A mammalian expression vector for expression and purification of secreted proteins for structural studies

Daniel J. Leahy, Charles E. Dann, Patti Longo, Benjamin Perman, Kasra X. Ramyar

Research output: Contribution to journalArticle

Abstract

A mammalian expression vector with features optimized for simple expression and purification of secreted proteins has been developed. This vector was constructed to facilitate X-ray crystallographic studies of cysteine-rich glycoproteins that are difficult to express by other means. Proteins expressed with this vector possess an N-terminal human growth hormone domain and an octahistidine tag separated from the desired polypeptide sequences by a tobacco etch virus protease recognition site. Advantages of this vector are high levels of expression, simple detection and purification of expressed proteins, and reliable cleavage of the fusion protein. Cotransfection of this vector with a dihydrofolate reductase gene allows amplification of expression levels with methotrexate. Over one dozen cysteine-rich secreted proteins have been expressed in sufficient quantity for structural studies using this vector; the structure of at least one of these proteins has been determined. (C) 2000 Academic Press.

Original languageEnglish (US)
Pages (from-to)500-506
Number of pages7
JournalProtein Expression and Purification
Volume20
Issue number3
DOIs
StatePublished - Jan 1 2000

Keywords

  • CHO cells
  • Electroporation
  • SRα
  • pSGHV0

ASJC Scopus subject areas

  • Biotechnology

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