Recombinant proteins from variants

In E. Coli bacterial lysates, the proteome (i.e., the full array of proteins produced) was analyzed by isoelectric focusing and mass spectrometry.97 A comparison of capillary electrophoretic separation and slab gel separation of a recombinant monoclonal antibody demonstrated that the precision, robustness, speed, and ease-of-use of CE were superior.98 Seventy-five proteins from the yeast ribosome were analyzed and identified by capillary electrophoresis coupled with MS/MS tandem mass spectrometry.99 Heavy-chain C-terminal variants of the anti-tumor necrosis factor antibody DE7 have been separated on capillary isoelectric focusing.100 Isoforms differing by about 0.1 pi units represented antibodies with 0,1 or 2 C-terminal lysines. 

Although it has long been thought, based on genetic mutant and biochemical evidence, that aurones are derived from chalcones, the biosynthetic mechanism has only recently been clarified, and some aspects of the enzymatic process still await in vivo proof An mRNA from A. majns, specifically expressed in the petal epidermal cells, has been shown to encode a recombinant protein with aureusidin synthase (AUS) activity.AUS is a variant polyphenol oxidase (PPO) that can catalyze conversion of either 2, 4, 6, 4-etrahydroxychalcone (naringenin chalcone) or 2, 4, 6, 3,4-pentahydroxychalcone to... 

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. 

It is evident from the discussions above that mass spectrometric method in combination with enzymatic digestion offers a convenient approach to the characterization of GM-CSF and its variants. ESI-MS method demonstrated a mass accuracy of better than 0.01% for a recombinant protein. The mass spectral data of the enzymatic digest of GM-CSF and its variants allow the precise determination of the molecular weights of the peptides, leading to the identification of sites of covalent modifications, the disulfide bonding pattern, and confirmation of the cDNA-derived sequence of the protein. 

Capillary isoelectric focusing coupled to mass spectrometry has gained popularity in recent years (by analogy to two-dimensional gel electrophoresis). Additional information obtained from mass spectrometry includes not only precise molecular-weight determination but also the possiblity for peptide sequencing. Analysis of hemoglobin variants, recombinant proteins, and monoclonal antibodies have been demonstrated [1,7,8]. 

---