Functional expression and display of an antibody Fab fragment in Escherichia coli: study of vector designs and culture conditions.
Several different vector designs are currently being used to display and express Fab molecules in Escherichia coli, but their relative efficiency in phage display and protein expression cannot be compared from the published data. We systematically investigated which vector design most effectively displays and expresses Fab molecules in E. coli using, as a model system, a human Fab against tetanus toxoid (tt). Three different vectors were used in this study: pFab1 where the VL-CL and VH-CH1 genes were driven by two promoters in two separate expression cassettes, and pFab2 and pFab3 that both contain one dicistronic expression cassette with two translation initiation sites and either VH-CH1 before VL-CL or VL-CL before VH-CH1, respectively. The display of tt-Fab on the surface of phage and the expression of tt-Fab protein in E. coli were compared for the aforementioned vectors. Our results showed that the pFab3 vector was most effective in Fab display. A 10-fold increase in the expression of secreted Fab was observed in pFab3 when compared with vectors pFab1 and pFab2. Further experiments were conducted using pFab3 to optimize expression levels using different strains of E. coli and various culture conditions. The highest expression of tt-Fab was obtained using the pFab3 vector in host strain JM105 with an induction temperature at 37 degrees C and IPTG concentration of 0.1 mM.
Department of Immunotherapeutics, Purdue Pharma LP, 6 Cedar Brook Drive, Cranbury, NJ 08512, USA.
Susanne
S
Corisdeo
Baiyang
B
Wang
D001435:Bacteriophages / Q000235:genetics; D003001:Cloning, Molecular; D003470:Culture Media / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D005822:Genetic Vectors; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D007544:Isopropyl Thiogalactoside / Q000737:chemistry; D009841:Oligonucleotides / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D013745:Tetanus Toxoid / Q000276:immunology
D003470:Culture Media; D007140:Immunoglobulin Fab Fragments; D009841:Oligonucleotides; D011994:Recombinant Proteins; D013745:Tetanus Toxoid; D007544:Isopropyl Thiogalactoside