Expression of caltrin in the baculovirus system and its purification in high yield and purity by cobalt (II) affinity chromatography.
Direct protein extraction from animals is the only approach available to obtain caltrin, calcium transport inhibitor. Here we report the expression and purification of caltrin, previously shown to hinder the influx of calcium into epididymal spermatozoa. Cloning of the caltrin gene into the pCDNA3.1 V5/His-TOPO vector and the subsequent ligation of the caltrin-His sequence into the transfer vector pBacPAK9 allowed the expression of recombinant caltrin using the baculovirus expression vector system (BEVS). Recombinant His-tagged caltrin was purified utilising both nickel (II)-nitrilotriacetic acid (Ni(2+)-NTA) and cobalt (II)-carboxymethylaspartate (Co(2+)-CmAsp) immobilised metal affinity chromatography (IMAC). Using the BEVS, caltrin-His was identified in the supernatant and in the cell lysate, suggesting that caltrin is a secreted protein. Based on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot results, purified recombinant caltrin-His was ascertained to be approximately 14.5kDa. Purification under the Co(2+) system yielded significantly purer protein samples when compared to the Ni(2+) system. Furthermore, Co(2+) was observed to bind the recombinant caltrin-His protein with higher efficiency and specificity and to yield a higher total protein concentration. Collectively, our results indicate that the Co(2+) system would be a better approach for purifying caltrin-His proteins than the Ni(2+).
Phan
Tony C A
TC
Department of Orthopaedic Surgery, University of Western Australia, Nedlands, WA 6009, Australia. tonyphan@cyllene.uwa.edu.au
Nowak
Kristen J
KJ
Akkari
P Anthony
PA
Zheng
Ming H
MH
Xu
Jiake
J
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D015153:Blotting, Western; D002460:Cell Line; D002846:Chromatography, Affinity / Q000379:methods*; D003035:Cobalt / Q000737:chemistry* / Q000378:metabolism; D051379:Mice; D009532:Nickel / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D029607:Seminal Vesicle Secretory Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D018411:Spodoptera / Q000821:virology
D011994:Recombinant Proteins; D029607:Seminal Vesicle Secretory Proteins; D003035:Cobalt; D009532:Nickel