Innovative modular membrane adsorber system for high-throughput downstream screening for protein purification.
To develop the most efficient strategy for the purification of proteins, two types of adsorber membrane devices with different functionalities were designed and tested: 8-strips and single spin columns. The most suitable type of membrane adsorber and the optimal chromatographic loading/elution conditions for several target proteins from different biological matrices could be determined simultaneously in microliter scale. Ion exchange (IEX), metal chelate (MC), and Concanavalin A (Con A) modified membrane types were tested in the devices. Bovine serum albumin (BSA) and lysozyme were used as model proteins for investigations of the binding capacity and protein recovery percentage of the 8-strip anion exchange and the cation exchange membrane. The isolation of His(6)-tagged proteins, Bgl-His and GFP-His from fermentation broth and lysate, respectively, was performed using an 8-strip metal chelate affinity membrane loaded with different metal ions. Separation behavior of a ternary protein mixture (BSA, lysozyme, and Bgl-His) was studied in 8-strips IEX and metal chelate membrane chromatography. The Con A affinity devices were developed on the basis of metal chelate membrane spin columns loaded with Cu(2+) ions and investigated using glucose oxidase (GOD) as model protein. In summary, the advantages of the membrane adsorber technology, such as fast processing and easy scale-up, were utilized. The devices made it possible to load the membrane directly with preclarified fermentation broth or cell lysate and separate the protein of interest often in a single step.
Kökpinar
Oznur
O
Institut für Technische Chemie, Universität Hannover, Germany.
Harkensee
Daniela
D
Kasper
Cornelia
C
Scheper
Thomas
T
Zeidler
Robert
R
Reif
Oscar-Werner
OW
Ulber
Roland
R
D000327:Adsorption; D002614:Chelating Agents / Q000737:chemistry; D003208:Concanavalin A / Q000737:chemistry; D005949:Glucose Oxidase / Q000302:isolation & purification; D007474:Ion Exchange; D008567:Membranes, Artificial; D008970:Molecular Weight; D009113:Muramidase / Q000302:isolation & purification*; D012710:Serum Albumin, Bovine / Q000302:isolation & purification*; D013499:Surface Properties
D002614:Chelating Agents; D008567:Membranes, Artificial; D003208:Concanavalin A; D012710:Serum Albumin, Bovine; D005949:Glucose Oxidase; D009113:Muramidase