Determining the identity and structure of recombinant proteins.
In this unit peptide mapping protocols with separation of the constituent peptides by high-performance liquid chromatography (HPLC) analysis and by high-resolution SDS-PAGE are presented. Peptide mapping is ideally suited for comparative purposes--for example, combined analysis of the recombinant protein and its natural counterpart (or some other well-characterized standard). This unit also outlines the general strategy used to determine the linkage pattern of a monomeric recombinant protein containing two intramolecular disulfide bonds. The approach is an extension of peptide mapping, where the aim is to isolate and characterize peptides containing only a single disulfide bond. A two-dimensional electrophoretic method is also described in which the protein isoelectric point is displayed as a function of pH to yield an electrophoretic titration curve. This method is especially useful for checking for deamidation (e.g., of Asn to Asp) in which additional negative charge is introduced into the modified protein.
Denslow
N D
ND
University of Florida, Gainesville, Floria, USA.
Rose
K
K
Righetti
P G
PG
D002851:Chromatography, High Pressure Liquid / Q000379:methods; D004220:Disulfides / Q000737:chemistry; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D007526:Isoelectric Point; D010449:Peptide Mapping / Q000379:methods; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D004220:Disulfides; D011994:Recombinant Proteins