Structural analysis of recombinant protein prepared by semi-dry electroblotting after sodium dodecyl sulfate-polyacrylamide gel electrophoresis

Abstract

Proteins that were separated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis were electroblotted onto polyvinyliden difluoride membranes in procedures to prepare homogeneous recombinant proteins for direct N‐terminal sequence analysis. A semi‐dry blotting procedure was employed to immobilize protein bands on the membranes for subsequent sequence analysis. This method has been used routinely to evaluate the quality of recombinant proteins, which are present in crude cell extracts produced by different expression systems or under different expression conditions. N‐Terminal processing, amino acid misincorporation, as well as the inefficient secretion of recombinant proteins can be detected by direct N‐terminal sequence analysis of the purified electroblotted samples. Consequently, time‐consuming chromatographic procedures can be eliminated. These procedures are also especially valuable for determining degradation sites of a purified recombinant protein, as well as evaluating multiple gene products expressed by isolated cluster genes.