Protein-based materials expression

As stated in Chapter 9, The consilient approach to tissue engineering utilizes biology s own materials and mechanisms, concerned with tissue structure and function, to achieve tissue restoration. The key materials elements are three the capacity of the elastic protein-based material to match the elastic modulus of the tissue to be restored, a remarkable biocompatibility of the pure elastic protein-based material, and the facility to design into the protein-based polymer sequence any desired biologically active sequence, which, by virtue of the innocuousness of the elastic protein-based material, allows proper expression of the incorporated biologically active sequence. This provides the opportunity for the proper functional relationship of protein-based material to the cells and the extracellular matrix of the tissue to be restored. 

Protein based assays are influenced by the relative amount of protein in the cells, the so-called expression level. Expression of a specific protein varies according to different parameters, such as climate, soil, drought conditions, etc. If the expression level in a given sample is different from the expression level of the materials used as calibrators, additional uncertainty will impact the result expressed in mass fractions. While again, for individual and intact kernels other analytical approaches (pooled sampling procedures from GIPSA, see above) are possible that would not be affected by this phenomenon, this would not be applicable for processed samples. 

Similar to protein-based biopharmaceuticals, the standard battery of genotox-icity studies is not considered to be relevant for cell-based therapies unless there is a specific cause for concern regarding the nature of any expressed products that would indicate a potential interaction directly with DNA or other chromosomal material [50,52], The conduct of genotoxicity studies for assessing the genotoxic potential of process related contaminants is also not considered to be appropriate [50],... 

Non-natural amino acids can be incorporated into peptides and polypeptides via several different methodologies. Solid-phase peptide synthesis (SPPS) is a straightforward method for incorporation of non-natural amino acids and allows the incorporation of essentially any amino acid but is limited by the size of the peptides produced 18). Suppression-based strategies, both in vitro and in vivo, have been developed for site specific incorporation of diverse set non-natural amino acids into natural and synthetic polypeptides 19). Alternatively, auxotrophic expression hosts have been used for multisite incorporation of nonnatural amino acid in protein polymers, where multiple natural amino acids of one type can be replaced with non-natural analogues during protein biosynthesis (20, 21). Multisite incorporation of non-natural amino acids in the synthesis of protein polymeric materials facilitates chemical modification at multiple sites and can modulate the physical properties of the protein polymers (22). 

A summary of the industrial-scale process development for the nitrilase-catalyzed [93] route to ethyl (/ )-4-cyano-3-hydroxy-butyrate, an intermediate in the synthesis of Atorvastatin (Pfizer Lipitor) from epichlorohydrin via 3-hydroxyglutaronitrile (3-HGN) was recently reported (Figure 8.15) [94], The reaction conditions were further optimized to operate at 3 m (330 gL ) substrate, pH 7.5 and 27 °C. Under these conditions, 100% conversion and product ee of 99% was obtained in 16 h reaction time with a crude enzyme loading of 6% (based on total protein, 0.1 U mg-1). It is noted that at pH < 6.0 the reaction stalled at <50% conversion and at alkaline pH a slowing in reaction rate was observed. Since the starting material is of low cost and the nitrilase can be effectively expressed in the Pfenex (Pseudomonas) expression system at low cost, introduction of the critical stereogenic center... 

A number of proteomic studies on archival material have utilized Liquid Tissue (Expression Pathology, Inc., Gaithersburg, MD), a commercial protein extraction kit for FFPE tissue.4,9,25-28 This kit is also based upon HIAR techniques and shares a similar work flow to the methods already discussed. Thin, typically 5-10pM, sections are cut from paraffin tissue blocks, the paraffin is removed, and the tissue deparaffinized and rehydrated in alcohols and distilled water before microdissection. The cellular material is then suspended in Liquid Tissue buffer and heated at 95°C for 90 min. Trypsin is added, and the material is digested overnight at 37°C prior to reduction with DTT and analysis by LC-MS/MS.26... 

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