Production Of Recombinant Proteins And Labelled Proteins

For expression of recombinant proteins, exponentially growing cultures are infected at a high MOI to ensure synchronous infection of the majority of cells. The relationship between the amount of virus added (MOI) and the number of infected cells is not linear, but is described by the Poisson function (see Figure 7.1): thus an MOI of 1 infectious particle per cell is sufficient to infect only 63 % of the culture, and an MOI of 3 is sufficient to infect 95 %. In practice it is preferable to infect at a nominal MOI of 5 in order to take account of any assay variation. In a productive infection, cell division will cease shortly after virus addition, and cell metabolism and oxygen utilization will increase approximately twofold over the first day post-infection. As for virus production, it is essential to ensure adequate nutrient and oxygen supply by infecting at a reasonable cell concentration (approx. 2 X 106 cells/ml). The efficiency of small-scale systems can be increased by re-suspending cells in fresh medium at higher concentration (3-3.5 X 106 cells/ml) before infection, though this is rarely necessary. The optimum time for collection of product should be established by carrying out a time course evaluation. For most proteins, the preferred harvest time is between 48 h and 72 h post-infection, but some products require an earlier collection time because of the product degradation observed later in the infection cycle. Cells and supernatant are separated by centrifugation or filtration for downstream processing. For intracellular products, cells are harvested by centrifugation, washed, and lysed by homogenization, detergent, or other method in a small volume of buffer (generally 1-5 % of the culture volume). Detergents such as NP40 (0.5 % solution in buffer) are a very useful means of disrupting cells, because they give complete lysis of cells but leave nuclei intact. Cell debris and nuclei are removed by centrifugation, and the product is available in concentrated form for assay and purification.

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