Protein Folding Optimization in Yeast Display: Engineering Better Expression Systems

The success of yeast display campaigns ultimately depends on the structural integrity and functional quality of displayed proteins. While yeast provides sophisticated eukaryotic machinery for protein folding and quality control, there may still be challenges for expressing heterologous proteins. Examples of difficult situations include complex disulfide bond patterns, unusual structural features, or specific folding requirements. For developing therapeutic proteins, ensuring proper protein folding and implementing effective quality control measures is essential for identifying variants that will translate successfully to downstream applications.

The Protein Folding Challenge in Yeast Display

The process of protein folding begins with co-translational translocation into the endoplasmic reticulum, where nascent polypeptides encounter a oxidative environment containing molecular chaperones, folding catalysts, and quality control mechanisms. While this machinery is highly evolved and generally effective for yeast proteins, it was not designed to handle the diverse array of heterologous proteins that researchers attempt to display using yeast surface display technology.

The endoplasmic reticulum of yeast contains numerous molecular chaperones, including BiP, protein disulfide isomerases, and calnexin, which facilitate proper protein folding and disulfide bond formation. However, the specificity and capacity of these systems may not be optimal for all heterologous proteins, particularly those from mammalian or other non-fungal sources. The result can be the accumulation of misfolded proteins that may still be transported to the cell surface and displayed, where they can participate in non-specific binding interactions with altered specificity or affinity compared to correctly folded proteins.

One of the most common folding-related problems involves the formation of incorrect disulfide bonds, particularly in proteins containing multiple cysteine residues. The oxidizing environment of the endoplasmic reticulum promotes disulfide bond formation, but the presence of protein disulfide isomerases does not guarantee that the correct disulfide connectivity will be achieved for all proteins. This challenge is particularly acute for antibody fragments, which typically contain multiple disulfide bonds that are critical for maintaining proper structure and binding function.

Size constraints represent another significant challenge for protein folding in yeast display systems. While yeast can successfully fold and display many proteins, there appears to be an upper limit to the size and complexity of proteins that can be effectively processed through the secretory pathway and displayed on the cell surface. Large proteins or protein complexes may be subject to increased rates of misfolding, aggregation, or degradation, leading to reduced display levels or the presentation of truncated or otherwise compromised variants.

The yeast quality control machinery, while sophisticated, may not be optimally configured for all heterologous proteins. The endoplasmic reticulum-associated degradation (ERAD) pathway is designed to identify and eliminate misfolded proteins, but its specificity is tuned for yeast proteins and may not effectively recognize folding defects in proteins from other organisms. This can result in the escape of misfolded proteins from quality control surveillance, leading to their display on the cell surface where they may exhibit altered binding properties or reduced stability.

Signal Peptide Optimization: The Gateway to Proper Folding

Signal peptide optimization represents one of the most effective and widely applicable strategies for improving protein folding and display in yeast systems. The signal peptide plays a crucial role in directing nascent polypeptides to the endoplasmic reticulum and can significantly influence the efficiency of translocation, folding, and subsequent processing. Different signal peptides can have dramatically different effects on the expression and folding of the same target protein, making signal peptide optimization a critical component of any yeast display optimization campaign.

The most commonly used signal peptide in yeast display systems is derived from the α-factor mating pheromone, which includes both a signal sequence and a pro-peptide region that is removed during processing. However, this signal peptide is not optimal for all proteins, and significant improvements in expression and folding can often be achieved by testing alternative signal peptides. Systematic studies have identified several signal peptides that consistently outperform the α-factor signal for certain classes of proteins.

The optimization of signal peptides can be approached through both rational design and empirical screening strategies. Rational approaches involve the analysis of signal peptide sequences and structures to identify features that may be important for efficient translocation and folding of specific target proteins. Factors such as hydrophobicity, charge distribution, and cleavage site specificity can all influence signal peptide performance and can be systematically varied to optimize expression for specific applications.

Empirical screening approaches involve the construction of libraries containing multiple signal peptide variants and the selection of optimal combinations based on expression levels or functional activity. This approach can be particularly effective when combined with flow cytometry-based screening methods that allow for the rapid assessment of large numbers of signal peptide variants. The use of fluorescent reporters or functional assays can provide quantitative measures of signal peptide performance and enable the identification of optimal combinations for specific target proteins.

The choice of signal peptide can also influence the kinetics of protein folding and quality control in the endoplasmic reticulum. Some signal peptides may promote more efficient translocation, allowing more time for proper folding before quality control mechanisms are engaged. Others may influence the co-translational folding process by affecting the rate of translation or the interaction with ribosome-associated chaperones.

Chaperone Co-expression: Enhancing the Folding Environment

Chaperone co-expression strategies offer another powerful approach for improving protein folding in yeast display systems. The endoplasmic reticulum of yeast contains numerous molecular chaperones that assist in protein folding, but the levels and types of chaperones present may not be optimal for all heterologous proteins. The co-expression of additional chaperones or the overexpression of endogenous chaperones can significantly improve the folding and display of challenging proteins.

The most commonly employed chaperone for yeast display applications is BiP (Kar2p in yeast), the major ER-resident Hsp70 chaperone that assists in protein folding and quality control. Overexpression of BiP has been shown to improve the display of numerous proteins, particularly those that contain complex disulfide bond patterns or are prone to aggregation. The co-expression of BiP can be achieved through the use of multi-plasmid systems or by integrating additional copies of the BiP gene into the yeast genome.

Protein disulfide isomerases represent another class of chaperones that can be particularly beneficial for improving the folding of disulfide-containing proteins. The co-expression of PDI1 or other disulfide isomerases can help ensure proper disulfide bond formation and reduce the accumulation of misfolded variants with incorrect disulfide connectivity. The choice of specific disulfide isomerases may depend on the target protein, with some proteins benefiting more from certain isomerases than others.

The co-expression of multiple chaperones simultaneously can provide synergistic benefits for challenging proteins that require assistance with multiple aspects of the folding process. However, the overexpression of chaperones can also have negative effects, including competition for cellular resources, interference with normal cellular processes, or the stabilization of partially folded intermediates that might otherwise be properly folded or degraded.

The timing and level of chaperone expression must be carefully optimized to achieve maximum benefit without negative side effects. Inducible expression systems can provide temporal control over chaperone levels, allowing for the optimization of expression timing relative to target protein expression. The use of different promoter strengths can provide control over chaperone expression levels, enabling the identification of optimal ratios between target proteins and chaperones.

ER Retention Strategies: Extending Folding Time

ER retention strategies provide a complementary approach for improving protein folding by extending the time available for folding and quality control in the endoplasmic reticulum. The addition of ER retention signals, such as the KDEL sequence, to displayed proteins can increase their residence time in the ER and improve the efficiency of folding and quality control. While this approach may reduce the overall display levels due to retention in the ER, it can significantly improve the quality of displayed proteins and reduce the proportion of misfolded variants.

The implementation of ER retention strategies requires careful optimization of the retention signal and its placement within the fusion protein. The retention signal must be positioned such that it is accessible to the retention machinery while not interfering with the folding or function of the target protein. In some cases, the use of cleavable retention signals that are removed during processing can provide the benefits of ER retention while allowing normal transport to the cell surface.

The duration of ER retention can be controlled through the use of different retention signals with varying strengths or through the co-expression of factors that modulate the retention machinery. Longer retention times may improve folding quality but can also increase the risk of protein degradation or aggregation in the ER. The optimal retention time must be determined empirically for each target protein and application.

Temperature control can also be used in conjunction with ER retention strategies to further optimize folding conditions. Lower temperatures can slow the folding process and reduce the rate of misfolding, while extended retention times provide additional opportunities for proper folding to occur. The combination of temperature optimization and ER retention can be particularly effective for temperature-sensitive proteins or those with complex folding requirements.

Promoter Engineering and Expression Optimization

Promoter engineering represents another important strategy for optimizing protein expression and folding in yeast display systems. Different promoters can provide varying levels of expression, and the choice of promoter can significantly influence the folding efficiency and quality of displayed proteins. High-level expression driven by strong promoters may overwhelm the folding machinery and lead to increased rates of misfolding, while weak promoters may result in insufficient expression levels for effective screening.

The optimization of promoter strength often involves finding a balance between expression level and folding quality. Medium-strength promoters, such as the PGK1 or TDH3 promoters, often provide better results than very strong promoters like GAL1 for proteins that are challenging to fold. However, the use of inducible promoters like GAL1 may provide additional control over expression timing and levels. Ultimately, each heterologous protein will need to be tested using different promoters. Furthermore, each promoter may be engineered by adding tandem upstream activation sequences to strengthen or weaken promoter activity

The timing of protein expression relative to cell growth and other cellular processes can significantly influence folding outcomes. Expression during exponential growth phase may provide optimal folding conditions due to high metabolic activity and chaperone availability, while expression during stationary phase may reduce competition for folding resources. The use of inducible systems allows for precise control over expression timing and can enable the optimization of expression conditions for specific target proteins.

The rate of protein synthesis can also influence folding outcomes, with slower synthesis rates sometimes providing better folding efficiency for complex proteins. The use of promoters with different transcriptional kinetics or the modulation of translation rates through codon optimization can provide control over synthesis rates and improve folding outcomes for challenging proteins.

Ranomics: Your Partner in Protein Folding Excellence

The complexities of protein folding optimization in yeast display systems require deep expertise and extensive experience to navigate successfully. Ranomics brings this expertise to biotech and pharmaceutical companies through comprehensive protein engineering services that address every aspect of the folding challenge, from signal peptide optimization and chaperone co-expression to advanced quality control screening methods.

Our team has developed proprietary protocols for optimizing protein expression and folding in yeast display systems, incorporating the latest advances in signal peptide engineering, chaperone co-expression, and ER retention strategies. We understand that proper protein folding is critical for the success of therapeutic protein development programs, and we work closely with our biotech and pharma clients to ensure that selected variants will exhibit the structural integrity and functional properties required for downstream applications.

Ranomics' full-service approach includes not only the optimization of expression systems but also the implementation of sophisticated quality control measures to ensure that only properly folded variants are selected during screening campaigns. Our experience with both yeast display and mammalian display systems enables us to select the optimal platform for each specific protein target and to implement appropriate folding optimization strategies.

The protein folding challenge is particularly critical for pharmaceutical companies developing therapeutic antibodies and other complex proteins, where structural integrity directly impacts safety, efficacy, and manufacturability. Our team has extensive experience working with challenging protein targets, including antibody fragments with complex disulfide bond patterns, enzymes with specific cofactor requirements, and membrane proteins with unique folding challenges.

For companies seeking to maximize the value of their protein engineering investments, partnering with Ranomics provides access to the expertise and methodologies needed to overcome folding challenges and ensure the selection of high-quality variants. Our commitment to staying at the forefront of protein folding technology ensures that our clients benefit from the latest advances in expression optimization, chaperone engineering, and quality control methods.

Whether you're developing therapeutic antibodies, optimizing enzyme properties, or engineering novel binding proteins, Ranomics has the expertise and capabilities to help you achieve optimal protein folding and quality in your yeast display campaigns. Our comprehensive approach addresses every aspect of the folding challenge, from initial expression optimization through final variant validation, ensuring that your protein engineering projects achieve their full potential.