Peptide recombinant expression

The N-terminal region of fibrillin-1 (the first 29 residues after signal peptide) contains largely basic residues (estimated pi, 11.1). There is an N-terminal putative furin cleavage site at the N-terminus (RAKRjR residues 41-45). Recombinant secreted N-terminal fibrillin-1 can be furin processed (Raghunath et al., 1999 Ritty et al., 1999 Wallis et al., 2003). In our mammalian recombinant expression system, a significant level of... 

In contrast to prophylactic DNA-based vaccination that is used to prevent infectious diseases, therapeutic DNA-based vaccination is aimed to combat cancer [79]. Many tumor cells express TAAs, which are not expressed by normal cells (see Part V, Chapter 6). Therefore, these TAAs may serve as the molecular basis of recognition of tumor cells by the immune system. The DNA of TAAs is administered to the patient (e.g., intradermally), whereupon some of the patienfs cells are transfected and thereafter express TAA. Recombinant expression of TAA, especially from APCs in the skin, induces a systemic immune response towards the TAAs and, therefore, to all tumor cells expressing those molecules [80, 81]. Instead of using the DNA of the whole TAA, the DNA of tumor-specific peptides may also be used. 

Ribosomes needed for translation in the PURE system are isolated from E. coli using sucrose-density gradient centrifugation. The protein factors necessary for translation in E. coli are recombinantly expressed as His-tagged fusions, and purified to homogeneity. These include the factors for initiation (IFl, IF2, and IF3), elongation (EF-G, FF-Tu, FF-Ts), peptide chain release (RFl and RF3), ribosome recycling (RRF), methionyl-tRNA transformylase (MTF) for formylation of the initial Met-tRNA, and the 20 aminoacyl-tRNA synthetases (ARSs) for transfer RNA (tRNA) recy-... 

HPCE continues to become more widely used for the deteetion, separation, and quantification of peptides and proteins, since the use of capillaries greatly reduces sample volume and analysis time compared to conventional gel eleetrophoresis. This offers perfect qualification for the analysis of PTMs, but surprisingly only a small number of HPCE apphcations have been developed during the past years. Since these modifications represent phenomena that occur in vivo often in very small amounts, they can be easily missed. This may explain why in eertain cases recombinantly expressed proteins have an altered or absent aetivity compared with the naturally expressed proteins. 

In order to determine the selectivity of each peptide sensor substrate, assays with a panel of recombinantly expressed, activated kinases can be performed (Figure 1.7b). In this case, the concentration of substrate peptide is held constant at two to three times the determined K. For example, the p38 sensor depicted in Figure 1.6 was incubated with various MAP (mitogen-activated protein) and non-MAP kinases and fluorescence emission was monitored. Figure 1.7b demonstrates the selectivity of the p38 sensor for the target kinase [23]. Following this initial screen, kinase selectivity can be addressed directly in cell lysates. Cells can be stimulated to activate the kinase of interest and kinase activity can be determined in the presence and absence of a selective inhibitor of the kinase of interest. Residual activity in the presence of inhibitor would indicate sensor cross talk with other kinases. 

Tripet B, Yu L, Bautista DL, Wong WY, Irvin RT, Hodges RS. Engineering a de novo-designed coiled-coil heterodimerization domain for the rapid detection, purification and characterization of recombinantly expressed peptides and proteins. Protein Eng 1996 9(ll) 1029-42. 

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