By Larry DeLucas
This quantity of present subject matters in Membranes specializes in Membrane Protein Crystallization, starting with a evaluate of previous successes and normal tendencies, then extra discussing demanding situations of mebranes protein crystallization, mobilephone unfastened construction of membrane proteins and novel lipids for membrane protein crystallization. This ebook additionally comprises instruments to enchance membrane protein crystallization, method developments, and crystallization innovations used for photosystem I and its complexes, constructing Membrane Protein Crystallization as a wanted, useful reference for researchers.
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Extra info for Current Topics in Membranes, Volume 63
The so‐called Expression‐PCR (E‐PCR) was originally developed for prokaryotic cell‐free systems and this methodology is based on the amplification of a single gene within a two‐step PCR, while simultaneously supplying the PCR product with the necessary regulatory elements for transcription and translation and optional elements for purification (Merk, Meschkat, & Stiege, 2003). The T7 promoter and an additional sequence stretch for unhindered transcription, a hairpin sequence protecting the 50 ‐end of the mRNA against degradation, the epsilon sequence, and the ribosome binding site from T7 gene10, as well as a spacer sequence to the translation start codon ATG, are introduced upstream of the translated gene.
11A). Resulting E‐PCR products were applied directly to the linked transcription‐translation procedure for cell‐free protein synthesis. Radiolabeled in vitro translation products were separated on an SDS gel and ICOS receptor synthesis in the insect cell lysate was analyzed. The results of this experiment demonstrated that expression of the ICOS receptor without a signal peptide at the N‐terminus resulted in the accumulation of the nonglycosylated protein within the lysate as expected (Fig. 11B).
RESULTS A. Extract Preparation High‐yield in vivo expression of biologically active recombinant protein is frequently achieved in cell lines derived from the fall army worm Sf or from the cabbage looper Trichoplusia ni (Vaughn, Goodwin, Tompkins, & McCawley, 1977). Recombinant baculoviruses containing the gene of interest are usually propagated in these insect‐derived cell lines. Particular insect cell lines, for example, Sf cells, grow well in suspension cultures and these cells can be easily scaled up for the large scale production of recombinant proteins.
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