Recombination clone selection

The MS techniques described previously for characterization of the final recombinant protein product can be applied at all stages during process development. MS might be used upstream to define clone selection, processing format, and purification steps, and downstream to characterize the final product, ascertain lotto-lot reproducibility, determine stability, and define the formulation of biopharmaceutical molecules. Presented here are some examples found either in the literature or from our own experience in which MS has been found to be a useful or necessary tool. Potential limitations of MS methods are discussed, and when appropriate, other analytical methods are mentioned that can be alternatives to MS and are also efficient tools for biopharmaceutical development. 

The floxed stop cassette of the integrated construct can be deleted in ES cells by transient transfection with a Cre-expressing plasmid (available, e.g., at http //www.addgene. org/) and subsequent low-density plating as selection for recombined clones is impossible. After electroporation, we plate 500-2,000 cells on feeders in a 10 cm dish and analyze 24 clones for recombination by PCR or Southern blot. At least two recombined clones should be analyzed for knockdown efficiency. 

Select one recombinant clone for the 2.0-kb insert and one for the 1.4-kb insert for each drug-resistant mutant and plate them in ampicillin L-broth plates. 

Rine, J., Hansen, W., Hardeman, E., Davis, R. W. (1983). Targeted selection of recombinant clones through gene dosage effects. Proc. Natl. Acad. Sci. USA, 80, 6750-6754. 

If there is a need to insert in some distance of the selection cassette additional sequences (e.g., a loxP site or a point mutation), then an attempt should be made to introduce these sequences together with restriction sites that can be used for Southern blot analysis. Alternatively, genomic DNA of homologous recombinant clones can be checked by PCR for the presence of that sequence. 

Kouprina,N., and Larionov, V. (2008) Selective isolation of genomic loci from complex genomes by transformation-associated recombination cloning in the yeast Saccharomyces cerevisiae, Nat Protoc. 3, 371-377. 

Select the recombinant clone for presence of the mini-X by streaking the colonies on LB/Tet plate. Colonies that are tetracycline resistant have retained the mini-X and can be used for next Fix step (see Note 5). 

Leem SH, Noskov VN, Park JE et al (2003) Optimum conditions for selective isolation of genes from complex genomes by transformation-associated recombination cloning. Nucleic Acids Res 31 e29... 

Cell-free translation system, used for the identification of cloned genes and gene expression, has been investigated extensively as a preparative production system of commercially interesting proteins after the development of continuous-flow cell-free translation system. Many efforts have been devoted to improve the productivity of cell-free system [1], but the relatively low productivity of cell-free translation system still limits its potential as an alternative to the protein production using recombinant cells. One approach to enhance the translational efficiency is to use a condensed cell-free translation extract. However, simple addition of a condensed extract to a continuous-flow cell-free system equipped with an ultrafiltration membrane can cause fouling. Therefore, it needs to be developed a selective condensation of cell-free extract for the improvement of translational efficiency without fouling problem. 

Nitrilases convert nitriles to the corresponding carboxylic acids and NH3 through a cysteine residue in the active site [50]. Because of their high enantio- and regio-selectivity, nitrilases are attractive as green catalysts for the synthesis of a variety of carboxylic acids and derivatives (Figure 1.10) [51,52]. Recently, a number of recombinant nitrilases have been cloned and characterized heterologously for synthetic applications [50,53,54]. 

---