Overproduction of Membrane Associated, and Integrated, Proteins using Saccharomyces cerevisiae

Abstract

Structural and functional eukaryotic membrane protein research continues to grow at an increasing rate, placing greater significance on leveraging productive protein expression pipelines to feed downstream studies. Bacterial expression systems (e.g., E. coli) are often the preferred system due to their simple growth conditions, relative simplicity in experimental workflow, low overall cost per liter of cell growth, and ease of genetic manipulation. However, overproduction success of eukaryotic membrane proteins in bacterial systems is hindered by the limited native processing ability of bacterial systems for important protein folding interactions (e.g., disulfide bonds), post-translational modifications (e.g., glycosylation), and inherent disadvantages in protein trafficking and folding machinery compared to other expression systems.

In contrast, Saccharomyces cerevisiae expression systems combine positive benefits of simpler bacterial systems with those of more complex eukaryotic systems (e.g., mammalian cells). Benefits include inexpensive growth, robust DNA repair and recombination machinery, amenability to high density growths in bioreactors, efficient transformation, and robust post-translational modification machinery. These characteristics make S. cerevisiae a viable first-alternative when bacterial overproduction is insufficient. Thus, this chapter provides a framework, using methods that have proven successful in prior efforts, for overproducing membrane anchored or membrane integrated proteins in S. cerevisiae. The framework is designed to improve yields for all levels of overexpression expertise, providing optimization insights for the variety of processes involved in heterologous protein expression.

1. Introduction

Although each heterologous protein expression system provides benefits and drawbacks, Saccharomyces cerevisiae is a versatile system that combines the advantages of prokaryotic and eukaryotic systems. S. cerevisiae are eukaryotic organisms that perform post-translational modifications (e.g., acetylation, N- and O-linked glycosylation, amidation, phosphorylation, disulfide bond formation, and others), have robust DNA repair and recombination machinery, overexpress protein in a cost-efficient manner, and are generally regarded as safe (GRAS). Yet, S. cerevisiae workflows are similar to prokaryotic organisms in terms of growth rate, ease of genetic manipulation, and growth requirements. These advantages have made S. cerevisiae a prominent means for heterologous overexpression of membrane associated (MAP) and membrane integrated (MIP) proteins (1–5).

The aim of this chapter is to provide a framework that requires minimal prior experience while facilitating optimization and success, via guidance and notes, for a wide variety of MAPs/MIPs by leveraging S. cerevisiae overexpression. Thus, a generic workflow, derived from prior experience, is outlined for an “average protein”. However, it is important to note that optimizing protein yield is an empirical process and, in part, unique to each MAP/MIP. When possible, optimization strategies and notes provide guidance on specific steps. The specific topics covered in this chapter are: gene cloning, generating competent S. cerevisiae cells, transforming S. cerevisiae cells, overexpressing MAPs/MIPs in S. cerevisiae, immunodetecting and fluorescent screening to assess expression level, and optimization strategies (Fig. 1).

Figure 1: Heterologous Expression of Membrane Proteins Workflow.

Workflow proceeds from top to bottom, between sections and left to right within a section. Four of the primary events for each process are represented as a summary of techniques covered in this chapter.

2. Materials

Unless indicated otherwise, all solutions in this chapter can be prepared and stored at room temperature (RT). Ultra-purified water (ddH₂O, resistivity of ~ 18 MΩ at 25 °C) should be used in all preparations to prevent potential contamination and promote consistency. All indicated pH values are determined at RT, unless indicated. Follow institutional guidelines for waste disposal of all materials and laboratory safety protocols for transporting larger net volumes of yeast culture between locations. Unless noted, specific kits and reagents can be obtained from vendors of choice. In some instances, a specific source is noted that performs well in our workflow.

2.1. Cloning Target MIP/MAP Gene into Yeast Expression Vector

1. Desalted DNA primers for gene insertion (see Note 1).

2. Desalted DNA primers for sequencing (see Note 1).

3. Yeast expression vector. This protocol will use the previously described p83γ-series of 2μ-derived plasmid (6).

4. DNA gel loading buffer.

5. Restriction enzymes, specific to vector (see Note 2).

6. DNA Ladder.

7. T4 DNA ligase and ligation buffer. Store at −20°C (see Note 2).

8. Mixed dNTPs. Store at −20°C.

9. High-fidelity DNA polymerase kit stored according to manufacturer’s protocol. Phusion® High-Fidelity DNA Polymerase (New England BioLabs) or Advantage 2® Polymerase Mix (Takara Bio) are preferred polymerase kits used in our studies.

10. 50X TRIS acetate EDTA (TAE) buffer: 2 M TRIS-HCl, pH 8.0, 1 M acetic acid, 50 mM EDTA.

11. Ethidium bromide (EtBr) solution: 2 mg/mL in 1X TAE or ddH₂O (see Notes 3–4).

12. Ethanol: molecular biology grade.

13. Super Optimal Broth (SOB) medium: autoclave before use.

14. Selection antibiotic: specific antibiotic and final concentration depends on the plasmid being used and method of selection.

15. Luria-Bertani (LB)-antibiotic glycerol stock solution: 50% (v/v) glycerol in LB-antibiotic medium (see Note 5).

16. Agarose: molecular biology grade.

17. Escherichia coli cells: Omnimax™ cell line, or equivalent.

18. LB: prepare as broth or with agar.

19. SOB with catabolite repression (SOC) medium: autoclave before use.

20. PCR purification kit.

21. Gel extraction kit.

22. Miniprep kit.

2.2. Preparing Competent S. cerevisiae Cells

1. S. cerevisiae cells: W303.1B strain (MATa ade2–1 ura3–1 his3–11 his3–15 leu2–3 leu2–112 trp1–1 can1–100).

2. Yeast extract peptone dextrose (YPD) agar plates: autoclave YPD agar for 15 minutes. Pour YPD agar into plates. Store plates at 4 °C.

3. YPD broth: sterile filter (0.2 μm) before use.

4. YPD-glycerol broth: YPD broth fortified with 15% (v/v) glycerol (see Note 6).

2.3. Transforming Competent S. cerevisiae Cells

1. Sterile DMSO: molecular biology grade.

2. PEG 3350 lithium acetate TRIS-EDTA (PLATE) solution: 40% (w/v) PEG 3350, 0.1 M lithium acetate, 10 mM TRIS-HCl, pH 7.0, 1 mM EDTA. Prepared with ddH₂O and sterile filtered (0.2 μm) before use (see Note 7)

3. Complete supplement mixture-His (CSM-His) plates: 0.77 g/L CSM-His, 20 g/L low melting agar,10 g/L glucose, 3.0 g/L ammonium sulfate, 1.7 g/L yeast nitrogen base (without amino acids or ammonium sulfate). Prepared with ddH₂O. Autoclave for 15 minutes, then pour plates (~20 plates per 500 ml).

4. TRIS-EDTA (TE) buffer: 10 mM TRIS-HCl, pH 9.0, 150 mM EDTA.

5. Sheared salmon sperm DNA: 10 mg/ml. Store at −20 °C.

2.4. S. cerevisiae Growth and Protein Expression

1. 10X raffinose solution: 10% (w/v) raffinose in pre-autoclaved RT ddH₂O. Sterile filter (0.2 μm) before use.

2. 20X amino acid free yeast nitrogen base (YNB) solution: 13.4% (w/v) yeast nitrogen base (no amino acids) in ddH₂O. Sterile filter (0.2 μm) before use. Store at 4 °C.

3. 20X galactose solution: 40% (w/v) galactose in ddH₂O. Autoclave ddH₂O and cool to RT prior to adding galactose. Sterile filter (0.45 μm) after galactose is added to RT ddH₂O (see Note 8).

4. Glucose solution: 40% (w/v) glucose in ddH₂O. Autoclave for 15 minutes prior to use.

5. 4X yeast extract peptone galactose (YPG) solution: yeast extract 8%, peptone 16%, and galactose 8% (w/v for all). Heat ddH₂O to dissolve extract and peptone. Autoclave mixture and let cool before adding galactose from the 20X stock. Mix.

6. Resuspension buffer: 50 mM TRIS-HCl, pH 7.5, 10% (v/v) glycerol, 150 mM NaCl, 10 mM EDTA, pH 8.0. Store at 4 °C.

7. Antifoam 204 (see Note 9).

8. 10X CSM-His media: 7.9 g/L in warmed ddH₂O. Sterile filter (0.2 μm) before use. Store at 4 °C.

9. Synthetic complete without histidine (SC-HIS) media: 37.5 ml 10X CSM-His, 10% (w/v) raffinose, 18.8 ml 20X YNB, and 9.4 ml 40% (w/v) glucose

2.5. Protein Expression Validation Via Immunodetection and Fluorescent Screening

1. Coomassie protein stain.

2. Antibody conjugated to fluorescent probe: specific antibody determined by target protein.

3. 30% (w/v) Acrylamide/bis-acrylamide: 29:1 acrylamide:bis-acrylamide. Store at 4 °C.

4. 4X Laemmli sample buffer: 200 mM TRIS-HCl, pH 6.8, 8% (w/v) SDS, 40% (v/v) glycerol, 50 mM EDTA, 0.08% (w/v) bromophenol blue. Mix thoroughly and aliquot 960 μl into tubes. Store at −20 °C. Add 40 μl β-mercaptoethanol to aliquots before use.

5. Ammonium persulfate solution: 10% ammonium persulfate (w/v) in ddH₂O. Store at −20 °C.

6. Protein standards (e.g., Precision Plus Protein Kaleidoscop from Bio-Rad).

7. N,N,N,N′-tetramethyl-ethylenediamine (TEMED).

8. Blotting paper: thin and thick variants.153

9. Western blot transfer buffer: 25 mM TRIS-HCl, pH 8.3, 192 mM glycine, 20% (v/v) ethanol.

10. Stacking gel buffer: 0.5 M Tris-HCl, pH 6.8.

11. Resolving gel buffer: 1.5 M Tris-HCl, pH 8.8.

12. 10X TRIS buffered saline (TBS): 1.5 M NaCl, 0.1 M TRIS-HCl, pH 7.4.

13. 2X solubilization buffer: 100 mM TRIS-HCl, pH 7.4, 800 mM NaCl, 10% (v/v) glycerol, add detergent specific for POI (see Note 10). Store at 4 °C.

14. 1X TBS with tween (TBST): add 0.05% (v/v) Tween-20 to TBS.

15. SDS-PAGE running buffer: 25 mM TRIS-HCl, pH 8.3, 0.192 M, glycine, 0.1% (w/v) SDS.

16. Wash buffer: 1x TBS.

17. Blocking solution: 5% (w/v) milk in TBST.

18. 0.2 μm PVDF membrane.

2.5. Required Equipment

Purchasing the large pieces of equipment below for exclusive overexpression in yeast cells is not required. If equivalent equipment is already used for bacterial expression, it can accommodate yeast overexpression as well, if proper protocols are in place to prevent cross-contamination. However, it is strongly encouraged to keep flasks and other small non-disposable materials specific to yeast or bacterial expression systems.

1. Polyacrylamide gel electrophoresis chamber and power supply.

2. Polyacrylamide gel casting chamber.

3. Agarose electrophoresis chamber and power supply.

4. Thermocycler.

5. Water bath.

6. Semi-dry electrophoretic transfer cell.

7. Fluorescent microplate reader.

8. Bead beater or cell disrupter such as an Avestin Emulsiflex®.

9. 0.5 mm glass beads (if using a bead beater).

10. Optical bottom 96 well plates.

11. Centrifuges and centrifuge tubes.

12. Fermentor/bioreactor (if pursuing fermentation).

13. UV transilluminator or gel imager.

14. Incubator shakers.

3. Methods

Although this chapter’s emphasis is on MAP/MIP expression, S. cerevisiae is also extensively used to produce eukaryotic cytosolic and secretory proteins that do not produce well in bacterial systems. For example, proteins requiring post-translational modifications for function, proteins with low solubility in bacteria, or proteins that fold incorrectly in bacterial systems (7). Recent findings that S. cerevisiae produces prokaryotic MIPs more efficiently than prokaryotes further underscores the system’s versatility (8).

However, alternative yeast expression systems are also available for MAP/MIP production, including Kluyveromyces lactis and Pichia pastoris (reclassified to Komagataella phaffii). K. lactis is prevalent in the food industry largely because of its ability to upscale efficiently and its GRAS FDA status. K. lactis produces heterologous proteins from a wide range of organisms and is a Crabtree-negative yeast, meaning it does not produce ethanol during respiratory conditions. Since K. lactis does not lose carbon to ethanol production, its biomass formation and recombinant protein titer are generally higher than S. cerevisiae titers. K. lactis also has lower hyperglycosylation events than S. cerevisiae, making proteins generated in K. lactis less immunogenic than those made in S. cerevisiae. K. phaffii is like K. lactis in that it also exhibits lower glycosylation than S. cerevisiae and is Crabtree-negative. Of these three yeasts, K. phaffii is the only methylotrophic yeast. Methylotrophic yeasts require specialized equipment to handle but can grow with methanol as a carbon source. Both K. phaffii and K. lactis are associated with high yields of secretory and cytosolic proteins. However, these yeast systems suffer from decreased access to vectors and other genetic tools, making S. cerevisiae the current predominant yeast for heterologous overexpression in research settings. Still, if protein expression fails in S. cerevisiae, K. lactis and K. phaffii are viable alternative eukaryotic expression systems to consider. For a more detailed comparison between these systems, please refer to the recent review (9).

3.1. Cloning Target Gene(s) Into Yeast Expression Vector(s)

Inserting a gene of interest (GOI) into a plasmid can be done in a variety of ways. Popular approaches, not discussed in this chapter, include ligase-independent and GAP repair cloning, which are suited to high throughput screening methodologies. Focused expression of a single MAP/MIP is amenable to a traditional restriction cloning approach and will be the method of choice for this chapter’s gene cloning. If standard restriction digest cloning does not meet the needs of a specific project, alternative gene cloning methods should be explored (10–12). For this chapter, the “p83γ“ vector (Fig. 2), designed by our lab, will serve as the backbone plasmid, or shuttle vector. This plasmid was selected because it can propagate in yeast or bacterial lines and has favorable characteristics for protein overexpression including an N-terminal 3C protease cleavable maltose binding protein (MBP) tag, C-terminal 8X histidine tag, HIS3 selection marker, and expression is driven by a galactose inducible promoter.

Figure 2: p83γ Protein Expression Plasmid.

The p83γ plasmid is a chimeric shuttle vector derived from pRS423-Gal1 modified to contain an N-terminal, 3C cleavable, maltose binding protein tag with a C-terminal, non-cleavable, 8X polyhistidine tag. This plasmid contains the 2μ origin of replication, HIS3 selection marker, ampicillin resistance marker to facilitate bacterial cloning, and the strong GAL1 promoter with Cyc terminator.

After vector selection, DNA primers for gene insertion must be designed to match restriction sites present in the plasmid’s multiple cloning site (MCS). For this example, restriction sites that match the MCS, and are not present in the GOI, correspond with the XmaI and XhoI restriction enzymes at the N- and C-termini, respectively (see Note 11). Primers should be designed with a section matching the GOI, a restriction site, and an overhang region of 5–6 base pairs. The overhang region increases digestion efficiency (13). To ensure successful termination, add two stop codons to the primer immediately following the last codon of the GOI (see Notes 12–13).

1. Prepare 50 µl PCR reactions using a high-fidelity DNA polymerase (e.g. Q5 or Phusion® polymerases) in a tube that fits the thermocycler (see Notes 14–15). Components for each reaction should be added to the tube in the following order (or match instructions provided with the polymerase kit of choice):

   • ddH₂O: 36.5 μl.
   • 5X HF buffer: 10 μl.
   • 10 mM dNTP solution mix: 1.0 μl.
   • 100 μM forward primer: 0.5 μl.
   • 100 μM reverse primer: 0.5 μl.
   • Template DNA: 1.0 μl (see Note 16).
   • High-fidelity polymerase: 0.5 μl.

2. Gently mix with a pipette before securely placing reaction in a thermocycler.

3. Carefully close thermocycler lids. Ensure that lids do not compromise the reaction tube’s seal or physical integrity, then program and run the following protocol (or related protocol specific to polymerase and GOI size being used).

   1. Denature at 98 °C for 30 seconds (initial denaturation).
   2. Denature at 98 °C for 10 seconds (cyclic denaturation).
   3. Anneal primers at 3 °C above melting temperature of the lowest melting primer.
   4. DNA extension at 72 °C (30 seconds per 1000 amplicon bases).
   5. Repeat steps b-d for a total of 35 cycles.
   6. Final extension at 72 °C for 10 minutes.
   7. Hold at 4 °C until convenient for PCR analysis.

4. Analyze PCR products via gel electrophoresis.

5. Prepare an electrophoresis sample by mixing 4 μl of PCR reaction, 1 μl of 10X loading dye, and 5 μl ddH₂O.

6. Prepare an agarose gel (0.7% w/v) using 1X TAE buffer, adding 5 μl of stock EtBr (2 mg/mL) to the heated gel solution (see Note 3).

7. Run gel at 100V until the gel loading dye front has migrated approximately ¾ the gel’s length. Include an appropriately sized DNA ladder to verify whether amplicons match expected sizes. If they match, proceed to step 8 (see Note 17).

8. Purify PCR products with a PCR clean-up kit, using manufacturer’s protocol (general PCR clean-up protocol follows):

   1. Gently mix PCR products with 5X binding buffer.
   2. Load onto a DNA binding column held in a collecting tube.
   3. Spin tube to bind DNA and remove buffer.
   4. Discard flow-through.
   5. Wash DNA binding column with wash buffer (~750 μl).
   6. Spin and discard flow-through.
   7. Repeat steps e-f.
   8. Perform a final spin to remove residual buffer.
   9. Move DNA column into a clean nuclease free collection tube.
   10. Add elution buffer (10–30 μl) to column (see Note 18).
   11. Incubate at RT for 2 minutes.
   12. Spin down elution buffer, collecting purified DNA in flow-through.
9. After purification, PCR products must be digested with restriction enzymes (see Notes 15, 19). For efficiency, vector and PCR products should be digested concurrently in separate tubes. A protocol, using NEB enzymes, is below:

   1. In separate tubes, add either 25 μl purified PCR product or 25 μl vector, at 40 ng/μl, leave 5 μl of each undigested to serve as controls in step 14.
   2. Add to each tube:

      • ddH₂O: 18 μl.
      • 10X NEB CutSmart® Buffer: 5 μl.
      • Restriction enzyme A, corresponding to forward primer amplification: 1 μl.
      • Restriction enzyme B, corresponding to reverse primer amplification: 1 μl.
   3. Mix thoroughly and incubate for 2 hours at 37°C.
10. Once digestion is finished, DNA must be gel-purified (see Note 20).

    1. Prepare samples by mixing 6 μl of 10X loading dye into each digestion reaction.
    2. Prepare vector and purified PCR product controls by mixing 2 μl of 10X loading dye and 13 μl ddH₂O with 5 μl of undigested vector or purified PCR product.
    3. Run samples on an agarose gel (0.7% w/v), with a DNA ladder, as before.
    4. Visualize DNA using a UV transilluminator (see Note 21).
    5. Determine which bands have been digested by comparison to undigested controls and excise bands of interest with a clean blade (see Note 22–23).
    6. Determine mass of each gel slice containing a band of interest.
    7. Utilize a gel extraction kit to purify isolated bands, according to manufacturer’s protocol. General protocol below:
    8. Add 100 μl of gel dissolving buffer for every 100 mg of agarose.
    9. Ensure lids are tightly closed and dissolve agarose at 50°C until completely dissolved (~10 minutes).
    10. Once dissolved, add one volume of pure isopropanol to each tube, and gently invert several times.
    11. Add 750 μl of DNA-containing solution to a DNA binding column and spin, discarding flow-through. Repeat until all DNA is bound to column.
    12. Wash bound DNA with 500 μl of agarose dissolving buffer to remove residual agarose, spin, and discard flow-through.
    13. Wash DNA binding column with wash buffer (~750 μl).
    14. Spin and discard flow-through.
    15. Repeat steps m-n steps once more.
    16. Spin again to remove residual wash buffer.
    17. Move DNA column into a clean nuclease free collection tube.
    18. Add 10–30 μl elution buffer to column (see Note 18).
    19. Incubate at RT for 2 minutes.
    20. Spin down elution buffer collecting purified DNA in flow-through.
11. Once digested PCR products and vectors are purified, PCR products must be inserted into vectors via a ligase reaction (see Note 15). Successful ligation reactions require complete digestion, from both restriction enzymes, of PCR products and vectors. The ratio of purified PCR product to purified vector is also important. At low ratios, vectors may self-ligate, but at a 10-fold molar excess, most vectors do not self-ligate (see Note 24).

    1. Calculate the amounts of PCR product and vector needed for ligation:

       $$\begin{array}{l}PCR product mass \left(ng\right)\\ =target ratio*\frac{PCR product length \left(base pairs\right)}{Vector length \left(base pairs\right)}*Vector mass \left(ng\right)\end{array}$$
    2. Set aside 5 μl of digested vector for a ligation control that will be analyzed later.
    3. After amounts of PCR product and vector have been determined, prepare a 20 μl ligation reaction in a microcentrifuge tube by adding the following reagents, in order:

       • ddH₂O: 17 – (vector volume + PCR product volume) μl.
       • Purified vector solution: volume dependent on above equation.
       • Purified PCR product solution: volume dependent on above equation.
       • 10X ligation buffer: 2 μl.
       • DNA ligase: 1 μl.
    4. Perform overnight ligation reaction at RT.
    5. Heat inactivate ligase at 65 °C for 10 minutes.
    6. Store at 4°C, until needed.
12. After ligation is complete, ligated vector can be used to transform competent E. coli cells. Transformation is performed as follows:

    1. Place a tube of competent E. coli cells from a −80°C freezer on ice (see Note 25).
    2. Begin incubating selective LB-agar plates at RT (see Note 26).
    3. As competent cells thaw, make three 200 μl aliquots in separate tubes.
    4. To each tube add either 5 μl of ligation reaction (experimental condition), 5 μl of purified digested vector (negative control), or 5 μl of original vector (positive control) (see Note 27).
    5. Mix DNA throughout tubes by flicking tubes.
    6. Incubate tubes on ice for 10–30 minutes.
    7. Heat-shock cells by placing them in a water bath (42 °C) for exactly 30 seconds.
    8. Immediately place tubes back on ice for 2 minutes.
    9. Pre-heat SOC medium (815 μl per transformation) in a water bath.
    10. Add 800 μl of pre-warmed (42 °C) SOC medium to each transformation tube.
    11. Horizontally incubate tubes on a shaking incubator (250 RPM) for 1 hour at 37 °C.
    12. Add 15 μl of pre-warmed SOC medium to RT plates.
    13. Place 25 μl of transformed cells into 15 μl of SOC media on agar plates.
    14. Evenly distribute cells using sterilized beads or a cell spreader.
    15. Incubate plates overnight at 37 °C.
    16. Compare control and experimental plates (see Note 28).
13. After successful transformation, vector containing GOI should be expanded and isolated to make stocks for future use. Common ways to preserve the new vector are to make glycerol or vector stocks.

    1. Fill two cell culture tubes with 5 ml of LB-antibiotic medium. Inoculate both tubes with half of a single E. Coli colony, using good sterile technique.
    2. Incubate cells overnight in a shaking incubator at 37 °C and 250 RPM.
    3. Pellet cells by centrifuging at 3,000 x g and 4 °C for 10 minutes.
    4. Resuspend one tube using 2 mL of LB-antibiotic glycerol stock solution and make 250 µl aliquots of the suspension.
    5. Snap freeze aliquots and immediately store at −80 °C. This glycerol stock, containing the GOI, is stable for years at −80 °C.
    6. With the second culture tube, isolate vector via a miniprep kit, according to manufacturer-supplied protocol. General protocol below:
    7. Use 250 μl of resuspension buffer to resuspend cell pellet and transfer to a microcentrifuge tube.
    8. Add 250 μl of lysis buffer and mix by inversion to release DNA from cells.
    9. Add 350 μl of neutralization buffer and mix by inversion.
    10. Perform a 10-minute high-speed spin to pellet cellular debris.
    11. Transfer supernatant to a DNA binding column held in a collecting tube. Centrifuge and discard flow-through.
    12. Add 500 μl of DNA binding buffer to remove other materials that may be bound to the column, spin the column, and discard flow-through.
    13. Wash DNA binding column with wash buffer (~750 μl).
    14. Spin tube again and discard flow-through.
    15. Repeat steps n-m once more.
    16. Perform a final spin to remove residual buffer.
    17. Move DNA column into a clean nuclease free collection tube.
    18. Add elution buffer (10–30 μl) to column (see Note 18).
    19. Incubate at RT for 2 minutes.
    20. Spin down elution buffer, collecting purified DNA in flow-through.
    21. Store purified vector at −20 °C.
14. Successfully purified vectors must be verified for proper gene insertion before transforming S. cerevisiae cells for protein overexpression. Verification is done in a variety of ways but, in general, a screen preceding a set of sequencing reactions is a robust and efficient combination for vector verification. That process is outlined below:

    1. Perform a double restriction digest on vectors expected to contain GOI.
    2. Screen vectors by running an agarose gel with digested vector, original vector (undigested, pre-ligation), purified vector (with suspected GOI insertion), GOI amplicon from first PCR reaction, and a DNA ladder (see Note 29).
    3. After screening positively, send isolated vectors and sequencing primers specific for vector backbone to be sequenced (see Notes 30–31).
    4. Keep agar plates containing colonies that were transformed with ligated vector in case a mutation or incomplete GOI insertion has occurred. If sequencing results show an error, remaining colonies may have correct GOI insert.
    5. If sequencing analysis indicates that the GOI has been correctly inserted into the backbone, the glycerol stock set in the −80 °C should be marked as a sequence confirmed stock. Otherwise, it should be discarded.
    6. Glycerol stocks can generate extra vector, when needed. It is also important to keep a purified vector stock in case glycerol stocks become contaminated.

3.2. Generating Competent S. cerevisiae Cells

Awareness of mutations that alter the functional state of S. cerevisiae has recently increased (14,15). Companies selling S. cerevisiae for protein overexpression have quality controls in place to ensure that customers receive cells with high expression capabilities. However, if expression levels are consistently low, it may be beneficial to replace cell stocks. Generating competent cells requires vigilant sterile technique because they are prepared without strong selective pressure, increasing the risk of contamination. Labs that routinely work with both E. coli and yeast are particularly susceptible to contamination. If attentive to sterility, producing competent S. cerevisiae cells is a straightforward process. Despite the relative ease of preparing competent yeast cells, poor preparations can have a strong effect on transformation efficiencies and overexpression. A common error that lowers cell survival and transformation efficiencies is harsh preparation conditions of glycerol stocks. Yeast cells undergo less damage and cell death when slowly frozen in cryoprotectants, relative to being quickly frozen in liquid N2, or placed directly in a −80 °C freezer (16).

1. Prepare a sterile tube containing 10 mL of sterile YPD broth and inoculate it with cells of desired strain for protein expression (e.g., W303.1B cells).

2. Perform an overnight growth in a shaking incubator at 220 RPM and 30 °C.

3. Quadrant streak a YPD plate with cells from the growth.

4. Incubate for 48 hours at 30 °C.

5. Prepare three sterile aerated culture tubes with 5 mL of sterile YPD broth in each. Inoculate each tube with a single colony from plates in step 3 (see Notes 32–33).

6. Incubate culture tubes for 24 hours in a shaking incubator at 220 RPM and 30 °C.

7. Centrifuge growths for 10 minutes at 3,000 x g.

8. Discard supernatants and resuspend each pellet with 5 ml of 15% (v/v) glycerol-YPD broth.

9. Make 500 μl aliquots of resuspended cells in cryovials until all cells have been used.

10. Slowly freeze cells by placing cryovials in an insulated cryobox containing Styrofoam peanuts or cardboard shreds, then place it in a −80 °C freezer.

3.3. Transforming S. cerevisiae Cells with Expression Plasmid

Common transformation errors affecting downstream applications are contamination (unintentional genetic material or other microorganisms), harsh storage conditions, or buffer pH drift. Other frequent errors include transforming cells outside of log phase or performing a heat shock for an insufficient duration. To prevent lowered transformation efficiencies, ensure good sterile technique, thorough cleaning and autoclaving of equipment between uses, gentle handling and storage of competent cells, check pH of PLATE solutions, and monitor heat shock conditions.

1. Thaw one cryovial containing competent S. cerevisiae cells on ice.

2. Centrifuge cells at 3,000 x g and 4 °C for 10 minutes.

3. Resuspend pellet using 150 μl of PLATE solution (see Note 34).

4. Add 20 ng of vector DNA per every 1000 vector nucleotides (i.e., a 5kb vector requires ~100 ng of vector) and 5 μl of sheared salmon sperm (10 mg/ml) to suspension (see Note 35).

5. Mix well, either by repeated inversion or gentle vortexing.

6. Add 10 μl of DMSO and mix again.

7. Perform a 15-minute RT incubation.

8. Heat shock cells for 20 minutes at 42 °C.

9. Use a microcentrifuge to pellet cells and decant supernatant.

10. Gently resuspend pellet with 200 μl of TE buffer.

11. Plate 50 μl of suspension on one CSM-His plate and remaining 150 μl on another.

12. Incubate plates for 2 days at 30 °C.

3.4. Overexpressing MAPs/MIPs in S. cerevisiae Cells

Protein overexpression in yeast cells should remain consistent between batches that follow the same protocol, with protein yields increasing after optimization. If expression levels become inconsistent, improper preparation of materials for that growth is a potential issue. For example, overheating galactose or raffinose solutions causes isomerization and results in decreased protein expression.

1. Prior to starting a large growth, a preculture outgrowth should be performed. Preculture outgrowths aid in time efficiency and protein yield.

   1. Inoculate a culture tube containing 5 ml SC-His media with a single colony from the transformation selection plate.
   2. Incubate culture tube in a shaking incubator for 4 hours at 220 RPM and 30 °C.
   3. Autoclave a 1 L baffled flask containing 270 mL ddH₂O and let it cool to RT.
   4. Add 18.8 ml of sterile 20X YNB solution, 37.5 ml 10X CSM-His media, and 18.8 ml 40% (w/v) glucose to the RT flask.
   5. Inoculate with 5 ml starter culture from step 1a.
   6. Incubate in a shaking incubator at 220 RPM and 30 °C for 24 hours.
2. To maximize protein yields, smaller preculture growths must be scaled up.

   1. Autoclave 15 – 1 L baffled flasks containing 270 mL ddH₂O and let cool to RT.
   2. Add 18.8 ml of sterile 20X YNB solution, 37.5 ml 10 X CSM-His media, 18.8 ml 40% (w/v) glucose, and 37.5 ml 10% (w/v) raffinose to the RT flask (see Note 36).
   3. Inoculate each flask with 10 ml of culture from step 1f.
   4. Incubate flasks in a shaking incubator at 220 RPM and 30 °C for 24 hours.
   5. After 24 hours of growth, 600 nm culture optical densities should fall within a range of 15–20 and have glucose concentrations below 0.1% (indicated by pink coloration in ade2–1 S. cerevisiae mutants). If these conditions are not met, continue growth until they are (see Note 37).

3. Once optical densities and glucose conditions are met, induce overexpression in each flask by adding 125 ml of 4X YPG medium to each flask. Continue incubating at 220 RPM and 30 °C for 16 hours.

4. Alternatively, cultures can be fermented in a bioreactor (steps 4–5).

   1. Prepare fermentor for culture by performing the following steps (specific to a 10L working volume):

      • Add 500 μl of antifoam 204 and 5.1 L of ddH₂O to fermentor.
      • Calibrate pH probes with pH standards (pH 4.0 and pH 7.0).
      • Clean dissolved oxygen (DO) probe and replace its electrolyte solution.
   2. Prepare fermentor vessel for autoclaving.

      • Disconnect tubing, shut clamps, and place protective caps as needed.
      • Position sterile lines in 50 ml vials and seal with foil.
      • Wrap all non-sterile connections with foil.
   3. Autoclave for 1 hour (see Note 38).
   4. Cool to RT by incubating at RT or attaching to a cooling unit.
   5. Add sterile components to fermentation vessel: 350 ml of 20X YNB solution, 700 ml of 10X CSM-His media, 175 ml of 40% (w/v) glucose, and 700 ml of 10% (w/v) raffinose.
   6. Calibrate DO to 0%.
   7. Set temperature to 30 °C
   8. Set agitation rate from 200 to 350 RPM, scaling with a DO reading of 20–90%.
   9. Allow air to flow at 2.5 L/minute.
   10. After reaching these settings, calibrate DO to 100% (see Note 39).
   11. Inoculate fermentation vessel as described above (step 1, A-F)
   12. Grow S. cerevisiae for 24 hours.
5. Induce culture by adding 2.5 L of 4X YPG media to fermentation vessel.

   1. Set air flow rate to 5 L/minute to compensate for decreases in DO that occur by adding non-equilibrated media to vessel (see Note 40).
   2. Maintain other settings as before and continue incubating for 16 hours.

6. After incubating for 16 hours, in either shaker incubators (steps 1–3) or in a fermentor (steps 4–5), cells can be harvested and prepared for protein analysis.

7. Centrifuge cells in pre-weighed containers at 3,000 x g and 4 °C for 10 minutes.

8. Weigh pellets in their containers.

9. Resuspend pellets using 60 ml of resuspension buffer per 80 g of pellet mass.

10. Lyse cells immediately or store in a −20 °C freezer until needed.

3.5. Protein Preparation, Immunodetection, and Fluorescent Screening

A critical step before performing experiments is to verify that overexpressed protein is the MAP/MIP being pursued. SDS-PAGE followed by immunoblotting with a protein-specific antibody are simple and common methods for verifying protein identity. These may require optimization for a specific MAP/MIP (e.g., SDS-PAGE resolving gel percentage). Most potential complications are avoidable minor issues (e.g., bubbles affecting transfer or readouts).

1. If frozen cells are used, begin thawing them on ice before proceeding.

2. Prepare 12% (w/v) SDS-PAGE gels (make 4):

   1. Mix and pour resolving gel, using the following components, in order:

      • ddH₂O: 6.6 ml.
      • 30% (w/v) acrylamide mix: 8 ml (see Note 41).
      • 1.5 M Tris-HCln pH 8.8: 5 ml.
      • 10% (w/v) SDS: 0.2 ml.
      • 10% (w/v) ammonium persulfate: 0.2 ml.
      • TEMED: 8 µl.
   2. Add water on top of gel to prevent dehydration.
   3. Allow resolving gel to set.
   4. Decant water on top of resolving gel.
   5. Mix and pour stacking gel, using the following components (in order):

      • ddH₂O: 3.4 ml.
      • 30% acrylamide mix: 0.83 ml.
      • 1.5 M Tris-HCl, pH 8.8: 0.63 ml.
      • 10% (w/v) SDS: 50 µl.
      • 10% (w/v) ammonium persulfate: 50 µl.
      • TEMED: 8 µl.
   6. Carefully place comb into gel to avoid trapping bubbles in wells.
   7. After gels set, they can be stored at 4 °C, until needed.
3. Lyse cells with a bead beater or Emulsiflex (C3 or C5).

   1. Bead beater settings: maximum speed for 5 cycles of 1 minute beating followed by a 1-minute pause. Fill approximately half the canister with cells, then add 0.5 mm pre-chilled glass beads until full.
   2. Emulsiflex settings: 28,000 PSI for 3 passes.

4. Centrifuge lysed cells for 1 hour at 7,500 x g and 4 °C. Collect supernatant, setting aside a 30 µl sample for a gel (see Note 42).

5. Resuspend pellets using volume of ddH₂O that was present before centrifugation. Save a 30 µl sample to run on an SDS-PAGE gel.

6. Centrifuge supernatant from step 5 at 101,000 x g at 4 °C for 1 hour. Membranes and MAPs/MIPs will be present in pellet produced by this spin.

7. Decant new supernatant. Save a 30 µl supernatant sample to run on an SDS-PAGE gel.

8. Prepare membrane pellets for storage. Using a stir bar at moderate speeds, resuspend membrane pellets with 16 ml of solubilization buffer per gram of pellet (see Notes 43–44). Resuspension may require several hours.

9. Store resuspended membrane pellets at −20 °C.

10. Prepare samples for SDS-PAGE.

    1. Normalize sample volumes with resuspension buffer before loading (see Note 45).
    2. Add 10 µl of 4X Laemmli buffer to 30 µl of normalized samples (see Note 46).
    3. If screening a single construct, prepare SDS-PAGE gel lanes by loading 20 µl samples as follows:
       Lane 1: protein standard (5 µl).
       Lane 2–5: samples from cell lysate, resuspended cell pellet, high-speed spin supernatant, and solubilized membrane pellet, respectively.
       Lanes 6–10: repeat as above.
    4. If screening multiple constructs or conditions, run 2 identical gels per construct or condition and prepare SDS-PAGE gels by loading 20 µl samples as follows:
       Lane 1: protein standard (5 µl).
       Lane 2–5: samples from the first set of conditions or first construct’s cell lysate, resuspended cell pellet, high-speed spin supernatant, and solubilized membrane pellet, respectively.
       Lane 6: protein standard (5 µl).
       Lane 7–10: samples from the second set of conditions or second construct’s cell lysate, resuspended cell pellet, high-speed spin supernatant, and solubilized membrane pellet, respectively.
    5. Perform electrophoresis at 140 V for 1 hour or until dye front indicates adequate separation for target MAPs/MIPs.
11. Prepare to transfer protein to a PVDF membrane.

    1. Cut 2 pieces of thin blot papers, thick blot papers, and a single PVDF membrane for each gel, matching filter paper and membrane sizes to gels.
    2. Wet filter paper in 1X transfer buffer.
    3. Quickly submerge membrane in pure methanol to activate.
    4. Rinse in 1X transfer buffer prior to transferring (see Note 47).
    5. Place transfer components on semi-dry transfer apparatus in the following order: thick blot paper, thin blot paper, membrane, SDS-PAGE gel, thin blot paper, thick blot paper (see Note 48).
    6. If one construct is being analyzed only use half of gel (lanes 1–5 or lanes 6–10) during transfer (see Note 49).
    7. If two constructs or sets of conditions are being analyzed, only transfer one of the identical gels (see Note 49).

12. Transfer proteins to membrane at 0.33 amps and 25 V for 30 minutes, per gel.

13. Coomassie stain identically prepared gels to visualize proteins present in samples.

    1. Transfer gels to separate containers.
    2. Use enough staining solution to cover gels (~20–25 ml).
    3. Incubate on a rocker until desired intensity is achieved (~15–20 minutes).
    4. Gels can be destained to improve imaging by exchanging stain for ddH₂O.
    5. After destaining, image gels and store in fresh ddH₂O at 4 °C.
    6. If gels indicate protein degradation (unexpected bands of decreasing size), protease inhibitors can be added to all steps involving protein extraction and isolation.
14. Perform immunodetection on transferred proteins.

    1. Make the 5% (w/v) milk in 1X TBST blocking buffer (see Note 50).
    2. Transfer PVDF membranes to separate containers with blocking buffer. Prevent dehydration or antibody binding can be altered.
    3. Block for 1 hour at RT on a rocker. If background noise is obfuscating results, consider blocking overnight at 4 °C on a rocker to increase signal.
    4. Prepare antibody, according to manufacturer’s instructions (see Note 51).
    5. Place enough antibody working solution in separate containers to prevent dehydration during incubation.
    6. Transfer membranes to their corresponding containers and incubate at RT for 1 hour on a rocker (see Note 52).
    7. Wash 3 times with TBS buffer, for 15 minutes each, on a rocker at RT (see Note 53).
    8. Image and analyze membranes in comparison to Coomassie stained gels, noting expression levels (indicated by fluorescence intensity and blot size), approximate mass, and localization pattern of target MAPs/MIPs.
15. Optional. A screening protocol that can determine overexpression potential of a specific vector, if it is expressing target MAPs/MIPs as fluorescent fusion proteins, has been described (1). This technique is performed as follows:

    1. Culture S. cerevisiae cells as described above (section 3.8, steps 1–3 or steps 4–5). Do not induce protein expression in one of the cultures. This culture is used to measure background fluorescence generated by vector.
    2. Collect 10 ml from a culture that was induced and 10 ml from a culture that was not.
    3. Centrifuge for 10 minutes at 3,000 x g and 4 °C.
    4. Resuspend pellets in 200 µl of resuspension buffer.
    5. Place 200 µl from each suspension into a 96-well black Nunc optical bottom plate.
    6. Read fluorescence of each suspension and from a fluorescent fusion protein standard (GFP in this example) with a known concentration. (see Notes 54–56).
    7. Estimate MAP/MIP concentrations using the observed fluorescence values: Use the following equation to estimate protein concentration (mg/ml) in whole cells (F. = fluorescence):

       $$\frac{\text{F}.\text{ of induced cells}-\text{F}.\text{ of noninduced cells}}{\text{F}.\text{ of the standard}}*\left[\text{standard}\right]=\left[\text{whole cell GFP}\right]$$
    8. This can predict mg/ml of cell culture by dividing the whole cell concentration by 40 (8 ml cell culture/0.2 ml resuspension volume) (see Note 57).
    9. The recorded value for percent fluorescence due to GFP in S. cerevisiae membranes, when overexpressing MAPs/MIPs is 60%. Therefore, the value from step h must be multiplied by 0.6. The result is the concentration (mg/ml) of fusion proteins in cell membranes.
    10. Final MAP/MIP concentrations are calculated with a MAP/MIP to fluorescent protein molar mass ratio and the concentration of fusion proteins in cell membranes (final value in step IV), using the following equation:

        $$\frac{\text{Molar mass of POI}}{\text{Molar mass of GFP}}\text{*}\left[\text{cell membrane fusion proteins}\right]=\left[\text{POI}\right]\left(\text{mg}/\text{ml}\right)$$

3.6. Optimizing Membrane Protein Overexpression in S. cerevisiae.

One of the simplest methods to increase MAP/MIP yields is to adjust growth conditions, even minor adjustments can result in large expression differences. Growth condition modifications maximize protein production without changing equipment needs, sizes, or amounts. If protein yield is sufficient but larger protein amounts are needed, quantities or volumes of flasks can be increased (see Note 58). One need that can differ between MAPs/MIPs is nutrient composition. If given proper nutrients, in sufficient quantities, cells express proteins more efficiently. Thus, adjustments to media may improve yields. Minimal medias maintain vector stability and are often a good starting point for expression (17). Increasing media concentrations increases yield if cells receive proper nutrient ratios but at low enough amounts that other processes are favored over MAP/MIP production. If cells receive an insufficient nutrient composition, switching media types may improve results (see Notes 59–60).

Carbon source composition can also be modified to improve protein expression. The above protocols use a total 2% (w/v) carbohydrate concentration, distributed evenly between raffinose and glucose. This carbohydrate strategy generally keeps glucose levels under the GAL1 repression threshold but variations of this strategy, such as using alternative carbohydrates (e.g., maltose, lactose, etc.), using only glucose, or altering total carbohydrate concentration, may increase yields.

Oxygen availability, temperature, and chemical chaperones also influence protein production (17–20). Oxygen concentrations are linked to energy and metabolism which alter resource usage, vector replication, or partitioning. For growths performed in incubators, oxygen levels are controlled by a combination of culture volume, flask size, flask type, antifoam usage, and shake rate. Larger flasks, baffled flasks, higher shake rates, and including antifoam increase oxygen availability. Growths in fermentors increase oxygen accessibility by increasing agitation rate, increasing air flow, or utilizing gas mixtures with different oxygen concentrations. Some MAPs/MIPs express better with low oxygen, while others produce better with increased oxygen levels. Low growth temperatures may improve yields for toxic MAPs/MIPs but lower temperatures are otherwise associated with decreased protein production. Lastly, chemical chaperones (e.g., glycerol) can improve MAP/MIP protein folding and decrease protein degradation rates, potentially increasing target protein yields.

If altering growth conditions does not improve yields, modifying the expression vector may be necessary. Simple modifications such as exchanging a tag from one terminus to the other or changing tag type can increase yields to satisfactory levels (see Note 61). Expression tags (e.g., galectin, FLAG, maltose binding protein, Smt3, and fluorescent proteins) are commonly used to increase expression levels and protein folding of MAPs/MIPs. Recent studies have demonstrated that GFP tags can detoxify membrane proteins, increasing protein yields (21). Of the fluorescent proteins, a recent study demonstrated that mVenus increases membrane protein expression in mammalian cells more than other fluorescent proteins (22).

The addition of an N-terminal Kozak sequence immediately before the start codon may also increase expression levels. Kozak sequences are initiation sites for most mRNA transcripts and can improve the probability of ribosomes recognizing start codons. Traditionally, the yeast Kozak sequence AAAAA has been considered the most efficient sequence but there is recent evidence indicating that other Kozak sequences may be more efficient (23,24).

Another optimization strategy is to exchange the backbone vector for one that uses a different promoter or selection marker. For example, ADH2 is an ethanol-inducible promoter that allows expression during stationary phase and does not require inductants since S. cerevisiae produces ethanol as glucose is consumed (25,26). Constitutive promoters should be avoided because uncontrolled MAP/MIP production can result in toxicity issues. However, if expressing a MAP/MIP results in selective pressures against its own production, lower and uniform protein expression via a constitutive promoter may be beneficial.

Changes in target gene codon usage can also increase yields. Each expression system has a specific pool of t-RNA molecules that can affect protein manufacturing. If a specific codon is overrepresented in the GOI during overexpression, the complementary t-RNA may become depleted, slowing addition of amino acids to nascent peptides. Codon optimization tools have been developed to account for t-RNA depletion and most are free to use. Codon optimization can dramatically improve protein yields, with some reports suggesting > 1,000-fold improvement after codon optimization (27). 1,000-fold improvement from codon optimization is atypical but illustrates that potential changes in expression are substantial for codon-optimized constructs (see Note 62). Lastly, changing cell lines can benefit protein expression but that strategy may require changes to selection methods, growth conditions, and/or vectors.