Introduction
Post-translational modifications (PTMs) are critical in determining the structure, stability, and functionality of proteins expressed in biological systems. PTMs involve the enzymatic alteration of proteins during and after protein synthesis, leading to changes that can profoundly impact protein properties and behavior. For proteins produced using the methylotrophic yeast Pichia pastoris as an expression host, understanding and controlling PTMs is essential for ensuring proper protein folding, biological activity, and efficacy for downstream applications.
This article focuses on PTMs in recombinant proteins expressed in Pichia pastoris, and the associated implications and strategies for optimizing PTM profiles to achieve desired protein qualities. Pichia has emerged as an attractive alternative to other common protein expression systems due to advantages such as high yields and ease of genetic manipulation. However, Pichia exhibits differences in its PTM pathways compared to mammalian cells. Consequently, the PTM profiles of Pichia-expressed proteins must be carefully evaluated and controlled to avoid functionality issues resulting from improper modifications.
The following sections provide an in-depth examination of the significant PTMs occurring in Pichia-expressed proteins, the effects of PTMs on protein properties, current approaches for controlling PTMs through system engineering and process optimization, real-world examples of effective PTM management, and future directions for advancing PTM control in proteins produced using this vital expression host.
Types of Post-Translational Modifications in Pichia
The predominant PTMs in recombinant proteins expressed by Pichia pastoris include glycosylation, phosphorylation, acetylation, methylation, and disulfide bond formation. These modifications have significant implications for the expressed proteins’ structure, activity, and pharmacokinetic behavior.
Glycosylation
Glycosylation involves the enzymatic attachment of glycan (carbohydrate) structures to certain amino acid residues of the protein. Pichia glycosylates proteins mainly via N-linked glycans attached to asparagine residues, while O-linked glycans attached to serine/threonine residues occur less frequently. Compared to mammalian cell glycosylation, N-glycans in Pichia are typically high in mannose content while lacking terminal galactose and sialic acid residues. These differences can impact protein immunogenicity, serum half-life, and bioactivity.
Phosphorylation
Phosphorylation is the addition of phosphate groups to amino acid residues like serine, threonine, and tyrosine. This PTM can regulate protein activity, stability, trafficking, and protein binding interactions. While the basic mechanisms are conserved, the specific phosphorylation sites and stoichiometry can vary for proteins expressed in Pichia compared to mammalian cells. These differences can alter protein signaling, enzyme kinetics, and biological function.
Acetylation and Methylation
N-terminal acetylation and methylation occur frequently in Pichia-expressed eukaryotic proteins. N-terminal acetylation modifies the alpha-amine of the first amino acid, while methylation occurs on lysine and arginine residues. These modifications can impact protein stability, folding, macromolecular complex formation, and subcellular localization. However, patterns differ between Pichia and mammalian cell systems.
Disulfide Bond Formation
Disulfide bonds between cysteine residues help stabilize the tertiary structure of many proteins. While Pichia forms disulfide bonds within the endoplasmic reticulum like mammalian cells, differences in the redox environment and enzyme systems can lead to non-native disulfide bond arrangements that disrupt proper protein folding.
Implications of Post-Translational Modifications on Protein Functionality
By altering the structure, charge, hydrophobicity, and molecular recognition properties of proteins, PTMs profoundly influence various aspects of protein behavior and function:
Protein Stability
PTMs affect interactions between amino acids that stabilize or destabilize protein conformations. Glycosylation and disulfide bonds generally enhance stability, while other modifications like phosphorylation can reduce strength by introducing repulsive charges. Suboptimal PTMs in Pichia-expressed proteins can, therefore, reduce shelf life.
Biological Activity
PTMs directly impact the active sites and binding interfaces involved in the biological activity of proteins. This can enhance or disrupt molecular interactions and signaling. Glycosylation and phosphorylation commonly influence protein bioactivity.
Immunogenicity
Differences in PTM profiles between Pichia and mammalian cell expression can introduce novel epitopes recognized as foreign by the immune system. This impacts antibody production and clearance rates. Glycosylation differences are a significant factor for increased immunogenicity risk with Pichia.
Pharmacokinetics
PTMs affect how proteins interact with factors influencing their absorption, distribution, metabolism, and excretion (ADME). For instance, glycosylation increases hydrodynamic volume, alters protein solubility and stability, and protects against proteolysis, directly influencing pharmacokinetics. Therefore, pharmacokinetic profiles can differ for proteins with non-mammalian PTMs.
Therefore, strategic engineering of PTMs is critical for obtaining the desired protein qualities, as discussed next.
Strategies for Controlling and Enhancing Post-Translational Modifications in Pichia
Various approaches can be employed to optimize PTM profiles in Pichia expression systems:
Strain Engineering
Protein glycosylation patterns in Pichia can be engineered via overexpression or knockout of genes involved in glycan biosynthesis. This allows the attachment of complex mammalian-type glycans to achieve structures that avoid immunogenicity risks.
Culture Condition Optimization
Environmental conditions like temperature, pH, dissolved oxygen, nutrients, and growth phase affect cellular metabolism and the post-translational enzymatic machinery. Optimization of culture conditions can thus improve desired PTMs.
Media Composition
Media composition influences PTMs by providing substrates/cofactors needed for modifications, altering cellular metabolism, or changing physical parameters like redox potential. Media can be designed to enhance PTM fidelity.
Induction Strategies
Modulating factors like inducer concentration, induction timing, temperature shifts upon induction, and induction duration impact cellular physiology and shape PTM formation. This allows targeted enhancement of desired modifications.
PTMs can be optimized through these strategies to suit the specific protein application and performance requirements.
Challenges and Future Directions in Post-Translational Modification Control
While existing strategies have enhanced control over PTMs in Pichia, several challenges remain:
- Achieving uniform and homogenous PTM profiles at large production scales remains difficult. Differences across fermentation batches are observed.
- Current glycoengineering approaches cannot reproduce the full range of complex glycan structures typical of mammalian PTMs. Further glycoengineering is needed.
- The interactions between various PTMs make precise control over individual modifications challenging. Combined modulatory strategies are required.
- Certain PTMs like tyrosine sulfation are yet to be engineered effectively in Pichia. Further expansion of the PTM repertoire is needed.
Future innovations to address these challenges include:
- Novel screening systems and “-omics” analysis to better characterize and control batch-to-batch variability.
- I am combining glycoengineering with in vitro enzymatic glycan remodeling as a hybrid approach to obtain complex, humanized glycans.
- Artificial intelligence-guided bioprocess optimization for multivariate precision control over interacting PTMs.
- Engineering of expanded enzymatic machinery from mammalian cells for efficient synthesis of rare/novel eukaryotic PTMs like sulfation.
Conclusion
Post-translational modifications profoundly impact the functionality of proteins produced in Pichia pastoris. Controlling and engineering PTMs through approaches like strain engineering, bioprocess optimization, and coexpression enables the generation of Pichia-expressed proteins with enhanced properties for stability, bioactivity, and in vivo performance. Advances in glycoengineering, combined PTM control strategies, and enzymatic diversification will enable unprecedented precision over PTMs, cementing Pichia as a versatile, efficient platform for industrial and therapeutic protein production.
