(188a) Use of Free-Solution Conjugate Electrophoresis (Fsce) as a Highly Sensitive Method to Differentiate Genetically Engineered Protein Polymers with Slight Differences in Length or Charge Distribution

We have used genetic engineering to create a series of non-natural, repetitive polypeptides (?protein polymers?) that are water-soluble and adopt random-coil conformations in solution. These protein polymers were produced by expression in E. coli bacteria, with molecular weights ranging from 9 kDa to 36 kDa and with designed sequences consisting of Gly, Ala, Thr, Ser, and positively charged Arg amino acids.

Here we demonstrate that Free-Solution Conjugate Electrophoresis (FSCE) in fused silica capillaries is a highly sensitive technique that can discriminate between protein polymers that have only slight differences in length and charge. Commonly used methods to assess protein purity after expression, such as RP-HPLC, MALDI-TOF, and SDS-PAGE have not shown sufficient resolution to reliably separate these protein polymers. In FSCE, a protein polymer is conjugated to a DNA oligonucleotide and analyzed by free-solution electrophoresis. The hydrodynamic drag of the protein polymer causes a mobility shift in the migration velocity of the conjugated DNA compared to free DNA. Polydisperse samples are easily profiled, with high resolution, using this method.

The same proteins were expressed in a vector containing an N-terminal decahistidine tag for affinity purification and a separate expression vector encoding a C-terminal octahistidine tag. Although all proteins appeared pure using the standard techniques mentioned above, we show that using FSCE we can identify multiple truncated protein products that resulted from incomplete expression of the full-length protein when expressed with only the N-terminal affinity tag. We also demonstrate that FSCE is able to distinguish between protein polymers with identical overall charges but with the charges in different positions along the chain, due to ?end-effects? lending more weight to charges at the ends of the protein.