In vitro modification

For the purpose of this chapter, enzyme-catalyzed modifications of proteins will be divided into two groups hydrolytic and nonhydrolytic reactions. Generally speaking, post-translational reactions occurring in vivo are catalyzed by highly specific enzymes under rather restricted conditions in contrast with in vitro modifications which are carried out under less specific conditions. 

Decreased biotin affinity ([22,31] in vivo modification of gene [28] and in vitro modification of protein (e.g. nitro-streptavidin) [32])... 

Boussard P, Devleeschouwer MJ, Dony J. In vitro modification of antimicrobial efficacy by protamine. Int J Pharm 1991 72 51-55. 

Kling D, Kunkle J, Roller AS, et al. 1978. Polychlorinated biphenyls In vivo and in vitro modifications of cholesterol and fatty acid biosynthesis. J Environ Pathol Toxicol 1 813-828. 

Studies of PTM have been benefited from the recent advancements in mass spectrometry, the introduction of new software and Internet-based MS data search facilities, computer-assisted topology prediction for a variety of PTMs (visit http // ca.expasy.org/), chemical synthesis of modified peptides and proteins, development of modified peptide specific antibody, in vitro modification techniques, exploitation of other eukaryotic cells such as insect cells for protein expression [4, 5, 16, 32], and progress in affinity purification of modified proteins. 

In vitro modification of proteins, producing a variety of well-defined amino acid derivatives, can occur as a result of deliberate manipulation or by inadverent modification during isolation or subsequent handling. In either case, the derivatives, if properly identified, can provide useful information about that protein or peptide. Many are stable to acid or alkali, thus facilitating their determination, and all, at least in theory, should be recoverable from total enzymatic digests. This recovery can be facilitated if the substituent contains a radioactive nuclide or has some other prominent chemical characteristic such as absorption in the visible region of the spectrum. 

Orehek J, Douglas JS, Bouhuys A (1975) Contractile responses of the guinea pig trachea in vitro modification by prostaglandin synthesis-inhibiting drugs. J Pharmacol Exp Ther 194 554-564... 

During development of chicken skeletal muscle cells, the marked increase in Ca transport of sarcoplasmic reticulum, observed both in vivo and in vitro systems, can be interpreted mainly as the result of an increase in the concentration of Ca transport ATPase. Changes in the fatty acid composition of muscle membranes developed in vivo occur with a balance between chain length and unsaturation, without affecting significantly their Ca permeability. In cultured muscle cells their fatty acid composition can be manipulated to a great extent by lipid supplementation of the culture medium. The effects of these in vitro modifications in lipid composition on the calcium transport function of muscle membranes should be investigated. 

The enzymes forming new glycosidic linkages are involved in the synthesis of hemicelluloses and exist in all plants. They are, however, not yet available for in vitro modifications of hemicelluloses. Plant polysaccharide synthetases are still poorly characterized and only a few of the enzymes participating in the synthesis of hemicelluloses have been isolated and characterized (41). Furthermore, even fewer of them have been cloned and produced in another host organisim in significant amounts. 

Stem cell-derived blood cells could provide potentially unlimited and on-demand source of therapeutic cells for a variety of clinical applications, including bone marrow transplantation, adoptive T cell and dendritic cell therapies, as well as for blood transfusions, for example, platelet or red blood cell therapy. Some of these applications, for example, bone marrow transplantation, has been used for decades and have revolutionized modern medicine. Yet, current paradigms of isolating donor or patient cells for acute or future transplantation, with or without in vitro modification, are unsustainable in the face of high demand and immediate needs. The true impact of these therapies could only be realized if blood lineage cells (hematopoietic stem and progenitor cells, dendritic cells, T cells, red blood cells, platelets, etc.) are available on-demand and as ready-to-use therapeutics. 

---