E. coli-expressed human neurotrophin-3 characterization of a C-terminal extended product

Publisher Summary In the development of proteins as therapeutic agents, it is crucial to ensure the homogeneity of the potential products; however, over-expression of recombinant proteins in E.coli often leads to translational errors as well as post-translational modifications. This chapter describes the E.coli expression of human neurotrophin-3 (NT-3), a member of the nerve growth factor (NGF) family of neurotrophic factors, and discusses the characterization of this higher molecular weight product, and shows that it is due to misreading through the UGA codon by tryptophan incorporation. The chapter shows that when the cloned DNA is terminated with the codon UGA, both the mature protein and a higher molecular form are produced in the inclusion bodies. Examination of the endoproteinase Lys-C map of the higher molecular weight material demonstrates that the peptides corresponding to the C-terminus are absent and a new peptide is generated. N-terminal analysis of the peptide gives IGRTWGSADK. Therefore, the C-terminus of the protein has been extended with the peptide WGSADK. This sequence matches perfectly with the translated cloned DNA sequence, before another in-frame termination codon (UAA) is met. The chapter explains that the higher molecular weight form of NT-3 is caused by reading through of the UGA stop codon by tryptophan incorporation.