Tight attachment of chitin-binding-domain-tagged proteins to surfaces coated with acetylated chitosan.
Several excellent procedures for trapping tagged proteins have been devised, but many of these are expensive, cannot be used outside a limited pH range, fail to work in the presence of chaotropic agents, or are difficult to use. The chitin binding domain (CBD) of Bacillus circulans chitinase, which binds to chitin matrices prepared from inexpensive reagents isolated from crab shells, is an alternative tag that can be used under a variety of pH and denaturing conditions. Kits based on the interaction between the CBD and the chitin beads are available commercially. Here, we show that simultaneous treatment of microtiter plates with chitosan, a deacetylated form of chitin, and acetic anhydride produces a surface-bound film of chitin that also interacts tightly with the CBD. Chitin-coated microtiter well plates captured a CBD-tagged heterodimeric human glycoprotein hormone analog directly from mammalian cell culture media, even when present in trace amounts. Binding to the surface was stable in sodium dodecylsulfate and reversed only partially at low pH or in 8M urea at 37 degrees C. This technique appears well suited to surface attachment and permits biochemical or other analyses of molecules that can be tagged with a CBD.
Bernard
Michael P
MP
Department of Obstetrics and Gynecology, UMDNJ-Robert Wood Johnson (Rutgers) Medical School, Piscataway, NJ 08854, USA.
Cao
Donghui
D
Myers
Rebecca V
RV
Moyle
William R
WR
D000089:Acetic Anhydrides / Q000737:chemistry; D000107:Acetylation; D000906:Antibodies / Q000276:immunology; D001407:Bacillus / Q000201:enzymology / Q000235:genetics; D002686:Chitin / Q000378:metabolism*; D002688:Chitinases / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D048271:Chitosan / Q000737:chemistry* / Q000378:metabolism; D006063:Chorionic Gonadotropin / Q000235:genetics / Q000276:immunology; D007457:Iodine Radioisotopes; D011485:Protein Binding; D017434:Protein Structure, Tertiary / Q000235:genetics; D021122:Protein Subunits; D011863:Radioimmunoassay / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000032:analysis* / Q000235:genetics / Q000276:immunology
D000089:Acetic Anhydrides; D000906:Antibodies; D006063:Chorionic Gonadotropin; D007457:Iodine Radioisotopes; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; D002686:Chitin; C031800:acetic anhydride; D048271:Chitosan; D002688:Chitinases