A system for stable indirect immobilization of multimeric recombinant proteins.
BACKGROUND
To perform an ELISA or for panning phage particles which display recombinant proteins, one of the reactants is immobilized on solid phase. Immobilization in ELISA is generally performed by passive adsorption of ligands to plastic. However, protein is denatured during the adsorption process. This may result in low efficiency interaction between ligands and receptors which depend on native structures. In contrast, indirect immobilization has been shown to prevent protein denaturation.


OBJECTIVE
The aim was to develop a system that allows efficient and stable indirect immobilization of a variety of recombinant multimeric proteins to solid phase.


RESULTS
A new vector was constructed which allows the expression of up to three proteins linked by the Jun/Fos leucine zipper. Purification of the resulting protein was achieved by Ni+ affinity chromatography utilizing the 6xHis-ABP (albumin binding protein) protein fused to the N-terminus of the Jun polypeptide. The high binding affinity of ABP to rat serum albumin (RSA) was exploited for indirect immobilization of recombinant proteins to solid phase. In an enzyme linked assay, the binding of ABP to immobilized RSA was shown to be 10-1000 times more efficient than other immobilization systems. Using the ZZ IgG binding domain of staphylococcal protein A as bait, the RSA-ABP immobilization system was successfully used to screen and enrich IgG Fc encoding DNA fragments from a cDNA phage library.


CONCLUSIONS
The newly designed vector termed pJuFoexpress allows production and purification of multimeric protein complexes linked by the Jun/Fos leucine zipper. Without chemical modifications, the recombinant proteins can be immobilized indirectly to solid phase. The immobilization results in the stable display of native, biologically active proteins which can be used in ELISA and phage display systems.
Grob
P
P
Institute of Virology, University of Zurich, Switzerland.
Baumann
S
S
Ackermann
M
M
Suter
M
M
D000818:Animals; D001435:Bacteriophages; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005803:Genes, Immunoglobulin; D016762:Genes, fos; D016758:Genes, jun; D005822:Genetic Vectors; D007128:Immunoglobulin Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007074:Immunoglobulin G / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019151:Peptide Library; D010957:Plasmids / Q000235:genetics; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000378:metabolism; D012709:Serum Albumin / Q000378:metabolism
D007128:Immunoglobulin Fragments; D007074:Immunoglobulin G; D007135:Immunoglobulin Variable Region; D019151:Peptide Library; D011993:Recombinant Fusion Proteins; D012709:Serum Albumin