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Biophysical and Biochemical Characterization of Peptide, Protein, and Bioconjugate Products
Published in Sandeep Nema, John D. Ludwig, Parenteral Medications, 2019
Tapan K. Das, James A. Carroll
Simply put, the primary structure of a protein consists of its amino acid sequence. For recombinant proteins, the amino acid sequence can be predicted from the cDNA used in its production. This basic attribute of a protein determines the entirety of its biophysical and biochemical properties under given conditions. The amino acid sequence of a protein determines its ability to fold properly and thus determines its ability to maintain its function. Therefore, a change in the primary structure, depending on its location, may have a range of effects on a protein’s activity, from no effect to a very large impact. The amino acid sequence can also impact the chemical and physical stability of a protein, even when there is no measurable impact on activity. Thus, confirming the amino acid sequence of a protein is fundamental to understanding its overall structure and properties.
General Introductory Topics
Published in Vadim Backman, Adam Wax, Hao F. Zhang, A Laboratory Manual in Biophotonics, 2018
Vadim Backman, Adam Wax, Hao F. Zhang
Following translation on a ribosome, a protein must undergo posttranslational modifications and folding. Protein folding is an extremely complex process, as the three-dimensional structure of proteins is of crucial importance to their function. Chaperones are special protein complexes that help proteins fold correctly. High density of intracellular environment, called molecular crowding, helps increase the rate of folding. Without crowding, if left to their own devices, the time required for proteins to fold would be dramatically increased. Indeed, approximately 30% of the space inside a cell is physically occupied by macromolecules. Due to steric interactions and volume exclusion, most of the remaining space is excluded for macromolecular interactions. As a result, as little as 1% of cell volume remains for macromolecular processes such as mRNA diffusion and protein folding to take place. An increase or decrease in the available volume is expected to significantly change many cellular processes.
Protein structure prediction
Published in A. K. Haghi, Lionello Pogliani, Devrim Balköse, Omari V. Mukbaniani, Andrew G. Mercader, Applied Chemistry and Chemical Engineering, 2017
Proteins play a key role in almost all biological processes. Proteins are important for maintaining the structural integrity of cell, transport, storage, regulation, signaling, immunity, and act as a catalyst. Proteins are made up of small peptides and these peptides are actually chains of amino acids. Proteins fold themselves in different conformations and these conformations are responsible for functions of protein. An accurate structural characterization of proteins is provided by X-ray but there are limitations of these methods. In spite of that, these methods are time-taking methods and labor intensive. Nowadays, protein structures are created computationally and deposited in Protein Data Bank (PDB) (http://www.rcsb.org). On the basis of structures, proteins are divided into four major groups.
Bioprocessing of recombinant proteins from Escherichia coli inclusion bodies: insights from structure-function relationship for novel applications
Published in Preparative Biochemistry & Biotechnology, 2023
Kajal Kachhawaha, Santanu Singh, Khyati Joshi, Priyanka Nain, Sumit K. Singh
In-vitro protein folding is done by decreasing the denaturant concentration of the solubilized IB, thereby driving the equilibrium toward the native folded state of the protein. However, the transition from the solubilized, unfolded, to the natively folded form of the protein is associated with instances of protein misfolding and aggregation.[120] As such, aggregation and protein misfolding constitute the biggest challenge during protein refolding as it impacts the final product’s overall process economics and quality. Protein refolding is a strong function of the initial concentration of the unfolded protein and follows first-order kinetics. On the contrary, protein aggregation is a higher-order process and is accelerated at the higher initial concentration of the proteins.[121] Thus, protein refolding is strongly favored under dilute conditions (approx. 0.01–0.1 mg/mL), wherein the molecular collisions between the unfolded and folding intermediates are minimized. The stability of the protein folding intermediates is enhanced by the addition of several molecular excipients (folding enhancers and aggregation suppressors) such as sucrose, L-arginine, ethyl ammonium nitrate (EAN), trifluoroethanol, cyclodextrins, and alginates.[122]
Using chemical chaperones to increase recombinant human erythropoietin secretion in CHO cell line
Published in Preparative Biochemistry and Biotechnology, 2019
Mehri Mortazavi, Mohammad Ali Shokrgozar, Soroush Sardari, Kayhan Azadmanesh, Reza Mahdian, Hooman Kaghazian, Seyed Nezamedin Hosseini, Mohammad Hossein Hedayati
Chemical chaperones are small molecules that assist molecular chaperones to fold a protein in endoplasmic reticulum also integrate into the protein structure to protect its folding in the secretory pathway. Molecular chaperones and chemical chaperones collaborate with each other to reduce misfolded protein response and enhance protein secretion.[5,15] Such collaboration happens as a result of the increased activity of molecular chaperones after treatment of the cells by chemical chaperone. Also chemical chaperones cooperate with molecular chaperones by adjust their activity.[16] Chemical chaperones are from different groups of components, including polyols such as glycerol, methylamines such as trimethylamine N-oxide (TMAO), sugar, and amino acid derivatives. It is worth mentioning that media optimization is currently the most important plan in recombinant protein production using CHO cell line. The existing challenges in bioprocessing tasks such as low yield and aggregation can be studied and resolved to improve protein production using chemical chaperones through handling molecular chaperones.[3]
Light Harvest: an interactive sculptural installation based on folding and mapping proteins
Published in Digital Creativity, 2018
Jiangmei Wu, Susanne Ressl, Kyle Overton
Proteins are large molecular chains with hundreds to thousands of atoms that adopt unique and perfect 3D structures through a unique self-folding process, placing chemical groups in just the right places to catalyze and build cellar functions. The process by which the atoms in a protein find their correct location to fold has fascinated structure biologists for decades, as how a protein folds and unfolds has important implications in medicine. Structural biologists use X-ray crystallography (Rupp 2010) to map the folded 3D structures of proteins through complex data collection, analysis and interpretation processes. X-ray crystallography allows the generation of very high resolution images of the molecules of life. To record high resolution X-ray diffraction images of protein crystals (Figure 1), large circular particle accelerators, synchrotrons, are used. A synchrotron emits high energy X-rays that are used to shoot protein crystals and record their diffraction images for subsequent structure determination. To study a protein folded 3-D structure, hundreds of X-ray diffraction images must be collected. A typical X-ray diffraction pattern often has thousands of spots corresponding to electron locations of atoms in a protein, and each of these spots must be measured and mapped using mathematical and computational tools to calculate a protein’s electron density map (Figure 2). These maps are then used to determine the 3-D coordinates of each atom’s positions in a given folded protein structure.