Genetic Modification to Expressed Gene

By the use of suitably designed primers, many modifications can be made to the gene of interest. These may include the addition of restriction endonuclease (RE) sites to assist cloning into plas-mids, plus linker sequences of the appropriate length to ensure their restriction prior to cloning; purification/detection tags (if not included in the plasmid) and a terminal stop codon. In addition, the expression of human immunodeficiency virus Type 1 (HIV-1) IIIB gp120 and gp160 in both COS and Chinese hamster ovary (CHO) cells may be very poor unless the native signal sequence has been replaced by that from human tissue plasminogen activator (TPA) or herpes simplex virus (HSV) type 1 glycoprotein D (Chapman et al. 1991; Lasky et al. 1986). Berman et al. (1988b) highlighted the importance of the signal sequence in the optimal targeting of the recombinant protein to the cellular secretory machinery and hence to the ambient medium. This not only aids protein purification, but is also less likely to lead to proteolytic degradation or aggregation of the product into poorly soluble high-molecular-weight bodies. If the homologous signal sequence is not effective in a specific expression system, consideration should be given to its replacement. In mammalian cell expression systems, the human interleukin-2 and TPA signal sequences are particularly effective and widely employed (Sasada et al. 1988; Chapman et al. 1991; Planelles et al. 1991). Codon usage effects are a major impediment to the efficient expression of many non-mammalian genes and the systematic replacement ('codon-optimization') of the native gene codons with codons chosen to reflect more closely the codon preference of highly expressed human genes has been shown substantially to increase the expression levels of, for example, both HIV-1 env and gag genes and to remove the constraints of cis- and trans-acting elements on their regulation (Haas et al. 1996; zur Megede et al. 2000). Protein production may also be initiated or enhanced by the removal of certain structural elements or regions, such as fusogenic domains, membrane anchors, multimerization domains, N-linked glycans and the cleavage sites between subunits (Fussenegger et al. 1999; Liljeqvist & Stahl 1999; Etchevery 1996). However, it is essential that such modifications neither remove nor disable important epitopes required in the final product, and it is recommended that these constructs are characterized for desired functionality at the earliest possible stage of development. Enhanced production of HIV env gene expression in mammalian cells was seen following removal of the cytoplasmic and transmembrane regions of gp41 from the full-length precursor polyprotein (gp160) to produce gp140 (Berman et al. 1988a; Berman et al. 1989; Willey et al. 1988; Jeffs et al. 2004, 2006), and on the removal of portions of both the N- and C-termini and V1 and V2 (but not V3) hypervariable loops (Pollard et al. 1992; Wyatt et al. 1993; Jeffs et al. 1996). Enhanced production of a C-clade gp140 from stable CHO-K1 lines was also achieved following ablation of the gp120/41 primary cleavage site, and in addition the proportion of oligomeric over monomeric forms was increased (Jeffs & Viera, personal observations).

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