Biophysical and Biochemical Analysis of Recombinant Proteins
For a recombinant protein to become a human therapeutic, its biophysical and biochemical characteristics must be well understood. These properties serve as a basis for comparison of lot-to-lot reproducibility; for establishing the range of conditions to s
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Biophysical and Biochemical Analysis of Recombinant Proteins Tsutomu Arakawa and John S. Philo
Introduction For a recombinant protein to become a human therapeutic, its biophysical and biochemical characteristics must be well understood. These properties serve as a basis for comparison of lot-to-lot reproducibility; for establishing the range of conditions to stabilize the protein during production, storage, and shipping; and for identifying characteristics useful for monitoring stability during long-term storage. A number of techniques can be used to determine the biophysical properties of proteins and to examine their biochemical and biological integrity. Where possible, the results of these experiments are compared with those obtained using naturally occurring proteins in order to be confident that the recombinant protein has the desired characteristics of the naturally occurring one.
Protein Structure Primary Structure Most proteins which are developed for therapy perform specific functions by interacting with other small and large molecules, e.g., cell-surface receptors, binding proteins, nucleic acids, carbohydrates, and lipids. The functional properties of proteins are derived from their folding into distinct three-dimensional structures. Each protein fold is based on its specific polypeptide sequence in which different amino acids are connected through peptide bonds in a specific way. This alignment of the 20 amino acids, called a primary sequence, T. Arakawa, Ph.D. (*) Department of Protein Chemistry, Alliance Protein Laboratories, 3957 Corte Cancion, Thousand Oaks, CA 91360, USA e-mail: [email protected] J.S. Philo, Ph.D. Department of Biophysical Chemistry, Alliance Protein Laboratories, 3957 Corte Cancion, Thousand Oaks, CA 91360, USA
has in general all the information necessary for folding into a distinct tertiary structure comprising different secondary structures such as α-helices and β-sheets (see below). Because the 20 amino acids possess different side chains, polypeptides with widely diverse properties are obtained. All of the 20 amino acids consist of a Cα carbon to which an amino group, a carboxyl group, a hydrogen, and a side chain bind in L configuration (Fig. 2.1). These amino acids are joined by condensation to yield a peptide bond consisting of a carboxyl group of an amino acid joined with the amino group of the next amino acid (Fig. 2.2). The condensation gives an amide group, NH, at the N-terminal side of Cα and a carbonyl group, C = O, at the C-terminal side. These groups, as well as the amino acyl side chains, play important roles in protein folding. Due to their ability to form hydrogen bonds, they make major energetic contributions to the formation of two important secondary structures, α-helix and β-sheet. The peptide bonds between various amino acids are very much equivalent, however, so that they do not determine which part of a sequence should form an α-helix or β-sheet. Sequence-dependent secondary structure formation is determined by the side chains. The 20 amino acids co
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