Proteins, affinity chromatographic production

As noted above, whole-cell MALDI-TOF MS was intended for rapid taxonomic identification of bacteria. Neither the analysis of specific targeted bacterial proteins, nor the discovery of new proteins, was envisioned as a routine application for which whole cells would be used. An unknown or target protein might not have the abundance or proton affinity to facilitate its detection from such a complex mixture containing literally thousands of other proteins. Thus, for many applications, the analysis of proteins from chromatographically separated fractions remains a more productive approach. From a historical perspective, whole-cell MALDI is a logical extension of MALDI analysis of isolated cellular proteins. After all, purified proteins can be obtained from bacteria after different levels of purification. Differences in method often reflect how much purification is done prior to analysis. With whole-cell MALDI the answer is literally none. Some methods attempt to combine the benefits of the rapid whole cell approach with a minimal level of sample preparation, often based on the analysis of crude fractions rather... 

Intracellular Recombinant Production and Affinity Chromatographic Purification of Proteins... 

Table 1 Comparison of the recombinant production and affinity chromatographic purification of GFP from B. megaterium [30]. Different affinity tag fusion forms of GFP were produced in II. megaterium WH323. Purification was performed using affinity chromatography. Amounts of purified GFP-Strep were determined using a Bradford protein assay kit (Bio-Rad Munich Germany) and BSA (Perbio Rockford USA) as standard. Amounts of purified GFP-His, His-TEV-GFP, Strep-Xa-GFP and Strep-TEV-GFP were calculated via their relative fluorescence per mg protein...

Reliable and reproducible procedures are needed to purify auxin receptor protein for antibody production and for further biochemical characterization. Two auxin affinity methods have been reported [11,17], but such methods have yielded indifferent results in our hands [22]. This communication describes auxin receptor purification using commercially available chromatographic materials and the initial characterization of monoclonal and polyclonal antibodies produced against the resulting preparations. 

Affinity complexation — Many proteins have affinities for other molecules that can be exploited to alter their retention characteristics in IEC. For example, some enzymes may be combined with synthetic substrates, cofactors, or products.1315 The same principle can be applied to other protein/receptor systems. One well-characterized example is the change in chromatographic behavior of fructose 1,6-diphosphatase in the presence of its negatively charged substrate... 

It is produced in recombinant CHO cells cultured in a medium free of animal-derived components. The BeneFix production process involves an ultrafiltration/diafiltration step, followed by four chromatographic steps ion exchange (Q resin), pseudo-affinity (Cellufine sulfate resin), hydroxyapatite, and affinity (immobilized Cu2+ ions). After these chromatographic steps, there are membrane processes (nanofiltration for viral clearance and diafiltration for solvent exchange), after which the purified protein is formulated (Edwards and Kirby, 1999). 

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