Recombinant purification

Shibata, T., R. P. Cunningham, and C. M. Radding. 1984. Homologous pairing in genetic recombination Purification and characterization of Escherichia coli recA protein. 

Recovery nd Purifica.tion. The production of EH Lilly s human insulin requires 31 principal processing steps of which 27 are associated with product recovery and purification (13). The production process for human insulin, based on a fermentation which yields proinsulin, provides an instmctive case study on the range of unit operations which must be considered in the recovery and purification of a recombinant product from a bacterial fermentation. Whereas the exact sequence has not been pubUshed, the principle steps in the purification scheme are outlined in Figure la. 

Fig. 2. Outline of possible steps in the recovery and purification sequence for recombinant tissue plasminogen activator derived from recombinant CHO...

Stability and Purification of the Recombinant Protein. There are no hard and fast rules specifying, eg, whether a recombinant protein is available in a soluble state in the cell. In some cases, the expression system must be engineered by in vitro mutagenesis to optimize overall yield of the protein. 

The methods involved in the production of proteins in microbes are those of gene expression. Several plasmids for expression of proteins having affinity tails at the C- or N-terminus of the protein have been developed. These tails are usefiil in the isolation of recombinant proteins. Most of these vectors are commercially available along with the reagents that are necessary for protein purification. A majority of recombinant proteins that have been attempted have been produced in E. Coli (1). In most cases these recombinant proteins formed aggregates resulting in the formation of inclusion bodies. These inclusion bodies must be denatured and refolded to obtain active protein, and the affinity tails are usefiil in the purification of the protein. Some of the methods described herein involve identification of functional domains in proteins (see also Protein engineering). 

Human growth hormone was originally manufactured by isolation of the natural product from human pituitaries and subsequent purification of the protein. Since 1985, manufacture of hGH has been almost exclusively by recombinant DNA technology. 

Several of these IFNs of mouse and human lymphocytes and fibroblasts are available commercially and have been best prepared in quantity by recombinant DNA procedures because they are produced in very small amounts by the cells. The commercial materials do not generally require further purification for their intended purposes. [Pestkas, Interferons and Interferon standards and general abbreviations. Methods Enzymol, Wiley Sons, 119 1986, ISBN 012182019X Lengyel, Biochemistry of interferons and their actions, Ann Bev Biochem 51 251-282 7982 De Maeyer and De Maeyer-Guignard, Interferons in The Cytokine Handbook, 3rd Edn, Thomson et al. Eds, pp. 491-516 7998 Academic Press, San Diego, ISBN 0126896623.]... 

Another application shows the preparative purification and polishing of a therapeutic fusion protein with a humanized recombinant IgG protein. The fusion protein was expressed by the fermentation of baby hamster kidney cells. The filtered culture supernatant (155 liters) contained 2.2 g of IgG and 75.5 g of total protein. After the immunoglobulins were isolated by expanded bed adsorption and rebuffering, the IgG fraction was bound to Fractogel EMD SOj (M). This column achieved baseline separation of complete antibodies (fusion protein) from small amounts of antibodies lacking the fusion part. The resulting highly purified IgG fraction (110 ml) was diluted to 150 ml and... 

An improved method of producing recombinant aequorin was devised based on the fact that the expression of the peak amount of apoaequorin in bacterial cells occurs several hours before the secretion into culture medium (Shimomura and Inouye, 1999). The cells containing apoaequorin in the periplasmic space, before secretion, are extracted under a very mild condition and, at the same time, converted into aequorin. The purification of the extract by two steps of column chromatography yields a high-purity preparation of recombinant aequorin. 

Deschamps, J. R., Miller, C. E., and Ward, K. B. (1995). Rapid purification of recombinant green fluorescent protein using the hydrophobic properties of an HPLC size-exclusion column. Protein Expression and Purification 6 555-558. 

Illarionov, B. A., et al. (2000). Recombinant obelin cloning and expression of cDNA, purification, and characterization as a calcium indicator. Method. Enzymol. 305 223-249. 

Inouye, S., et al. (1989). Overexpression and purification of the recombinant calcium-binding protein, apoaequorin./. Biochem. 105 473-477. 

Li, L. (2000). Gonyaulax luciferase gene structure, protein expression, and purification from recombinant sources. Method. Enzymol. 305 249-258. 

Masuda, H., et al. (2003). Chromatography of isoforms of recombinant apoaequorin and method for the preparation of aequorin. Protein Expression and Purification 31 181-187. 

Shimomura, O., and Inouye, S. (1999). The in situ regeneration and extraction of recombinant aequorin from Escherichia coli cells and the purification of extracted aequorin. Protein Expression and Purification 16 91-95. 

Stults, N. L., et al. (1992). Use of recombinant biotinylated aequorin in microtiter and membrane-based assays Purification of recombinant aequorin from Escherichia coli. Biochemistry 31 1433-1442. 

Verhaegen, M., and Christopoulos, T. K. (2002). Recombinant Gaussia luciferase. Overexpression, purification, and analytical application of a bioluminescent reporter for DNA hybridization. Anal. Chem. 74 4378-4385. 

In particular, the availability of such bacterial biocatalysts in the form of recombinant expression systems [136] in combination with simplified purification protocols opened up this methodology for large-scale applications [204]. 

Figure 5.6 Positive-ion electrospray spectrum obtained from the major component in the LC-MS analysis of a purified recombinant 62 kDa protein using a Cig microbore 50 X 1 mm column and a flow rate of 50 p.lmin . The starting buffer (buffer A ) was 0.1% TEA in water, while the gradient buffer (buffer B ) consisted of 0.1% TEA in acetonitrile-water (9 1 vol/vol). The running conditions consisted of 0% B for 5 min, followed by a linear gradient of 100% B for 55 min. Reprinted from J. Chromatogr., B, 685, McAtee, C. P., Zhang, Y., Yarbough, P. O., Fuerst, T. R., Stone, K. L., Samander, S. and Williams, K. R., Purification and characterization of a recombinant hepatitis E protein vaccine candidate by liquid chromatography-mass spectrometry , 91-104, Copyright (1996), with permission from Elsevier Science.

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