Protein Engineering Technology: Methods, Applications, and Advances in Biopharma

Protein engineering is a multidisciplinary field that involves the design, modification, and optimization of proteins to achieve desired functions, properties, or novel characteristics. It combines principles from molecular biology, biochemistry, computational biology, and synthetic biology to create proteins with improved stability, functionality, or novel characteristics.

Fundamentals of Protein Engineering

Protein engineering involves understanding the structure-function relationships in proteins and making structural modifications to achieve desired functional outcomes. A protein's three-dimensional structure is derived from its amino acid sequence and determines its functional activity. Through protein engineering, scientists create proteins with specialized structural features that enable them to perform specialized functions better than their natural counterparts.

Protein engineering uses directed evolution, rational design, and hybrid methods as its primary techniques. The application of these strategies individually or in combination enables the achievement of specific protein engineering goals, including optimization of enzymatic activity, improvement of stability in extreme environments, and enhancement of substrate selectivity.

Strategies in Protein Engineering

Rational Design

Rational design is a more targeted approach to protein engineering based on knowledge of protein structure and function. In rational design, mutations are introduced based on a thorough understanding of the protein's structure, often obtained through X-ray crystallography, NMR spectroscopy, or cryo-electron microscopy. Computational techniques, such as molecular dynamics simulations, are also used to predict the effects of mutations on protein stability, folding, and activity.

Key Steps in Rational Design:

• Structure Determination: The first step in rational design is to determine the high-resolution structure of the protein of interest. This can be accomplished using techniques such as X-ray crystallography, NMR, or cryo-EM.
• Identification of Target Sites: Based on the structure, key residues or regions that are important for the function of the protein are identified. These may be active site residues, residues exposed on the surface for substrate binding, or residues that affect stability.
• Mutation and Optimization: Specific amino acid substitutions, insertions, or deletions are introduced to improve protein properties such as binding affinity, stability, or specificity for a given ligand.
• Experimental Validation: The designed protein is synthesized and tested for the desired activity or function. If the design does not achieve the desired result, the process is iterated by further modifying the protein.

Directed Evolution

Directed evolution is a robust method in protein engineering that emulates the process of natural evolution. This technique creates genetic diversity through random mutagenesis, enabling the selection of proteins with enhanced or entirely new functions. It is especially valuable when the relationship between a protein's structure and its function remains unclear. The process begins with the random generation of genetic diversity, often achieved through techniques such as error-prone PCR (polymerase chain reaction) or DNA shuffling. Following this, variants exhibiting desirable traits are identified through screening or selection. Directed evolution is versatile and can be applied to a broad spectrum of protein targets, including enzymes, antibodies, and receptors.

Key Steps in Directed Evolution:

• Generation of Mutant Libraries: This step involves the introduction of random mutations into the gene encoding the protein of interest. Various methods, such as error-prone PCR, chemical mutagenesis, or DNA shuffling, can be used to generate a diverse library of protein variants.
• Screening and Selection: Once the mutant library has been generated, the next step is to screen or select the variants that have the desired characteristics. This process can be performed using high-throughput screening techniques, such as fluorescence-activated cell sorting (FACS), or using selection strategies in which only the functional proteins are allowed to survive or proliferate.
• Amplification and Iteration: Once the best-performing variants are identified, their DNA is amplified and subjected to further rounds of mutation and selection. Multiple rounds of directed evolution may be required to achieve the desired level of improvement in protein function.

Figure 1. General Protein Engineering Processes. A library can be established through either rational design aided by computational simulation and/or crystal structures, or via random mutagenesis or directed evolution. With the assistance of efficient screening methods, variants with different characteristics such as enhanced catalytic activity and increased stability to heat, acid, or alkali can be selected and analyzed. If no candidate shows the desired feature, it may be necessary to regenerate the library and repeat the process until the desired variant is obtained. (Li et al., 2020)

Hybrid Approaches

The combination of directed evolution and rational design often proves to be the most efficient method for protein engineering. This technique merges directed evolution's capability of producing diverse mutants with rational design's focused knowledge-based alterations.

For example, directed evolution can be used to generate a diverse library of protein variants, and then rational design can be used to refine the best-performing variants. Alternatively, rational design can be used to introduce key mutations that guide the evolution of more effective variants.

A prominent example of hybrid approaches is the engineering of enzymes for industrial applications. In many cases, directed evolution is used to create a diverse set of enzyme variants, and rational design is used to stabilize these enzymes or enhance their activity under specific conditions.

Tools and Technologies in Protein Engineering

Protein Microarrays

Protein microarrays are used to screen large numbers of protein variants quickly and efficiently. They can be used in directed evolution or rational design to identify potential candidates for further study.

High-Throughput Screening

High-throughput screening (HTS) is an essential tool for evaluating large libraries of protein variants. Techniques such as fluorescence-activated cell sorting (FACS) or enzyme-linked immunosorbent assays (ELISA) allow for the rapid evaluation of protein activity, binding affinity, and stability.

Computational Tools

• Rosetta: Rosetta is a comprehensive computational suite for protein modeling, structure prediction, and design. It is one of the most widely used tools for de novo protein design and optimization.
• AlphaFold: AlphaFold is an artificial intelligence (AI)-driven tool developed by DeepMind that has revolutionized the field of protein structure prediction.

Expression Systems

To synthesize engineered proteins, researchers rely on expression systems that enable the production of proteins in a laboratory setting. These systems use cells to express the genes that encode the desired proteins, which are then purified for further study or application.

• Bacterial Systems: Escherichia coli (E. coli) is one of the most commonly used expression systems due to its fast growth rate and high yield of recombinant proteins.
• Yeast Systems: Saccharomyces cerevisiae, a species of yeast, is a eukaryotic expression system capable of performing more complex post-translational modifications than bacterial systems.
• Insect Cells: Insect cells, particularly Spodoptera frugiperda (Sf9) cells, are used in the baculovirus expression vector system (BEVS), which is commonly used to produce complex, glycosylated proteins.
• Mammalian Systems: Mammalian cell systems are crucial for the production of proteins that require human-like post-translational modifications, such as glycosylation, phosphorylation, and proteolytic cleavage.

Analytical Techniques

Once proteins are synthesized, a variety of analytical techniques are used to characterize their structure, purity, and functionality. These techniques help validate the success of protein engineering efforts and ensure that the proteins meet the required standards for their intended application.

• Mass Spectrometry: Mass spectrometry (MS) is a powerful analytical technique used to measure the molecular weight and composition of proteins, providing insight into their modifications and purity. MS is widely used in proteomics, drug discovery and biomarker identification. It plays a critical role in the characterization of engineered proteins, including confirmation of their size, structure, and post-translational modifications.
• Surface Plasmon Resonance (SPR): SPR is a technique used to measure binding interactions between proteins and other molecules, such as ligands or other proteins, in real time. SPR is widely used in drug discovery, antibody characterization, and enzyme kinetics. It is also valuable in the study of protein-protein and protein-DNA interactions.
• Circular Dichroism (CD): Circular dichroism is a technique used to assess the secondary structure of proteins, providing insight into their folding state. CD is widely used in protein folding studies, stability assessment, and structural analysis of recombinant proteins. It plays an important role in confirming the structural integrity of engineered proteins.

Applications of Protein Engineering

Protein engineering has found widespread applications across various fields, including biotechnology, pharmaceuticals, and environmental sciences.

Therapeutic Protein Development

Protein engineering is essential for the creation of therapeutic proteins such as monoclonal antibodies, hormone analogues and enzymes. By modifying protein structure, researchers can increase protein stability while improving binding affinity and reducing immunogenicity. Engineered antibodies such as trastuzumab (Herceptin) have transformed breast cancer treatment. Researchers are using protein engineering to create enzyme-based treatments for rare genetic diseases, such as enzyme replacement therapy for lysosomal storage disorders.

Industrial Enzymes

Industrial enzymes for food production, biofuels, detergent production and paper processing are often developed through protein engineering. Enzymes are optimized through directed evolution and rational design for improved stability at elevated temperatures, enhanced catalytic efficiency, and improved chemical and solvent tolerance.

Agricultural Biotechnology

In agriculture, protein engineering is used to create crop varieties that are resistant to disease, pests and environmental stresses. Scientists have incorporated insecticidal proteins from Bacillus thuringiensis (Bt) into genetically modified crops to protect them from insect pests without the use of chemical pesticides. Protein engineering is helping to create drought-tolerant crops by designing proteins that enable plants to better respond to stressors caused by water shortages.

Biocatalysis

The field of protein engineering uses biocatalysis as a key application by using natural catalysts such as protein enzymes for chemical reactions. Engineered biocatalytic enzymes facilitate the creation of pharmaceutical products, green chemicals and biofuels. Through protein engineering, scientists are able to optimize enzymes for industrial reactions by increasing their stability, activity and specificity.

Biosensors

Scientists use protein engineering to create biosensors with the ability to detect specific biological or chemical substances. Engineered proteins act as precise receptors that bind target molecules, allowing scientists to develop sensors for medical diagnostics, environmental monitoring and food safety applications.

Successful Modified Proteins with Protein Engineering Technology

Protein engineering technology has enabled the creation of numerous modified proteins with profound applications across medicine, industrial biotechnology, agriculture, and environmental science. These engineered proteins are tailored to exhibit enhanced or entirely novel properties that do not exist in natural proteins, such as greater stability, higher efficiency, or entirely new functional abilities. The following examples highlight some of the most successful modified proteins, each marking a significant advancement in their respective domains.

Recombinant Human Insulin

The first major breakthrough in insulin production came with the development of recombinant DNA technology. In 1978, researchers engineered E. coli bacteria to produce human insulin by inserting the gene for insulin into the bacterial DNA. The engineered bacteria were then grown to express human insulin, which was harvested, purified and used for therapeutic purposes. This process enabled the mass production of human insulin, eliminating the need for animal-derived insulin and making it safer and more effective for patients.

Recombinant insulin has become the standard of care for diabetes worldwide, dramatically improving patient outcomes. The ability to further modify the insulin molecule has led to the development of insulin analogues with altered pharmacokinetics, such as long-acting insulin (e.g., Lantus) and rapid-acting insulin (e.g., Humalog), which provide even greater glycemic control.

Figure 2. Bacterial production of human insulin. Insulin consists of an A chain and a B chain, which are linked by disulphide bonds. In the initial process for insulin manufacture, synthetic DNA coding for the A and B chains was inserted into two plasmids, which were then separately introduced into Escherichia coli. The A and B chains were purified from the two bacterial cultures and then recombined to give the full insulin molecule. (Johnson, 2003)

Enzymes for Industrial Biocatalysis (e.g., Subtilisin)

Subtilisin, a protease enzyme derived from Bacillus subtilis, has been engineered to improve its stability, activity and resistance to harsh chemical environments. Protein engineering techniques, including directed evolution and rational design, have been used to modify the amino acid sequence of subtilisin to enhance some of its properties; for example, one study has demonstrated recombinant subtilisin with potent antibacterial activity against several pathogens. In the detergent industry, the engineered versions of subtilisin have increased resistance to high temperatures and alkaline conditions, making them more effective in industrial cleaning processes.

Monoclonal Antibodies

Monoclonal antibodies are another highly successful class of engineered proteins that have had a transformative impact on cancer therapy. Monoclonal antibodies are designed to specifically bind to a target antigen, such as a protein expressed on the surface of cancer cells, to induce an immune response or inhibit tumor growth.

The development of monoclonal antibodies began with the use of hybridoma technology, in which animal B cells were fused with myeloma cells to create immortal cell lines capable of producing large quantities of a single, specific antibody. Over time, protein engineering has further improved these antibodies by humanizing them (minimizing the immunogenicity of murine antibodies in humans) and optimizing their affinity for target antigens. Rituximab, for example, is engineered to target the CD20 antigen on B cells and is used to treat cancers such as non-Hodgkin's lymphoma. Herceptin (trastuzumab), designed to target the HER2/neu receptor, is used to treat breast cancer.

Avidin-Biotin System (Avidin)

Protein engineering has been used to modify avidin to improve its properties for specific applications. For example, streptavidin, a modified form of avidin derived from Streptomyces avidinii, has been engineered to have a reduced tendency to induce immune responses in humans. In addition, biotinylated proteins can be used to target specific tissues or cells by linking biotin to therapeutic agents or gene-editing tools such as CRISPR.

Creative Biostructure offers professional and comprehensive protein engineering solutions designed to meet the diverse needs of your research and development projects. Our advanced techniques, combined with state-of-the-art technology, enable precise modifications and optimizations of proteins to enhance their functionality, stability, and efficacy. Contact us today to learn more about how our tailored services can accelerate your research.

References

1. Johnson IS. The trials and tribulations of producing the first genetically engineered drug. Nat Rev Drug Discov. 2003;2(9):747-751.
2. Li C, Zhang R, Wang J, Wilson LM, Yan Y. Protein engineering for improving and diversifying natural product biosynthesis. Trends in Biotechnology. 2020;38(7):729-744.