In my research, I have developed a range of innovative and non-standard approaches to protein expression and purification, particularly for challenging targets. Here are the key skills and methods I’ve employed:
Use of Diverse Expression Hosts
- Yeast Systems: Leveraged Saccharomyces cerevisiae and Pichia pastoris for producing eukaryotic proteins, such as human Copper Transporter (CTR) proteins and other membrane protein complexes. These systems are particularly advantageous for complex proteins requiring post-translational modifications.
- Bacterial Systems: Utilized Escherichia coli and Bacillus subtilis for the heterologous expression of proteins and protein complexes, including among others challenging membrane proteins like Complex I membrane subunits and subcomplexes, as well as the notoriously difficult to express nitrogenase proteins from Azotobacter vinelandii, achieving fully active enzyme expression.
Expression of Diverse Protein Types
- Soluble Proteins: Efficiently expressed and purified soluble proteins using optimized bacterial and yeast systems.
- Metalloproteins: Specialized in the expression and purification of metalloproteins, ensuring proper metal cofactor incorporation.
- Membrane Proteins and Complexes: Successfully produced and purified challenging membrane proteins and their complexes, using advanced techniques to maintain their stability and functionality.
- Periplasmic Proteins: Applied targeted strategies for the expression and isolation of periplasmic proteins, ensuring high yield and purity.
Construct Design and In Silico Stability Analysis
- Construct Design: Designed expression constructs with optimal codon usage, fusion tags, and cleavage sites to maximize expression efficiency and facilitate downstream purification.
- In Silico Stability Analysis: Utilized computational tools to predict protein stability, identify potential mutations, and design truncations to enhance protein expression and solubility.
- Predicting Mutations and Truncations: Applied predictive modeling to introduce beneficial mutations and truncations that improve protein folding, stability, and functionality.
Innovative Tagging and Detection Techniques
- Fluorescent Tagging: Integrated fluorescent tags, including Green Fluorescent Protein (GFP), non-GFP based fluorescent tags and lanthanide binding tags, enabling straightforward detection, localization, and purification. This approach simplifies monitoring protein expression and assessing purification efficiency.
- Affinity Tags: Employed His-tags for efficient purification via immobilized metal ion affinity chromatography (IMAC), ensuring high purity and yield.
- Cytochrome c Tag: Utilized the cytochrome c tag for convenient detection, quantification, and purification of target proteins, enhancing the overall efficiency of the process.
Optimization of Growth and Induction Conditions
- Design of Experiments (DoE): Utilized DoE methodologies to systematically optimize growth and induction conditions, maximizing protein yield and quality.
- Chaperone Expression: Co-expressed molecular chaperones to aid in the proper folding of complex proteins, enhancing their solubility and functionality.
- Cofactor Synthesis and Maturation Proteins: Integrated cofactor synthesis and maturation proteins into expression systems to ensure proper cofactor incorporation and maturation of target proteins.
- PTM Management: Managed post-translational modifications (PTMs) effectively to maintain protein functionality and stability.
- Temperature and Induction Optimization: Conducted detailed optimization of growth and induction conditions, such as using lower temperatures (15°C) for prolonged induction periods to enhance protein yield and quality in yeast systems.
- Detergent Screening: Performed extensive screening of various detergents (e.g., DDM, LDAO) to identify optimal solubilization conditions for membrane proteins, ensuring their stability and functionality.
Scalable Cell Culturing
- Scalable Cell Growth: Experienced in growing cells at various scales, from microplates to small cultures, flasks, and up to 180 L fermentors. This versatility allows for effective scaling of protein production processes from research to industrial levels. I have managed cell cultures efficiently across these scales, ensuring consistent growth conditions and optimizing yields to meet the demands of different project phases, from initial research to large-scale production.
Advanced Purification Techniques
- Fluorescence-Detection Size-Exclusion Chromatography (F-SEC): Used F-SEC to evaluate solubilization efficiency and protein stability post-extraction, facilitating the identification of optimal purification conditions.
- TEV Protease Cleavage and Non-standard IMAC: Implemented TEV protease to remove affinity tags post-purification, achieving tag-free proteins suitable for downstream structural and functional studies. Employed metals other than Ni and Co for Histaged proteins purification.
Structural and Functional Validation
- Mass Spectrometry: Applied mass spectrometry to confirm the identity and purity of purified proteins, ensuring they are suitable for biophysical characterization and drug discovery efforts.
- Biophysical Characterization: Prepared high-quality protein samples for structural studies, including X-ray crystallography, to elucidate the structure-function relationships of target proteins.