Multiple affinity domains for the detection, purification and immobilization of recombinant proteins.
Affinity systems based on specific molecular recognition are valuable tools for detection, purification and immobilization of recombinant proteins. Here, novel multipartite affinity fusion vectors were assembled and investigated to allow flexible binding and elution conditions. The rationale for the assembly of different combinations of affinity domains was to take advantage of the wide variety of molecular interactions of these domains for purification, solubilization, detection and immobilization. In total, seven different affinity tags representing five different types of tag-ligand interactions were studied: (i) monoclonal antibodies-peptides (T7-tag and FLAG peptide); (ii) streptavidin-peptide (Strep-tag); (iii) hexahistidyl-metal ions (His6-tag; (iv) bacterial receptors-serum proteins (staphyloccal protein A-Fc and streptococcal protein G-serum albumin); (v) streptavidin-biotin (in vivo biotinylated peptide). Selected tags were evaluated for the production and purification of Escherichia coli DNA polymerase I (Klenow fragment). On the basis of the results, a vector (pAff2c) was assembled using a novel combination of affinity domains: (i) an in vivo biotinylated peptide; (ii) a His6 sequence, and (iii) a highly soluble serum albumin binding region. Using these three affinities, a wide variety of conditions can be employed for both the binding and the elution steps.
Nilsson
J
J
Department of Biochemistry and Biotechnology, Kungliga Tekniska Högskolan, Stockholm, Sweden.
Larsson
M
M
Ståhl
S
S
Nygren
P A
PA
Uhlén
M
M
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D004256:DNA Polymerase I / Q000235:genetics / Q000302:isolation & purification; D004274:DNA, Recombinant; D012321:DNA-Directed RNA Polymerases / Q000235:genetics; D004926:Escherichia coli; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification*; D014764:Viral Proteins
D004274:DNA, Recombinant; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; D004256:DNA Polymerase I