Strategies for achieving high-level expression of genes in Escherichia coli.

Vectors for gene expression in mammalian cells

“…The placement of transcription termination signals between two transcription units, along with the designing of gene orientation, can minimize transcriptional interference. Prokaryotic transcription terminators are well characterized, and their incorporation in expression vectors has multiple beneficial effects on gene expression [29]. In eukaryotes, a consensus sequence consisting of ATCAAA(A/T) TAGGAAGA has been identified in the termination region of nine genes [30].…”

“…Another concern in the design of expression vectors involves the potential ability of the 5 0 -UTR to form secondary structure. GC-rich regions have the potential to form stable hairpin structures, which can inhibit translation initiation, a phenomenon that has been documented in eukaryotic [32] and prokaryotic [29] expression systems. One remedy to this potential problem is the removal of the 5 0 -UTR prior to the insertion of cDNAs into expression vectors, with the caveat that the 5 0 -UTR may contain translational enhancer elements, such as the SP163 element of the vascular endothelial growth factor mRNA [33].…”

“…An alternative view holds that the abundance of tRNAs is probably a consequence of and not a reason for codon bias and, furthermore, the primary reason for codon bias is selection for the accuracy of protein synthesis on the ribosome [35]. In E. coli, transcripts of heterologous genes enriched with codons that are rarely used by E. coli may not be translated efficiently, or may result in polypeptides containing misincorporated amino acid residues (reviewed in [29,36]). Similarly, mammalian codon usage can adversely affect translation efficiency of heterologous genes.…”

“…However, signal peptides can vary significantly in their ability to promote protein secretion. In fact, the presence of a signal sequence per se does not necessarily ensure protein secretion, as has been documented in both prokaryotic (reviewed in [29]) and eukaryotic cells (e.g., [43]). There are many examples of efficient signal peptides including those derived from erythropoietin [44], tissue plasminogen activator [45], interleukin-2 [46], albumin [47], and immunoglobulin sequences [48].…”

“…Fusion partners, have a wide range of applications in both prokaryotic [29,49] and eukaryotic [1,49] expression systems. Fusion moieties are used as affinity handles for the facile isolation and purification of proteins (Chapter 25); as reporters (Chapter 6) in studies of promoter activity or localization of proteins in cellular compartments; as protein dimerization domains; as immunogens for the production of antibodies; to target antibodies (Chapter 21); to increase expression, folding, solubility, and secretion of proteins; or to display polypeptides on the surface of cells for vaccine development, protein-protein interactions, drug screening, and other potential applications.…”