Large-scale bacterial fermentation and isolation of scFv multimers using a heat-inducible bacterial expression vector.
This protocol describes optimised large-scale bacterial fermentation conditions for recombinant single-chain Fv molecule (scFv) monomers and multimers (diabodies and triabodies). The heat-inducible bacterial secretion vector, pPOW3, utilising the temperature-regulated tandem lambda promoters is particularly suited to the large-scale fermentation of single-chain antibodies, providing low-cost recombinant protein synthesis. The protein expressed by this vector is secreted into the periplasm where it is found as both the soluble and insoluble protein that is associated with the cell membranes. A protein fractionation method for the rapid extraction and affinity purification of the soluble protein fraction and the urea solubilization and refolding of the insoluble protein fraction expressed from single-chain antibody (Ab) fragment gene constructs is described. This method is simple to perform and utilises inexpensive reagents to provide cost-effective protein synthesis.
Bayly
Ann M
AM
CSIRO Health Sciences and Nutrition, 343 Royal Parade, Parkville, Victoria, 3052, Australia.
Kortt
Alexander A
AA
Hudson
Peter J
PJ
Power
Barbara E
BE
D000818:Animals; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D006801:Humans; D007128:Immunoglobulin Fragments / Q000096:biosynthesis* / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000096:biosynthesis* / Q000235:genetics; D007158:Immunologic Techniques; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics; D013696:Temperature
D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D011994:Recombinant Proteins