An improved dual-expression concept, generating high-quality antibodies for proteomics research.
A novel, improved dual bacterial-expression system, designed for large-scale generation of high-quality polyclonal antibody preparations intended for proteomics research, is presented. The concept involves parallel expression of cDNA-encoded proteins, as a fusion with two different tags in two separate vector systems. Both systems enable convenient blotting procedures for expression screening on crude bacterial cell cultures and single-step affinity purification under denaturing conditions. One of the fusion proteins is used to elicit antibodies, and the second fusion protein is used in an immobilized form as an affinity ligand to enrich antibodies with selective reactivity to the cDNA-encoded part, common for the two fusion proteins. To evaluate the system, four cDNA clones from putative nuclear proteins from the non-biting midge Chironomus tentans were expressed. Antibodies to these cDNA-encoded proteins were generated, enriched and used in blotting and immunofluorescence procedures to determine expression patterns for the native proteins corresponding to the cDNAs. The four antibody preparations showed specific reactivity to the corresponding recombinant cDNA-encoded proteins, and three of the four antibodies gave specific staining in Western-blot analysis of nuclear cell extracts. Furthermore, two of the antibody preparations gave specific staining in immunofluorescence analysis of C. tentans cells. We conclude that the dual-vector concept presented offers a highly stringent strategy for the generation of monospecific polyclonal antibodies, which are useful in proteomics research.
Falk
Ronny
R
Department of Biotechnology, AlbaNova University Center, Royal Institute of Technology (KTH), SE-106 91 Stockholm, Sweden.
Agaton
Charlotta
C
Kiesler
Eva
E
Jin
Shaobo
S
Wieslander
Lars
L
Visa
Neus
N
Hober
Sophia
S
Ståhl
Stefan
S
D000818:Animals; D000906:Antibodies / Q000235:genetics* / Q000302:isolation & purification; D000917:Antibody Formation / Q000235:genetics*; D002683:Chironomidae / Q000235:genetics* / Q000378:metabolism*; D003001:Cloning, Molecular / Q000379:methods; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics; D015202:Protein Engineering / Q000379:methods; D040901:Proteomics / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis
D000906:Antibodies; D011993:Recombinant Fusion Proteins