Selective isolation and purification of tat protein via affinity membrane separation.
This work deals with the separation of Tat protein from a complex fermentation broth using an affinity membrane system. Tat is a regulatory protein that is critical for HIV-1 replication and thus a potential candidate for vaccine and drug development. Furthermore, Tat can facilitate transport of exogenous molecules across cell membranes and is implicated in pathogenesis of HIV dementia. Affinity membranes were prepared through coupling of avidin within a 4-stack membrane construct. Tat (naturally biotinylated) accessibility in the bacterial lysate feed was influenced by the presence of RNAse, protein concentration, and ionic strength. Enhanced accessibility translated to a marked increase in the overall product yield per pass. The purity of the membrane-isolated Tat was compared to that prepared via packed column chromatography through SDS-PAGE, Western blot, activity assay, and neurotoxicity studies. Tat protein produced via membrane separation yielded primarily monomeric forms of the oligopeptide sequence, whereas column chromatography produced predominately polymeric forms of Tat. These differences resulted in changes in the neurotoxicity and cellular uptake of the two preparations.
Hollman
Aaron M
AM
Department of Chemical and Materials Engineering, University of Kentucky, Lexington, Kentucky 40506-0046, USA.
Christian
David A
DA
Ray
Philip D
PD
Galey
David
D
Turchan
Jadwiga
J
Nath
Avindra
A
Bhattacharyya
D
D
D015153:Blotting, Western; D002846:Chromatography, Affinity / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005285:Fermentation; D015696:Gene Products, tat / Q000302:isolation & purification*; D015497:HIV-1 / Q000737:chemistry; D008567:Membranes, Artificial; D054322:tat Gene Products, Human Immunodeficiency Virus
D015696:Gene Products, tat; D008567:Membranes, Artificial; D054322:tat Gene Products, Human Immunodeficiency Virus