Unit

UNIT 7.3 Determining the Identity and Structure of Recombinant Proteins

  1. Nancy D. Denslow (peptide mapping)1,
  2. Keith Rose (disulfide linkage analysis)2,
  3. Pier Giorgio Righetti (two-dimensional titration curve analysis)3

Published Online: 1 MAY 2001

DOI: 10.1002/0471140864.ps0703s03

Current Protocols in Protein Science

Current Protocols in Protein Science

How to Cite

Denslow, N. D., Rose, K. and Righetti, P. G. 2001. Determining the Identity and Structure of Recombinant Proteins. Current Protocols in Protein Science. 3:7.3:7.3.1–7.3.26.

Author Information

  1. 1

    University of Florida, Gainesville, Floria

  2. 2

    University Medical Center, Geneva, Switzerland

  3. 3

    University of Milano, Milan, Italy

Publication History

  1. Published Online: 1 MAY 2001
  2. Published Print: MAR 1996

Abstract

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.