease and its substrate, followed by cell surface display of the substrate [5]. Co-expression is achieved by inserting the protease and substrate genes downstream of the GAL10 and GAL1 inducible promoters, which are arranged in a tail-to-tail fashion on the plasmid [6]. The N-termini of the proteins contain a signal sequence which directs the protease and
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substrate to the endoplasmic reticulum (ER) [5, 7]. At the C-termini of the proteins, the ER retention signal peptide (FEHDEL) retains the protein and its substrate within the lumen of the ER [8, 9]. By fusing the substrate with the Aga2 protein, as in preceding yeast display technologies, the substrate is trafficked to the outer membrane of the cell, where it is displayed via disulfide bonding to the lipid-anchored membrane protein Aga1 [7]. Co-expression of a protease and its substrate offers the possibility of screening both enzyme and substrate libraries. As with other display technologies, the phenotype-genotype linkage allows the researcher to recover variants that possess desired phenotypes by screening or selection followed by recovery and sequencing of the genes from the recovered clones. YESS allows each yeast cell to act as an independent vessel for a unique protease-substrate reaction, the progress of which is monitored by FACS (Fig. 1). The Aga2-substrate fusion protein contains epitope tags (e.g., FLAG, H6, or HA) on either side of the putative cleavage sequence. Reactions are probed by staining cells with fluorophore-labeled epitope tag-specific antibodies. Fluorescence from both N-terminal and C-terminal epitope tags indicates no cleavage has occurred;
Fig. 1 Overview of the Yeast Endoplasmic Reticulum Sequestration Screening (YESS) system. The cleaved or uncleaved substrates are transported to the yeast cell surface through the Aga2-Aga1 surface display system. After staining with anti-FLAG-PE and anti-6×His-FITC antibodies, cells exhibiting relatively high PE fluorescence, but little or no FITC fluorescence, are isolated through FACS, which indicates specific cleavage at only the desired substrate sequence
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N-terminal fluorescence indicates putative cleavage of the substrate; lack of fluorescence indicates promiscuous cleavage (Fig. 1). Repeated rounds of FACS are used to isolate clones with the desired catalytic properties. Through the use of selection and counter-selection, YESS can be utilized to increase proteolytic activity towards a target peptide while simultaneously reducing activity towards undesirable substrates [5]. Alternatively, substrate libraries can be screened to profile proteolytic specificity. Finally, libraries of enzymes can be screened against libraries of peptides in order to isolate novel combinations. Yi et al. performed “library-on-library” screening in order to isolate TEV protease (TEV-P) variants with novel substrate specificity [5]. The TEV-P gene was mutated with degenerate NNS codons in the S1 substrate-binding pocket. This library of TEV-P mutants was then co-transformed with variants of the TEV-P recognition sequence (ENLYFQ↓S) where the P1 position (Q) was randomized. Isolation of active protease-substrate pairs by FACS, followed by in vitro characterization, showed that two novel variants, TEV-PE10 and TEV-PH21, recognized new substrates ENLYFES and ENLYFHS with 5,000- and 1,100-fold specificity reversals, respectively, compared to TEV-P. The YESS system should be generally applicable to the directed evolution and analysis of proteases as well as other protein-modifying enzymes [5]. Hepatitis C virus protease and human Granzyme K protease displayed cleavage of their preferred substrates when expressed in the YESS system. Additionally, the catalytic domain of the human Abl1 tyrosine kinase was shown to phosphorylate a known substrate sequence fused to the C-terminus of Aga2.
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Materials All the solutions are prepared using deionized water. All reagents are stored at 4 °C unless indicated otherwise. Escherichia coli (E. coli) cells are grown at 37 °C (both plates and liquid media) unless indicated otherwise. Yeast cells are grown at 30 °C (both plates and liquid media) unless indicated otherwise.
2.1 Library Construction
1. The designed PAGE-purified primers were purchased from IDT DNA (IDT DNA, Coralville, IA, USA). 2. Phusion DNA polymerase (New England Biolabs). 3. Taq DNA polymerase (Life Technologies). 4. dNTPs (Fermentas). 5. E. coli JUDE-1(DH10B F′:: Tn10(Tetr)) competent cells, which have the Streptomycin resistance, were prepared by a previously published electrocompetent method [10].
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6. E. coli MC1061 [F− Δ(ara-leu)7697 [araD139]B/r Δ(codBlacI)3 galK16 galE15 λ− e14− mcrA0 relA1 rpsL150(strR) spoT1 mcrB1 hsdR2(r−m+)] competent cells, which have the Streptomycin resistance, were prepared by a previously published electrocompetent method [10]. 7. pTrc99A-MBP with an ampicillin marker was used as an expression plasmid for parental TEV-P and its S1 pocket saturation mutagenesis libraries [11]. 8. pMOPAC-12 with a chloramphenicol marker was used as an expression plasmid for parental TEV-P and its error-prone PCR mutagenesis libraries [12]. 9. pRK792 with an ampicillin marker was used as an expression plasmid for TEV-P and its variants. It was also used for protein expression of Maltose-binding protein (MBP)-glutathione S-transferase (GST) fusion complex [13]. 10. All restriction enzymes were purchased from NEB unless indicated otherwise. 11. Other than annotated, all chemicals were purchased from Sigma (Sigma-Aldrich, St. Louis, MO). 12. QIAprep Spin Miniprep kit (QIAGEN). 2.2 Yeast Transformation and Cell Sorting
1. The Saccharomyces cerevisiae strain, EBY100 (URA+, leu−, trp−), was a generous gift from Dr. Dane K. Wittrup (Massachusetts Institute of Technology, Cambridge, MA). 2. The pESD (Yeast Epitope tagging vector for Surface Display) plasmid (Fig. 2) was generated on the basis of the yeast surface display construct pCTCon2 (generously contributed by Dr. Dane K. Wittrup, Massachusetts Institute of Technology, Cambridge, MA) and the yeast epitope tagging vectors pESC-TRP vectors (generously contributed by Dr. Edward W. Marcotte, University of Texas at Austin). 3. YPD medium: 10 g/L Yeast extract, 20 g/L Peptone, and 20 g/L D-Glucose. 4. YNB-CAA-Glucose medium: 20 g/L glucose, 6.7 g/L yeast nitrogen base without amino acids (Thermo Fisher), 5.4 g/L Na2HPO4, 8.6 g/L NaH2PO4·H2O and 5 g/L casamino acids, pH 7.4. 5. YNB-CAA-Galactose medium: 20 g/L galactose, 6.7 g/L yeast nitrogen base without amino acids, 5.4 g/L Na2HPO4, 8.6 g/L NaH2PO4·H2O and 5 g/L casamino acids, pH 7.4. 6. Cell washing buffer A: 1× Phosphate buffered saline (PBS) buffer (Life Technologies, Carlsbad, CA, USA), containing 0.5 % bovine serum albumin (BSA) and 1 mM ethylenediaminetetraacetate (EDTA), pH 7.4.
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Fig. 2 Map of the vector pESD. (a) The pESD plasmid is constructed based on the pCTCon2 and the pESC-TRP vectors, which contains an ampicillin resistant gene, and a TRP1 gene responsible for the tryptophan biosynthesis. The pESD plasmid contains a GAL1/10 bi-directional promoter, leading to a simultaneous expression of protease and its substrates under the induction of galactose. (b) Salient features of the substrate and protease fusion proteins used in the YESS system. The protease gene is under the control of GAL1 promoter with an ER targeting signal sequence (MQLLRCFSIFSVIASVLA) and an ER retention sequence (FEHDEL) located at the N-terminal end and the C-terminal end, respectively. The ER targeting signal sequence leads the protease into the ER, and the ER retention sequence retains the protease in the ER. Under the control of GAL10 promoter, an Aga2 gene is fused with a five-part cassette which in order encodes the counter-selection substrate of protease, the FLAG tag sequence (DYKDDDDK), the designed substrate, the 6×His tag sequence (HHHHHH), and an ER retention sequence (FEHDEL). An ER targeting signal sequence is also anchored at the N-terminal end of the Aga2, facilitating the transportation of the Aga2-substrate cassette into the ER
7. Cell washing buffer B: 1× PBS, containing 0.5 % BSA, pH 7.4. 8. Cell washing buffer C: 1× PBS, pH 7.4. 9. Anti-FLAG-PE antibody (ProZyme). 10. Anti-6×His-FITC antibody (Genscript). 11. FACSAria II (BD Biosciences San Jose, CA, USA). 12. Zymoprep™ Yeast Plasmid Miniprep II kit (Zymo Research).
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2.3 TEV-P Purification and Characterization
1. The TEV-P and its variants were cloned into pRK792 vector for the protein purification. All the materials were prepared based on previously published protocol [13].
2.3.1 Purification of the TEV-P and Synthesis of Peptide Substrate
2. Substrate peptides, including TENLYFQSGTRRW, TENLYFE SGTRRW, and TENLYFHSGTRRW, are designed and purchased from Genscript. 3. KaleidaGraph software (Synergy Software).
2.3.2 Purification of the MBP-GST Fusion Complex
1. Luria Broth (LB) medium: 10 g/L Bacto-tryptone, 5 g/L yeast extract, 10 g/L NaCl, pH 7.5. 2. Cell lysis buffer: 50 mM Tris–HCl, 50 mM NaH2PO4, 200 mM NaCl, 25 mM imidazole, 10 % glycerol, pH 8.0, adding 2 mg/mL lysozyme, cOmplete EDTA-free protease inhibitor cocktail (1 tablet/50 mL, Roche, Basel, Switzerland), and 5 mM fresh DTT added immediately before use. 3. Wash buffer: 50 mM Tris–HCl, 50 mM NaH2PO4, 200 mM NaCl, 60 mM imidazole, pH 8.0, and 5 mM fresh DTT added immediately before use. 4. Elution buffer: 50 mM Tris–HCl, 50 mM NaH2PO4, 200 mM NaCl, 60 mM imidazole, pH 8.0, and 5 mM fresh DTT added immediately before use. 5. Storage buffer: 50 mM Tris–HCl, 1 mM EDTA, pH 7.5, and 5 mM fresh DTT added immediately before use. 6. Reaction buffer: 50 mM Tris–HCl, 1 mM EDTA, pH 7.5, and 2 mM fresh DTT added immediately before use. 7. Ni-NTA affinity resin (QIAGEN). 8. French Press (Thermo Scientific). 9. Thermomixer R (Eppendorf). 10. Phenomenex C18 reverse-phase column (Phenomenex). 11. Magic 2002 instrument (Micron Bioresources).
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Methods
3.1 Library Construction 3.1.1 Construction of the S1 Pocket Library of TEV-P
1. The designed primers, which contain a randomized NNS codon (N = A, T, G, or C; S = G or C) in place of the wild-type codon at T146, D148, H167, and S170, are used to amplify the TEV-P gene by splicing overlap extension PCR [14]. In addition, a KpnI restriction site and a PstI restriction site are introduced by the designed primers at the 5′ and 3′ ends of the whole PCR products, respectively. 2. The PCR products are purified and then digested with KpnI and PstI. The digested PCR products are purified through gel extraction (see Note 1).
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3. The pTrc99A-MBP vector is linearized with the same enzymes, KpnI and PstI, and also purified through gel extraction (see Note 1). 4. The purified PCR products are ligated into the linearized pTrc99A-MBP vector using T4 DNA ligase, followed by plasmid purification and desalting (see Note 2). 5. The ligation products are transformed into E. coli MC1061 competent cells, which are plated on ampicillin selective LB plates. 6. The library quality is determined, and the library DNA is extracted and saved for the following experiments (see Note 3). 3.1.2 Construction of the Error-Prone PCR Library of TEV-P
1. PAGE-purified primers are designed and purchased from IDT DNA. A SfiI restriction site is introduced by the designed primers at both the 5′ and 3′ ends of the whole PCR products. 2. The error-prone PCR is performed based on the previously published protocol using varied dNTPs concentrations [15]. 3. Both the PCR products and the pMOPAC12 plasmid are digested with SfiI at 50 °C for 6 h, and then purified through gel extraction. 4. The SfiI digested PCR products are ligated into the linearized pMOPAC12 vector using the T4 DNA ligase, followed by plasmid purification and desalting. 5. The ligation products are used to transform electrocompetent E. coli JUDE-1 cells, which are plated on chloramphenicol selective LB plates. 6. The library quality and error rate are determined, and the library DNA is extracted and saved for the following experiments (see Notes 3 and 4).
3.1.3 Incorporation of Mutagenesis Library into Yeast Construct
1. PCR amplify mutagenesis library with primers containing homology sequences of pESD plasmid for in vivo recombination. 2. PCR amplified mutagenesis library is gel purified, followed by additional product cleaning using Qiagen PCR purification kit (see Note 5). 3. The pESD plasmids are linearized with SalI and XhoI, followed by product cleaning using Qiagen PCR purification kit (see Note 5). 4. Purified mutagenesis library DNA and linearized pESD plasmids are mixed with different ratios for the following yeast cell transformation (see Note 6).
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3.2 Yeast Transformation and Cell Sorting 3.2.1 Yeast Transformation
3.2.2 Yeast Cell Sorting
1. The protease library is inserted downstream of the GAL1 promoter in pESD vector by homologous recombination following electroporation of Saccharomyces cerevisiae EBY100 (URA+, leu−, trp−) as previously described [16]. 2. If needed, the substrate library is inserted downstream of the GAL10 promoter in pESD vector using the similar strategy with the protease library incorporation (see Note 7). 1. Cells are grown to an OD600 of 2.0–3.0 in YNB-CAA-Glucose medium at 30 °C, and induced by changing to YNB-CAAGalactose medium with an initial OD600 of 0.5 (see Notes 8 and 9). 2. Following medium exchange, grow cells at 30 °C overnight, with shaking at a speed of 250 rpm. 3. For the first round of cell sorting, the total cells with the number equaling to ten times of the original library size are collected (see Note 10). 4. The collected cells are washed once with one volume of cell washing buffer A and twice with cell washing buffer B. During the washing steps, cells are kept at 4 °C. Cells are collected by centrifugation at 1,000 × g, 1 min. 5. The washed cells are then resuspended in cell washing buffer B with a density of 5 × 104 cells/μL, followed by incubation with fluorescently labeled anti-FLAG-PE antibody at a final concentration of 0.02 μg/μL (see Note 11). 6. Cells are then centrifuged at 4 °C at 1,000 × g for 1 min. The supernatant is discarded, followed by washing with cell washing buffer B (once) to completely remove the unbound antibodies. 7. The washed cells are labeled with the anti-6×His-FITC antibody at a final concentration of 0.02 μg/μL (see Note 11). 8. The antibody-labeled cells are washed twice with cell washing buffer C, and then resuspended in 1×PBS buffer for the cell sorting using FACSAria II (see Note 12). 9. The resuspended cells are analyzed using BD Biosciences FACSAria II flow cytometer. The gates are set on 575/30 nm emission filter for PE as well as 510/20 nm emission filter for FITC (see Note 13). 10. The sorting gate of the first round cell sorting is drawn to include as many target cells as possible (Fig. 3a) (see Note 14). 11. A smaller gate (Fig. 3b–e) is drawn to enrich the target cells in the following sorting rounds, which present high PE as well as low FITC signals (see Note 15).
3.2.3 Identification of Selected Mutants
1. After four rounds of cell sorting and resorting, the sorted cells are plated on the selective YNB-CAA-Glucose media plate, which lacks tryptophan.
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Fig. 3 The representative library sorting process. The cells containing the mutagenesis libraries are labeled with anti-FLAG-PE and anti-6×His-FITC antibodies. (a) Representative FACS data of the starting library; (b–e) Representative FACS data of the cells after the first, second, third, and fourth-round enrichment; (f) FACS data of the representative isolated single clone
2. After incubation at 30 °C for around 2 days, individual clones are picked up from the plates to grow in YNB-CAA-Glucose liquid medium. 3. The inoculated single clones are then induced with YNBCAA-Galactose medium, followed by staining with the antiFLAG-PE antibody and the anti-6×His-FITC antibody (see Note 16). 4. The labeled cells are re-analyzed using flow cytometry (Fig. 3f). 5. Based on the FACS results, the single yeast clones, which present the high PE signal accompanied with low FITC signals, are isolated. 6. The plasmid DNA of the isolated yeast clones is extracted and then transformed into E. coli for DNA enrichment (see Note 17). 7. The protease gene in the plasmid DNA is sequenced to gather the gene sequence information of the mutants.
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3.3 TEV-P Purification and Characterization
1. The pRK792 plasmid is used as the protein expression vector. TEV-P and selected variants were expressed and purified as previously described [13].
3.3.1 TEV-P Purification
2. All the purified enzymes were >95 % pure as determined by SDS-PAGE with Coomassie staining. 3. All the buffers contain 5 mM freshly prepared DTT to prevent TEV-P oxidation (see Note 18).
3.3.2 MBP-GST Fusion Protein Purification
To monitor the cleavage of fusion proteins by TEV-P or its variants, maltose-binding protein (MBP) and glutathione S-transferase (GST) protein are fused with an internal peptide linker containing ENLYFXS-H6, where X can be Q, H, or E. The H6 will facilitate the purification of the MBP-GST fusion complex using the Ni-NTA affinity resin. The respective fusions are designated MBP-ENLYFQSGST, MBP-ENLYFES-GST, and MBP-ENLYFHS-GST. 1. The MBP-GST fusion gene is cloned into the pRK792 vector and the protein is purified using Ni-NTA affinity resin (see Note 19). 2. The inoculated cells are grown in ampicillin selective LB media, and then induced with 1 mM IPTG when OD600 reaches 0.6. The induced cells are grown in a shaker at 20 °C for 20 h, 250 rpm. 3. The induced cells are collected by centrifugation at 8,000 × g for 15 min. Cell pellets are resuspended with cell lysis buffer (1:7 to 1: 10 ratio) in a glass beaker, adding 1 mg/mL lysozyme, 5 mM freshly prepared DTT, protease inhibitor cocktail (EDTA free, one tablet for 50 mL), 10 unit/mL DNAase. 4. The cell lysate is stirred with a stir bar for 1 h in the 4 °C cold room. The cells are further disrupted using a French Press. 5. The disrupted cell lysate is centrifuged at 30,000 × g for 1 h. The supernatant is then collected and filtered with 0.45 μm filter. 6. The supernatant is loaded into a column filled with Ni-NTA resin equilibrated with wash buffer. 7. The column is washed with at least 20-fold volume of wash buffer, and then followed by elution with elution buffer (see Note 20). 8. All the purified fusion protein should be >95 % pure as determined by SDS-PAGE with Coomassie staining.
3.3.3 TEV-P Characterization: Peptide Substrate Digestion
1. 5 μM to 6 mM of substrate peptide are incubated with 0.025–5 μM purified enzymes at 30 °C for 10–30 min. Reaction tubes were placed in a Thermomixer R (see Note 21). 2. The reactions are quenched with 0.5 % Trifluoroacetic acid (TFA) followed by freezing at −80 °C.
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3. All the enzymatic reactions are analyzed by HPLC on a Phenomenex C18 reverse-phase column using an acetonitrile gradient and a flow rate of 1 mL/min (see Notes 22 and 23). 4. The product-synthesizing rate is calculated upon the integration area at 280 nm (see Note 24). 5. The data is fitted to nonlinear regression of the MichaelisMenten equation using KaleidaGraph software. 3.3.4 TEV-P Characterization: Protein Substrate Digestion
1. The enzymatic reactions are performed the same way with the peptide substrate digestion. All the enzymatic assays were carried out in the reaction buffer using freshly purified enzyme. 2. 0.1 μg protease is mixed with 5 μg MBP-GST fusion protein substrate, which is reacted in microfuge tube on a Thermomixer R at 30 °C for 1 h. 3. The reactions are quenched with 0.5 % Trifluoroacetic acid (TFA) followed by freezing at −80 °C. 4. All the enzymatic reactions are analyzed by SDS-PAGE with Coomassie staining.
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Notes 1. To increase the efficiency of ligation and following transformation, the residual gel from the gel purification steps must be removed completely. A following DNA purification step using the Qiagen PCR purification kit can be used. 2. The ligation products need to be desalted to increase the efficiency of transformation through electroporation. 3. Library quality is evaluated by sequencing isolated plasmids. Twenty single clones are picked up from the pooled clones followed by plasmid DNA isolation using a QIAprep Spin Miniprep kit. 90 % correctness, which means 90 % of the sequenced plasmids contain the designed mutations, is required for a good library to be used for the following experiments. 4. The library error rate is calculated by the average number of mutations in 20 sequenced clones divided by the total number of residues of the native protease. 5. To enhance the transformation efficiency of the yeast cells, highly pure and concentrated DNA is preferred, with library DNA concentration above 0.5 μg/μL and the pESD plasmid concentration above 1 μg/μL. Generally, the mixed DNA volume should be less than 10 % of that of the competent cells in the electroporation cuvette.
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6. As for different mutagenesis libraries, the transformation DNA mix with varied ratios of the purified mutagenesis library against the linearized pESD plasmids should be tested to maximize the transformation efficiency. 7. The protease and the substrate genes in the pESD plasmid are under the control of the GAL1 and GAL10 promoters, respectively. The directed evolution of a protease against a substrate library, or a protease library against a substrate library, or a protease library against a specific substrate can be applied in the YESS system. The substrate library can be incorporated into the pESD vector using the similar strategy with the protease library. 8. The over-growth (OD600 higher than 3.0) of yeast cells before the induction will lower the protein expression and surface display efficiency. In addition, the initial OD600 of the induced cells should be kept lower than 1.0 but above 0.5. 9. Penicillin (final concentration of 100 units/mL) and streptomycin (final concentration of 100 μg/mL) can be added into the culture medium to avoid the bacterial contamination. 10. The total amount of cells that are collected for sorting in the first round should be ten times of the original library size to avoid the loss of the library diversity. 11. The anti-FLAG-PE antibody or the anti-6×His-FITC antibody is diluted in the cell washing buffer B for the antibody labeling. The antibody labeling process is performed at 4 °C for 15 min followed by room temp for 30 min. 12. A cell density concentration close to 5 × 106 cells/mL is preferred for loading into the FACSAria II for the cell sorting. 13. To avoid the interference of the PE signal to the FITC signal, a 510/20 nm emission filter for FITC is preferred. 14. The big sorting gate of the first round cell sorting can be drawn to be close to the major uncleaved cell population to lower the possibility of losing the target cells (Fig. 3a). 15. The total cells being analyzed and sorted for each round can be varied. It depends on the total number of the cells that are collected in the previous round. 16. 106 cells are enough for the FACS scanning for the single clones. 17. The plasmid DNA from the yeast cells is extracted using Zymoprep™ Yeast Plasmid Miniprep II kit. 18. The purified TEV-P is diluted in the storage buffer, with the concentration being kept lower than 0.5 mg/mL to avoid protein aggregation. 19. The MBP-ENLYFXS-H6-GST fusion gene is inserted into pRK792 construct, which contains an ampicillin resistant gene.
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20. The purified fusion protein is dialyzed with reaction buffer for the later enzymatic experiments. 21. All the enzymatic assays are performed using freshly purified enzyme. Kinetic assays are carried out in reaction buffer. 22. The reaction samples are centrifuged at 16,000 × g for 1 min. The supernatant is collected for the HPLC analysis. 23. The proteolysis products are confirmed using LC-MS (ESI), which was performed on a Magic 2002 instrument. 24. To minimize the product inhibitory effect, the substrate conversion percentage is controlled to be less than 5 % to approximate steady-state kinetics. References 1. Marnett AB, Craik CS (2005) Papa’s got a brand new tag: advances in identification of proteases and their substrates. Trends Biotechnol 23:59–64. doi:10.1016/j.tibtech.2004.12.010 2. Overall CM, Blobel CP (2007) In search of partners: linking extracellular proteases to substrates. Nat Rev Mol Cell Biol 8:245–257. doi:10.1038/nrm2120 3. Rawlings ND, Barrett AJ, Bateman A (2010) MEROPS: the peptidase database. Nucleic Acids Res 38:D227–D233. doi:10.1093/nar/ gkp971 4. Craik CS, Page MJ, Madison EL (2011) Proteases as therapeutics. Biochem J 435:1– 16. doi:10.1042/BJ20100965 5. Yi L, Gebhard MC, Li Q et al (2013) Engineering of TEV protease variants by yeast ER sequestration screening (YESS) of combinatorial libraries. Proc Natl Acad Sci U S A 110:7229–7234. doi:10.1073/pnas.1215994110 6. Johnston M, Davis RW (1984) Sequences that regulate the divergent GAL1-GAL10 promoter in Saccharomyces cerevisiae. Mol Cell Biol 4:1440–1448 7. Boder ET, Wittrup KD (1997) Yeast surface display for screening combinatorial polypeptide libraries. Nat Biotechnol 15:553–557. doi:10.1038/nbt0697-553 8. Hardwick KG, Lewis MJ, Semenza J et al (1990) ERD1, a yeast gene required for the retention of luminal endoplasmic reticulum proteins, affects glycoprotein processing in the Golgi apparatus. EMBO J 9:623–630 9. Pelham HR, Hardwick KG, Lewis MJ (1988) Sorting of soluble ER proteins in yeast. EMBO J 7:1757–1762
10. Makino T, Skretas G, Kang TH et al (2011) Comprehensive engineering of Escherichia coli for enhanced expression of IgG antibodies. Metab Eng 13:241–251. doi:10.1016/j. ymben.2010.11.002 11. Fisher AC, Haitjema CH, Guarino C et al (2011) Production of secretory and extracellular N-linked glycoproteins in Escherichia coli. Appl Environ Microbiol 77:871–881. doi:10.1128/AEM.01901-10 12. Jung ST, Reddy ST, Kang TH et al (2010) Aglycosylated IgG variants expressed in bacteria that selectively bind FcgammaRI potentiate tumor cell killing by monocyte-dendritic cells. Proc Natl Acad Sci U S A 107:604–609. doi:10.1073/pnas.0908590107 13. Tropea JE, Cherry S, Waugh DS (2009) Expression and purification of soluble His(6)tagged TEV protease. Methods Mol Biol 498:297–307.doi:10.1007/978-1-59745-1963_19 14. Varadarajan N, Cantor JR, Georgiou G et al (2009) Construction and flow cytometric screening of targeted enzyme libraries. Nat Protoc 4:893–901. doi:10.1038/nprot.2009.60 15. Drummond DA, Iverson BL, Georgiou G et al (2005) Why high-error-rate random mutagenesis libraries are enriched in functional and improved proteins. J Mol Biol 350:806–816. doi:10.1016/j.jmb.2005.05.023 16. Benatuil L, Perez JM, Belk J et al (2010) An improved yeast transformation method for the generation of very large human antibody libraries. Protein Eng Des Sel 23:155–159. doi:10.1093/protein/gzq002
