PubMed · 11663689
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JAW McDonald, Roger Lewin. 1976-11-11. Genetic engineering.. https://pubmed.ncbi.nlm.nih.gov/11663689/
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For structural and functional genomics programs, new high-throughput methods to characterize well-expressing and highly soluble proteins are essential. A faster and more convenient approach to screen expression conditions of recombinant proteins compared to classical in vivo systems is the Escherichia coli cell-free expression system. Here, we describe a rapid procedure to screen for expression and solubility of recombinant proteins using an E. coli cell-free extract. The results presented cover 24 open reading frames of unknown function from different micro-organisms. In order to screen different variables that may interfere with solubility, we expressed the recombinant proteins with a histidine6 tag, either N-terminal or C-terminal at two temperatures (25 degrees C and 30 degrees C). The identification of recombinant proteins is performed by the dot blot procedure using an anti-histidine tag antibody. We designed a rapid method that allows the characterization of soluble candidates from a large number of genes or from a large number of variants that is highly compatible with structural genomics expectations.
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Electroporation-based gene therapy has become a "hot field" for non-viral gene delivery. This review summarizes the progress made in intramuscular and intratumoral electrogenetransfer, which include new applications and modifications. The progress in dendritic cell (DC) and stem cell transfection by use of electroporation has also been discussed. Rapid progress during the past two years clearly demonstrates the great potential of this technology, but there are challenges faced by both in vitro and in vivo applications, which include how to enhance the transfection efficiency for intratumoral delivery, how to extend the duration of gene expression for intramuscular injection, and how to increase the survival rate for in vitro cell transfection. Resolving these issues will shed new light on this field.