Preparative scale purification of recombinant proteins to clinical grade by isotachophoresis.
An electrophoretic procedure based on isotachophoresis has been developed for protein purification on a preparative scale in the 10 to 500 mg range. The system is simple, uses well understood physical properties, does not need ampholyte spacers and is able to produce sterile products of clinical grade. We demonstrate the applicability of this apparatus for the purification of denatured recombinant proteins and complex mixtures of proteins. The system may also be used for both cationic and anionic purification of proteins in their native form. The system is scalable from analytical to preparative protein loads at consistently high protein yields and purity levels. Total protein loads may vary as much as 1000 fold with the use of interchangeable columns of varying diameter and constant length. At both preparative and analytical scales concentration of products at greater than 20 mg/ml are obtainable. Toxicological considerations are addressed with assays for endotoxin, acrylamide and SDS concentrations, as well as the prevention of covalent protein modification.
Stowers
A W
AW
Queensland Institute of Medical Research, Royal Brisbane Hospital, Australia. tonyS@qimr.edu.au
Spring
K J
KJ
Saul
A
A
D004586:Electrophoresis / Q000295:instrumentation / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D017780:Malaria Vaccines / Q000302:isolation & purification; D011489:Protein Denaturation; D015800:Protozoan Proteins / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000302:isolation & purification*; D012710:Serum Albumin, Bovine / Q000302:isolation & purification; D014614:Vaccines, Synthetic / Q000302:isolation & purification
D017780:Malaria Vaccines; D015800:Protozoan Proteins; D011994:Recombinant Proteins; D014614:Vaccines, Synthetic; C065352:rhoptry associated protein, Plasmodium; C073168:rhoptry-associated antigen-2, Plasmodium; D012710:Serum Albumin, Bovine