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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5344_Библиотеки_им_академика_М_И_Перельмана.pdf
Introduction to Pharmaceutical Biotechnology, Volume 2 (Second Edition)
10.19.2 Genetically encoded sensors
Dynamic regulation of genotype metabolism and evolution and screening of
preferred phenotypes rely heavily on genetically encoded sensors (GES), making
them indispensable in synthetic biology and metabolic engineering [72]. Their
applications span the construction and management of biosynthetic pathways,
enabling in situ monitoring with minimal interference, thereby revolutionizing
synthetic biology and microbial cell factories [73]. Specific types of GES, like
fluorescent biosensors, are promising for examining biochemical processes within
complex cellular mechanisms, enabling targeted, long-term monitoring and control
of cellular processes [72].
10.19.3 Protein-based logic gates
Protein-based logic gates are a promising field within synthetic biology, attempting
to integrate computational logic into biological systems through protein interactions. Protein logic gates are designed to regulate protein association, mimicking
electronic logic gates but utilizing biochemical interactions. Advances in protein
design are pushing towards a nanoscale programming language where molecules
serve as operands, facilitating computational operations within living cells [74].
Various forms like YES and AND gates have been developed, employing a range of
reporter proteins to demonstrate their functionality. The dynamic ranges of these
gates can be significantly large, indicating their potential for versatile applications.
Protein logic gates can control cellular functions post-translationally, opening new
avenues for modulating PPIs inside and outside cells. The de novo design of a wide
range of logic gates using heterodimeric molecules signifies the potential for creating
new protein-based control systems [75].
10.19.4 Gene circuits for dynamic control
The use of gene circuits to dynamically orchestrate cellular function is central to the
field of synthetic biology. Genetic circuit design has evolved significantly with
crucial works like the repressilator and the toggle switch setting a foundation. The
design aims to program new biological behaviors, dynamics, and logic control,
necessitating a structured approach for implementation [ 76]. The development of
multilayer genetic circuits for dynamic regulation has allowed for more complex
control of metabolic pathways in response to environmental factors and variations
in gene expression. Gene circuits show promise as a means of dynamically regulating
the redirection and balance of pathways in the production of useful compounds.
They have the potential to replace conventional treatments, representing a major
step toward the practice of customized and precision medicine [77]. A networkcentric approach in designing gene circuits allows the engineering of increasingly
complex Boolean logic circuits, which use transcriptional regulators for predefined
cellular functions [78].
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10.20 Engineering multi-functional proteins
Engineering multi-functional proteins is a dynamic field aimed at developing
proteins with enhanced or novel functionalities. Through various engineering
techniques, proteins can be tailored to exhibit multiple functionalities, which can
be employed in numerous applications like drug development, material science, and
synthetic biology [79].
10.20.1 Fusion proteins
Fusion protein engineering is a vital facet of synthetic biology and biotechnology,
facilitating the creation of chimeric molecules with enhanced or diversified functionalities. Fusion proteins are often constructed by linking distinct gene fragments. The
efficiency and functionality of the resultant chimeric protein can significantly be
affected by the nature and length of the linkers used [80]. They find extensive
application in therapeutics, for instance, in developing chimeric antigen receptor
(CAR) T cell therapies. Domain-by-domain optimization strategies have been
critical in enhancing the function of synthetic immunoreceptors through fusion
protein engineering. The design and construction of fusion proteins come with
challenges, like ensuring proper folding and functionality of the fused domains.
Recent progress has been made in the structural prediction of fusion proteins, aiding
in overcoming some of these challenges [81].
10.20.2 Protein scaffolds
Protein scaffolds are tools used in assembling multi-enzyme complexes, facilitating a
high degree of specificity and efficiency in enzymatic reactions. Protein scaffolds can
be constructed in various ways, with their components and construction methods
significantly impacting enzyme kinetics. They play a crucial role in coordinating
enzymatic complexes that power various metabolic pathways [82]. Better substrate
channeling may result from the construction of multi-enzyme complexes on protein
scaffolds, increasing the overall productivity of the enzymatic processes. Scaffold
proteins play a critical role in the use of methods like compartmentalization and
substrate channeling for the manipulation of multi-enzyme processes [83].
10.20.3 Modular protein design
Using known modules as building blocks, modular protein design is a method for
designing proteins with certain properties. This method is useful for designing proteins
with up to a particular number of amino acid residues in a single polypeptide chain
and simplifying protein structures that incorporate them. Computational approaches
that predict how these modules might be assembled to accomplish certain structural
and functional goals have greatly assisted the concept of modularity in protein design.
Designing modular architectures and developing new protein-building modules are
crucial to investigating the vast array of potential topologies that evolution has not yet
explored [84]. Computational protein design methods have been instrumental in
advancing modular protein design, enabling the design of proteins with various
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structural motifs and functionalities. De novo design is a particular approach within
modular protein design where proteins are designed from scratch without relying on
existing protein templates [84].
10.20.4 Dual-enzyme systems
Dual-enzyme systems can be construed as systems where two enzymes work together
either in tandem or synergistic manner to carry out a particular biochemical reaction
or a set of reactions. Such systems are vital in metabolic pathways and can be
engineered for biotechnological applications [85, 86].
10.21 Ethical and safety considerations
Like other fields of biotechnology, protein engineering presents a host of ethical and
safety considerations that must be thoroughly examined to ensure responsible
research and application [87].
10.21.1 Bioethics in protein engineering
Bioethics in protein engineering involves a range of considerations, including
potential impacts on human health, the environment, and social norms. The ethical
behavior in protein engineering research and applications is guided by the core
concepts of bioethics, including autonomy, beneficence, nonmaleficence, and fairness. Therapeutic proteins and enzymes developed by protein engineering have great
potential to improve human health. However, to ensure the subject’s safety, it is
necessary to thoroughly assess the potential risks to human health and their long-term
consequences. The release of engineered proteins into Nature might have unexpected
consequences. Evaluating these products for their ecological friendliness is crucial.
Protein engineering advances may have far-reaching implications for social justice,
including questions of equity and accessibility. It is crucial to build efficient regulatory
frameworks to ensure that protein engineering is practiced ethically and that the
benefits are shared fairly. Enzyme and therapeutic protein activity development is
another area of protein engineering’s purview. These features are exciting because they
might bring improvements to current medical research and treatment ideas. These
developments, however, need a careful assessment of ethical, safety, and social factors
to guarantee the technology is created and used responsibly [88].
10.21.2 Biosafety and environmental concerns
In protein engineering, biosafety and environmental considerations are critical
because they involve the possible dangers connected with the release and use of
altered proteins and organisms. Protein engineering has the potential to endanger
both human health and the environment. Unintended outcomes, such as developing
dangerous byproducts or unintentionally manufacturing poisonous compounds,
may pose concerns [89]. Advances in protein engineering and synthetic biology
provide further biosecurity problems, particularly as technology becomes more widely
available and complex. This covers the possibility of using genetically modified
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proteins improperly or harmfully. A strong regulatory structure is necessary to reduce
these dangers. These principles incorporate confinement and supervision methods to
promote practical applications while minimizing harm [90].
10.21.3 Intellectual property rights
Intellectual property rights (IPR) play a vital role in protein engineering and
biotechnology by protecting discoveries and providing financial incentives for
more studies. The patent system is an essential part of IPR, allowing creators to
protect their innovations (IPR) legally. The changed proteins and the procedures
used to create them are safeguarded alongside the results of genetic and proteomic
studies [91, 92]. Open communication of scientific findings and the defense of
inventors’ rights to advance science and improve human health must coexist in a
delicate balance. Maintaining this equilibrium is crucial to guarantee that the
advantages of protein engineering are extensively obtainable while also stimulating
creativity. The economic and legal components of intellectual property, including
the costs and advantages of patents, are critical for comprehending the more
significant influence of intellectual property on the diffusion of research tools and
the advancement of the subject [93, 94 ].
10.21.4 Regulatory frameworks
It is essential to have regulatory frameworks in place to guarantee the development
and deployment of protein engineering technologies safely. They encompass rules,
guidelines, and procedures established by authorities to ensure safety, efficacy, and
ethical practices in the field of protein engineering [95]. Regulatory frameworks
worldwide have struggled to keep pace with new technologies, particularly in the
field of genetic modification and genome editing, which are integral to protein
engineering. The rapid advancement in these technologies exerts pressure on existing
regulatory frameworks, necessitating their evolution to address the novel challenges
and opportunities posed by these advancements. In a QbD-centered regulatory
framework, the emphasis is on ensuring product quality through the design of the
process itself. This approach is crucial in therapeutic protein product and process
development, where the quality of the engineered proteins is vital. The regulatory
pathway for therapeutic proteins, a significant aspect of protein engineering, often
involves rigorous scrutiny by regulatory bodies like the U.S. Food and Drug
Administration (FDA). The approval process examines therapeutic proteins’ safety,
efficacy, and quality, ensuring they meet the stringent regulatory standards before
being introduced to the market [96].
10.22 Studies in protein engineering
The creation of therapeutic proteins is one area where protein engineering has
become more important in the medical industry. The intricate design and engineering of proteins enable the treatment of various diseases, often surpassing the
capabilities of traditional small-molecule drugs [96].
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10.22.1 Therapeutic proteins
Protein-based therapies engineered in the lab have caused major changes in the way
diseases are treated. By 2023, proteins are expected to make up half of the top ten
selling pharmaceuticals, with effectiveness on par with or perhaps exceeding that of
many currently used small molecule-based therapies [97]. After the first recombinant
protein-based treatment, Humulin, was authorized by the FDA in 1982, the market
for protein-based pharmaceuticals exploded to an estimated $400 billion with
hundreds more candidates approved and in clinical studies. The potential of
protein-based therapeutics lies in the versatility of proteins, which can act as catalysts,
signaling molecules, transporters, and more. Their high specificity and potency are
particularly advantageous, allowing them to execute complex functions owing to their
intricate three-dimensional structures [97]. Strategic structural and chemical modifications directly made to protein structures have been crucial in overcoming challenges
such as protein aggregation, degradation, and denaturation inherent to protein-based
therapeutics. These design strategies have significantly improved in vivo stability,
pharmacokinetics, cell permeability and reduced undesired immunogenicity. Protein
engineering has enabled the development of various therapeutic proteins, including
antibodies and enzymes. Antibodies, for instance, specifically target antigens, blocking
specific signaling pathways or inducing cell death. They can also serve as transporters
for targeted drug delivery, as seen in antibody–drug conjugates like trastuzumab
emtansine. Enzyme-based drugs, conversely, can replace deficient or absent enzymes
catalyzing the degradation or modification of therapeutically relevant targets, with
examples including PEG-asparaginase and laronidase. A specific example of engineered therapeutic protein is Superoxide Dismutase, designed for effective diagnostics,
biotherapeutics, and biocatalysts, highlighting the potential outcomes of protein
engineering [88]. The field still faces challenges related to product heterogeneity,
process monitoring, and analytics during the development and production of
therapeutic proteins. Addressing these challenges is crucial for ensuring the efficacy
and safety of protein-based therapeutics. Promising therapeutic protein techniques
have been developed for the treatment of cancer, with a focus on the pharmacological
profile and targeted therapy. The strengths and weaknesses of protein-based medicines
for cancer therapy are highlighted, which also provides a thorough overview of the
field’s present state and prospective future possibilities. Continuous efforts have been
invested to enhance the production and efficacy of protein therapeutics, addressing the
demand for these potent biomolecules in modern medicine [98].
10.22.2 Industrial enzymes
The manufacturing of many useful goods for industry, such as drugs, animal feed,
chemicals, cleaning agents, and biofuels, has been greatly facilitated by industrial
enzymes. Protein engineering is a crucial part of making these enzymes more
effective at their jobs. Post-translational enzyme modification, structure-assisted
protein tailoring, and computational modeling approaches have all contributed to
the development of methods for more effective biocatalyst manufacturing. Enzyme
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variations have been created via protein engineering that exhibit enhanced catalytic
activity, widened or changed substrate specificity, and enhanced or reversed stereoselectivity. The widespread interest in enzyme-based processing technologies stems
from their potential for environmentally responsible product creation in a variety of
industrial settings. The pharmaceutical, food and feed, chemical, detergent, and
biofuel industries are just a few that are beginning to realize the vast industrial
potential of enzymes as biocatalysts [99]. By demonstrating how easily the sequence
of a protein can be modified to produce enzymes with improved functional
properties like stability, specific activity, and inhibition, recent advances in protein
engineering have had a significant impact on the development of commercially
available enzymes into better industrial catalysts. The considerable progress
achieved over the last three decades in recombinant DNA technology and the tools
of protein engineering is producing solutions that answer the huge unmet demands
of consumers and markets [100].
10.22.3 Diagnostic proteins
The engineering of diagnostic proteins is a pivotal application of protein engineering, enabling the development of robust diagnostic tools for various diseases.
Reliable diagnostic tools have been developed using techniques from the area of
protein engineering. These genetically engineered diagnostic proteins have shown
remarkable potential in the pharmaceutical and enzyme industries, where they may
help identify and manage several diseases [88]. Protein engineering aims to create
diagnostically useful new proteins. Protein structural manipulation permits the
development of diagnostic proteins, which may aid in diagnosing diseases earlier,
leading to better health outcomes. Protein engineering that uses light to regulate the
movement of kinesin and myosin motors along microtubules has potential diagnostic applications. Engineered diagnostic proteins have applications in various
domains, including biotherapeutics and biocatalysts. Technologies such as created
natural protein variations, Fc fusion protein, and antibody engineering, which also
have diagnostic uses, have a major influence on the state of the art in protein
treatments today. Protein engineering magnifies the already impressive range of
molecular tasks performed by proteins that have evolved spontaneously. Proteins
with optimized three-dimensional structures may be powerful diagnostic tools,
offering a wide range of molecular functions that can be used in the detection,
monitoring, and treatment of diseases [101].
10.23 Single-molecule techniques in protein engineering
Single-molecule techniques in protein engineering are vital for elucidating the
intricate behaviors and properties of individual molecules which are often obscured
in bulk measurements. Among these techniques, atomic force microscopy (AFM)
stands out for its versatility and precision in characterizing molecular structures and
interactions [102].
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10.23.1 Atomic force microscopy
Providing three-dimensional topographic pictures and structural features of materials, AFM has emerged as a leading technology working at the single-molecule level.
The atomic force microscope, which was developed in 1986, is a powerful instrument
for studying things at the nanoscale. It uses a cantilever to scan surfaces. AFM has
developed into a potent nanoscopic platform that makes it possible to characterize a
wide variety of biointerfaces, both synthetic and biological. Such work has greatly
benefitted the study of protein molecular and hierarchical assembly, especially of
misfolded species that occur during protein aggregation, and the monitoring of their
dynamics at the nanoscale [103]. AFM, along with its variants and hybrid
techniques, offers molecular data to support studies in the biochemical area of
protein engineering. The single-molecule statistical approach offered by AFM finds
applications in the solving of molecular assemblages and the structural characteristics of functional nanomaterials inspired by amyloid. AFM-based single-molecule
force spectroscopy (AFM-SMFS) research has been developed owing to contributions from a range of domains, including developments in surface chemistry, issues
in protein engineering, and diverse data processing theories and methodologies.
These improvements provide light on the growth of AFM methods, resulting in the
instrument being a valuable resource for studying protein behaviors and interactions
at the single-molecule level [104].
10.23.2 Single-molecule FRET
It is critical to investigate the dynamics of biomolecular structure using the singlemolecule Forster Resonance Energy Transfer (smFRET) method. The basic idea
behind smFRET is that energy may be transmitted between two fluorophore
molecules that are near to each other. This allows for the monitoring of nanoscale
distances as well as changes within them. The primary applications of this method
are nanoscale examinations of the conformations and dynamics of biomolecular
structures, which are often carried out in real time. Fluorescence microscopes are
often employed in this procedure, and the molecules being studied may be surfaceimmobilized or allowed to spread freely. Individual FRET pairs are highlighted by
lasers and other bright light sources, resulting in fluorescence signals strong enough
for single-molecule detection [105].
10.23.3 Optical tweezers
In recent years, there has been a change in the focus of research into the alteration of
single molecules toward the use of optical tweezers. This method makes use of light’s
ability to impose forces and torques on individual molecules in order to directly
measure the forces and torques that are created by biological processes. Optical
tweezers may be traced back to the work that Arthur Ashkin did in the 1970s and
1980s. In this study, light was used to capture and manipulate micron-sized latex
spheres, bacteria, red blood cells, and organelles inside cells. This was the beginning
of the development of optical tweezers. The versatility of optical tweezers is
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highlighted by their capability to measure not only forces and displacements but also
torques and angles. A notable enhancement to the method includes integrating
single-molecule fluorescence detection capabilities. Optical tweezers have a wide
variety of uses, including but not limited to the investigation of protein–nucleic acid
interactions, the folding of proteins and RNA, and the operation of molecular
motors. The data from optical tweezer experiments offer vital insights, with data
reproducibility and variability challenges across different laboratories. To transcend
these challenges and optimize instrument operation, data extraction, and analysis
indicates promising opportunities for future advancements in the field [106, 107].
10.23.4 Patch-clamp technique
The patch-clamp technique allows for precisely measuring ionic currents flowing
through a cell’s plasma membrane. It can either monitor currents passing through
single ion channels or those traversing the entire plasma membrane. This method
has been ideal in studying the functions and dysfunctions of electrically
excitable cells and their networks [107]. Utilizing a glass electrode, the patch-clamp
technique establishes a tight seal on a cell’s surface, enabling the direct measurement
of membrane potential and the amount of current passing across the cell membrane.
It is the only technique that can reliably record electrical activity within a single
neuron which make it as an exceptional resource. The precise observation and
manipulation of ionic currents are only two of the many scientific uses for this
technique [108]. Three decades ago, the patch-clamp method was developed, and it
revolutionized the study of cellular physiology and biophysics. Researchers were
able to get a better understanding of the physiological role of a single protein, in this
instance an ion-permeable channel in the plasma membrane of a cell. This was a
tremendous step forward for neurology and allied disciplines, as it opened the door
to research of the cellular and molecular processes underlying neurological ailments.
This approach may be used to study many other things; for example, ionic currents
in the heart, the excitability of neurons, and the electrophysiological characteristics
of different cell types [109]. Research into the biological basis of electrical signaling
and the functional dynamics of ion channels would benefit greatly from these
findings. Despite its widespread use, the patch-clamp method has certain limitations,
such as the need for a high signal-to-noise ratio in recordings, the fabrication of
electrodes, and the isolation of myocytes. Ongoing technical improvements, however, are helping to enhance the method’sefficacy, broaden its applicability, and
overcome its existing limitations [109].
10.24 High throughput screening methods
High throughput screening (HTS) allows for the quick evaluation of many
compounds’ biological or biochemical activity, making it an essential tool in the
early stages of drug development. HTS aims to find promising compounds, or ‘hits,’
that may be further developed into helpful medicinal medicines [110]. To find
potential ways for drug development screening large libraries of chemicals against a
particular target or pathway of interest. Novel biomaterials and techniques for
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modifying them have proliferated in recent years intending to imitate the intricate
microenvironments of genuine tissues, thereby improving the dependability and
utility of HTS. The integration of NGS and ML is transforming the screening design
and workflows, boosting the effectiveness of HTS [110].
10.24.1 Fluorescence-activated cell sorting (FACS)
Using physical and fluorescent properties, cells may be separated using a technique
called fluorescence-activated cell sorting (FACS), which is a subset of flow
cytometry. High-purity enrichment of certain cell populations is made possible by
FACS’s ability to sort a population of cells into subsets depending on the sum
quantity of essential biomarkers produced by the cells [111]. It finds extensive
application in separating and isolating antigen-specific B lymphocytes in a highthroughput manner, among other uses, thereby promoting the development of
valuable reagents for immunological research. The technique utilizes fluorescent
labeling of cells followed by their detection and sorting based on individual cellular
characteristics, such as size and granularity, in addition to their fluorescence. FACS
is often used in conjunction with other emerging technologies like droplet-based
microfluidics to enhance its throughput and application scope [112].
10.24.2 Microfluidics-based assays
Microfluidics-based assays are instrumental in precision medicine, particularly in
oncology, providing a more complex understanding of tumor behavior in response
to treatments. Functional assays, which directly assess treatment responses on live
cells while also taking into account parameters such as tissue of origin, tumor
microenvironment, and immune response, are improved by microfluidic technology.
In order to measure how well a therapy is working, functional assays might be used.
They include a wide range of non-genomic cell-based tests. These assays aim to
create an evolving system where the effect of molecular changes, as well as
microenvironmental factors, can be captured over time, thus providing more robust
predictors for optimal treatment identification [113]. Microfluidic models only need
a small number of cells, therefore they may be used with small samples like those
obtained from patient-derived biopsies. By simulating the complex tumor microenvironment, which includes elements impacting treatment response such as
nutrition, waste products, chemokines, and diverse cell types, these tests have the
potential to better predict treatment responses than conventional ones.
Microphysiological systems and ‘organs on a chip’ are two recent developments
that aim to replicate tissue/tumor function and therapy response. Multiple human
diseases have been investigated using the technique, but recent years have seen
particularly promising results in the study of cancer [114].
10.24.3 Yeast surface display
Yeast surface display (YSD) is a technology that displays recombinant proteins on
the yeast cell surface, which finds applications across a broad spectrum of
biotechnology and biomedical fields. YSD is favored due to several advantages,
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including its safety status by the FDA and the capability of yeast cells for PTMs,
which is crucial for displaying complex proteins. The method includes joining
recombinant proteins genetically to a protein found in high quantities in cell walls.
This is very useful for protein engineering projects since it allows the proteins to be
shown on the surface of yeast cells [115]. YSD has been used in various domains,
including the creation of anti-cancer antibodies and the selection of binding proteins
from scaffold protein combinatorial libraries. Research into creating carrier-free
immobilized enzymes for biocatalysis includes several possibilities of inquiry into
improving the efficiency of YSD systems for biotechnological applications [116].
10.24.4 Mass spectrometry-based methods
Methods based on mass spectrometry (MS-based methods) are essential for
revealing details about biological and molecular mechanisms. Proteomic analysis
based on MS is a significant method for finding novel disease biomarkers. It
contributes to the molecular knowledge of diseases, essential for advancing
customized treatment, particularly concerning complicated diseases like inflammatory bowel disease and diabetes [116]. Secondary ion mass spectrometry (SIMS),
inductively coupled plasma mass spectrometry (ICP-MS), laser desorption ionization mass spectrometry (LDI-MS), and electrospray ionization mass spectrometry
(ESI-MS) are all types of MS-based procedures (ESI-MS). These methods are used
for studying individual cells. These techniques enable the investigation of single cells,
expanding our understanding of the variety and functioning of biological systems
[117]. Other technologies for improved characterization and analysis of complex
materials such as lignin include matrix-aided laser desorption ionization and timeof-flight-secondary ion mass spectrometry. Profiling the metabolomic abundance of
biological materials is an everyday use of MS-based approaches. Understanding the
fluctuations in metabolite concentrations under various biological situations is
essential for diagnosing and treating illness, and differential abundance analysis
may assist with this. Over the years, advancements in MS-based technologies have
made them well-suited for biomarker discovery. The specificity and sensitivity of MS
have significantly contributed to the expansion of the proteomics field, supporting
the discovery of biomarkers crucial for understanding disease pathology and
developing new diagnostic and therapeutic strategies. While MS-based methods
have the potential to advance biomarker discovery significantly, challenges such as
improper experimental design, lack of standardized procedures, and quality control
during sample collection and analyses can delay the reproducibility and clinical
relevance of discovered biomarkers. Ensuring rigorous experimental design, proper
sample collection, and validation of identi fi ed biomarkers are crucial for overcoming
these challenges [118].
10.25 Protein engineering for nanotechnology
Protein engineering for nanotechnology explores the intersection of protein biology
and nanotechnology, focusing on repurposing protein molecules as nanostructures
and nanoscaffolds. This field enables the design and creation of protein-based
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