Purification of recombinant proteins

Proteins produced by recombinant DNA technology are usually purified by means identical to those available for purification of traditional non-recombinant proteins extracted directly from 

Two features of recombinant production in particular can impact very significantly upon the approach subsequently taken to purify the recombinant product inclusion body formation and the incorporation of purification tags. The processes of inclusion body formation, recovery and recombinant protein renaturation have been considered in Chapter 5. Once the recombinant protein has been refolded, additional purification (if required) follows traditional lines. 

The columns are here designed specifically for a defined purpose. These columns are easier to use, faster and they require much less resources. Some of the columns include the NAP-25 or PD 10 desalting columns (from Amersham Biosciences), His Tag columns such as Ni-NTA spin column from Qiagen, His Bind from Novagen or His GraviTrap from GE Healthcare. 

The NAP-25 /PD-10 column contains Sephadex G-25 and is used for a rapid desalting or buffer exchange of nucleic acids, proteins and oligonucleotides. 

These columns are used for purification of recombinant fusion proteins tagged to 6XHis. The commercial columns contain the precharged Ni coupled to a tetradentate chelating absorbent such as the NTA (nitrilotriacetic acid), bound to a matrix which could be Sepharose or Cellulose. 

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The previous ELP fusions all are examples of protein purification in which the ELP is covalently connected to the protein of choice. This approach is suitable for the purification of recombinant proteins that are expressed to high levels, but at very low concentrations of ELP the recovery becomes limited. Therefore this approach is not applicable for proteins expressed at micrograms per liter of bacterial culture, such as toxic proteins and complex multidomain proteins. An adjusted variant of ITC was designed to solve this problem. This variant makes use of coaggregation of free ELPs with ELP fusion proteins. In this coaggregation process, an excess of free ELP is added to a cell lysate to induce the phase transition at low concentrations of... 

Lin, C.T., Moore, P.A., Auberry, D.L. et al. (2006) Automated purification of recombinant proteins combining high-throughput with high yield. Protein Expression and Purification, 47 (1), 16-24. 

Metal chelate affinity chromatography finds most prominent application in the affinity purification of recombinant proteins to which a histidine tag has been attached (described later). As protein binding occurs via the histidine residues, this technique is no more inherently useful for the purification of metalloproteins than for the purification of non-metalloproteins (a common misconception, given its name). 

Other common impurities, such as immunoglobulins and protein A, result from the immunoaffinity purification of recombinant proteins or MAbs.16 If affinity chromatography is used to purify an antigen, then an ELISA can be used to detect contaminating levels of MAbs leached from the column. An assay for the antibody needs to detect the antibody in the presence and absence of its specific antigen. 

Lucas, C., C. Nelson, M.L. Peterson, S. Frie, D. Vetterlein, T. Gregory, and A.B. Chen (1988). Enzyme-linked immunosorbent assays (ELISAs) for the determination of contaminants resulting from the immunoaffinity purification of recombinant proteins. J Immunol Methods 113(1) 113-122. 

Recently, the purification of recombinant protein acyl transferases was published. Future investigations will show whether these biocatalysts may also serve as tools for acylating proteins. Depending on their substrate tolerance the incorporation of non-natural acyl analogues could also be possible. 

Sassenfeld, H.M. and Brewer, S.J. (1984) Apolypeptide fusion designed for the purification of recombinant proteins. Bio/Technology, 2, 76-81. 

Downstream Processing or Purification of Recombinant Protein in Pharmaceutical Scale... 

Fig. 1. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) analysis of expression and purification of recombinant protein. Ten-microliter aliquots were withdrawn at each step of the purification and loaded on a 12% SDS-PAGE gel in a Mini Protean III cell gel electrophoresis unit (Bio-Rad). The detection was performed with Coomassie blue staining. MW, low range (14-98 kDa) molecular weight marker (Bio-Rad).

Castilho LR (2001), Development of a dynamic filter for integrated perfusion cultivation and purification of recombinant proteins from mammalian cells, Fortschritt-Berichte Series - Reihe 17 (Biotechnologie/Medizintechnik), VDI-Verlag, Diissel-dorf. 

Hochuli E, Bannwarth W, Dobeli H, Gentz R, Stuber D, Genetic approach to facilitate purification of recombinant proteins with a novel metal chelate adsorbent, Biotechnology, 6 1321-1325, 1998. 

Sarris, A. H., Broxmeyer, H. E., Wirthmueller, U., Karasavvas, N., Cooper, S., Lu, L., Krueger, J., and Ravetch, J. V. (1993) Human interferon-inducible protein 10 expression and purification of recombinant protein demonstrate inhibition of early human hematopoietic progenitors. J. Exp. Med. 178,1127-1132. 

Barnthouse, K. A., Trompeter, W., Jones, R., Inampudi, P., Rupp, R., and Cramer, S. M. (1998). Cation exchange displacement chromatography for the purification of recombinant protein therapeutics from variants. J. Biotechnol. 66, 125-136. 

Chono, H. (1998). Purification of recombinant proteins from E. coli using Streamline, lnt. Conf. Expanded Bed Adsorption, Napa Valley, CA, 1998, Abstr., p. 9.3. 

Sharma, S. K. (1997). Designer affinity purification of recombinant proteins. In Affinity Separations A Practical Approach (P. Matejtschuk, ed.), pp.197-218. Oxford University Press, Oxford. 

Based on the vectors for the intracellular production and purification of recombinant proteins, the vector system was further expanded with vectors for the extracellular recombinant protein production. Affinity tag aided protein purification from the cell-free growth medium was made possible by the addition of the corresponding His6- and StrepII-tags [20, 35]. 

Production, Export and Affinity Chromatographic Purification of Recombinant Proteins from the Growth Medium... 

Riggs, P. (2000) Expression and purification of recombinant proteins by fusion to maltose-binding protein. Mol. Biotechnol. 15, 51-63. 

Figure 24.10B shows an alternative method consisting of the replacement of the mammalian signal peptide for a signal peptide that works in bacteria. This enables the purification of the recombinant hGH from the periplasm of the bacterial cell, reducing the difficulties associated with the purification of recombinant proteins from the cytoplasm. To achieve this, once the mammalian signal peptide... 

The His tag248 is by far the most popular affinity tag for purification of recombinant proteins. Typically, the tag is composed of 6-10 consecutive histidines at either terminus of the protein of interest, often separated by a protease-cleavage site. The presence of a His tag enables the use of IMAC for purification. IMAC is a rapid... 

Although the introduction of fusion tags has many potential benefits for the production and purification of recombinant proteins, they may interfere with the final use of the protein therefore, they often need to be removed. Many plasmids are designed to introduce a protease recognition site between an introduced tag and the protein of interest. Commonly used proteases and their recognition sites are shown in Table 2. Generally,... 

Q. Expression and affinity purification of recombinant proteins from plants. Protein Expr. Purif. 2002, 25, 195-202. 

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