Optimisation and evaluation of a high-throughput mammalian protein expression system

Protein Expr Purif. 2005 Jul;42(1):111-21. doi: 10.1016/j.pep.2005.03.012. Epub 2005 Mar 31.

Abstract

Transient transfection of mammalian cells with episomal vectors is a very useful method for producing high levels of recombinant proteins. Transient systems remove the need for the laborious and time-consuming process of creating stable cell lines. Here, we describe the optimisation and evaluation of a high-throughput transient expression system in HEK293-EBNA cells. The process was developed for the expression of 10 constructs simultaneously in deep-well plates and subsequent purification using 96-well plate affinity chromatography. This enabled multiple combinations of different constructs, vectors, and expression conditions to be studied in parallel.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Carboxypeptidase B2 / biosynthesis
  • Carboxypeptidase B2 / genetics
  • Carboxypeptidase B2 / isolation & purification
  • Cell Culture Techniques / methods
  • Cell Line
  • Cell Proliferation / drug effects
  • Epstein-Barr Virus Nuclear Antigens / genetics
  • Gene Expression / genetics*
  • Genetic Vectors / genetics
  • Glutathione Transferase / biosynthesis
  • Glutathione Transferase / genetics
  • Glutathione Transferase / isolation & purification
  • Green Fluorescent Proteins / biosynthesis
  • Green Fluorescent Proteins / genetics
  • Green Fluorescent Proteins / isolation & purification
  • Histidine / genetics
  • Humans
  • Lymphocyte Specific Protein Tyrosine Kinase p56(lck) / biosynthesis
  • Lymphocyte Specific Protein Tyrosine Kinase p56(lck) / genetics
  • Lymphocyte Specific Protein Tyrosine Kinase p56(lck) / isolation & purification
  • Poloxamer / pharmacology
  • Protein-Serine-Threonine Kinases / biosynthesis
  • Protein-Serine-Threonine Kinases / genetics
  • Protein-Serine-Threonine Kinases / isolation & purification
  • Protein-Tyrosine Kinases / biosynthesis
  • Protein-Tyrosine Kinases / genetics
  • Protein-Tyrosine Kinases / isolation & purification
  • Recombinant Proteins / biosynthesis*
  • Recombinant Proteins / isolation & purification
  • Reproducibility of Results
  • Transfection / methods

Substances

  • Cyan Fluorescent Protein
  • Epstein-Barr Virus Nuclear Antigens
  • Recombinant Proteins
  • Poloxamer
  • Green Fluorescent Proteins
  • polyhistidine
  • Histidine
  • glutathione S-transferase T1
  • Glutathione Transferase
  • Protein-Tyrosine Kinases
  • Lymphocyte Specific Protein Tyrosine Kinase p56(lck)
  • Protein-Serine-Threonine Kinases
  • Carboxypeptidase B2