Use of the avidin (imino)biotin system as a general approach to affinity precipitation.
Affinity precipitation, especially secondary effect affinity precipitation, has repeatedly been suggested as a valuable technique for the biotechnical downstream process. The present lack of applications is related to the scarcity of predictable affinity macroligands and to the fact that rather high affinity constants are required in affinity precipitation (K(D) < 10(-10)). The latter are rarely found in nature, at least in the case of small affinity ligands (affinity tags), and are usually difficult to handle (complex dissociation) once one has found them. In this article we describe a new type of thermoresponsive affinity macroligand. The base polymer (poly-N-isopropylacrylamide, or PNIPAAm) is produced by chain transfer polymerization. As a consequence, the structure, as well as the solubility behavior, is very homogeneous (polydispersity < 1.2), whereas the average molecular mass is small (<5000 g/mol). In pure water, the base polymer shows sharp thermoprecipitation at 32.2 degrees C. Each oligomer carries a single amino end group, which allows easy and defined coupling of the affinity ligand, while preserving the ligand's activity to the highest possible degree. Herein, the oligomer was coupled to iminobiotin. The ensuing affinity macroligand has a high affinity to avidin (and avidin-tagged molecules) at elevated pH (<10), but releases the avidin easily at lower pH (approximately 4). The affinity macroligands were used to purify avidin from solutions containing large amounts of lysozyme as well as from cell culture supernatants containing 5% fetal calf serum. In both cases, pure avidin was recovered (residual protein contamination below the detection limit), with yields of >90%.
Garret-Flaudy
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Center of Biotechnology, Swiss Federal Institute of Technology Lausanne, 1015 Ecublens, Switzerland.
Freitag
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D000327:Adsorption; D000345:Affinity Labels / Q000737:chemistry* / Q000378:metabolism; D001360:Avidin / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D001710:Biotin / Q000031:analogs & derivatives / Q000737:chemistry; D011232:Chemical Precipitation; D004527:Egg Proteins / Q000737:chemistry; D004795:Enzyme Stability; D009113:Muramidase / Q000737:chemistry; D011108:Polymers / Q000737:chemistry* / Q000378:metabolism; D012965:Sodium Chloride / Q000737:chemistry; D012995:Solubility; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D000345:Affinity Labels; D004527:Egg Proteins; D011108:Polymers; D001360:Avidin; D012965:Sodium Chloride; D001710:Biotin; D009113:Muramidase; C028072:2-iminobiotin