However, IRES-driven cap-independent translation is less efficient than cap-dependent translation. The translation of second ORF downstream of IRES element is initiated through a cap-independent mechanism which is mediated through IRES sequences. When IRES element is introduced between two open reading frames (ORFs), the first ORF is translated via cap-dependent mechanism. Co-expression of heavy and light chains under the control of a single promoter can be achieved using either viral internal ribosome entry site (IRES) or viral 2A-self cleavage sequences. Several studies have shown that employing a single vector provides a tighter control over the expression ratio of LC/HC. Generally, the recombinant mAb expression is attained either by cotransfection of two separate vectors each encoding one chain of antibody or transfection of a single vector encoding both chains. Therefore the balanced ratio of the light to heavy chains has a great effect on the optimal production of recombinant IgG in mammalian cells. The most common types of mAbs in the market, immunoglobulin G (IgG), are comprised of two identical light chains (LC) and two identical heavy chains (HC) polypeptides. Īpart from designing an optimal expression cassette with the strong promoter, which is common for all recombinant proteins, there is another challenge for mAbs production that should be taken into account. CHO-derived elongation factor-1 promoter (CHEF-1) has been shown to be a successful system to obtain high level of expression in mammalian cells. Alternatively, CHO-specific constitutive promoters are good candidates. DNA methylations of CMV which is caused by epigenetic events can lead to a decrease in the production of antibody. Although CMV gives high level of gene expression, there are several reports demonstrating the susceptibility of this promoter to silencing. A commonly used promoter for driving recombinant protein expression in mammalian cells is human cytomegalovirus (CMV) major immediate-early (MIE). Īn essential component of an expression cassette is promoter which contributes to expression level and stability of transgenes expression. To improve the productivity of cell line, designing optimal expression vector plays a key role. The creation of stable high producer cell line is one of the most crucial factors to support high demand of market. Today, the majority of approved therapeutic mAbs are produced in Chinese hamster ovary (CHO) cell lines. The emergence of monoclonal antibodies (mAbs) as an effective therapeutic agent has opened a new approach for the treatment of various diseases. Our findings indicated that the CHEF promoter is a viable alternative to CMV promoter-based expression in F2A-mediated vectors by providing both higher expression and level of stability. Further analyses showed that both IRES-mediated vectors, expressed mAbs with correct size, whereas in antibodies expressed via F2A system heterogeneity of light chains were detected due to incomplete furin cleavage. Studying the stability of the F2A expression system in the course of 18 weeks, we observed that the cells having CHEF promoter maintained their antibody expression at higher level than those transfected with CMV promoter. However, this difference was less significant in IRES-mediated mAb expressing cells. Our results indicated that CHEF promoter-based expression of mAbs was 2.5 fold higher than CMV-based expression in F2A-mediated vectors. The efficiency of these promoters was evaluated by measuring level of expressed antibody in stable cell pools. To compare the strengths of CHEF with cytomegalovirus (CMV) promoter for mAb expression in CHO cells, four bicistronic vectors bearing either internal ribosome entry site (IRES) or Furin/2A (F2A) sequences were designed. A critical step in recombinant expression is the utilization of strong promoters, such as the Chinese Hamster Elongation Factor-1α (CHEF-1) promoter. In recent years, monoclonal antibodies (mAbs) have been developed as powerful therapeutic and diagnostic agents and Chinese hamster ovary (CHO) cells have emerged as the dominant host for the recombinant expression of these proteins.
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