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RESEARCH PRODUCT

Keyhole Limpet Hemocyanin (KLH): Slow In Vitro Reassociation of KLH1 and KLH2 from Immucothel®

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Abstract Following our in vitro reassociation of keyhole limpet hemocyanin subunits in the presence of high concentrations (100 mM each) of calcium and magnesium chloride (Harris et al., 1997a, Micron 28, 31–41; 1997b, Micron 28, 43–56), we have now extended our investigations by using a buffer system containing a lower concentration of the two divalent cations (10 mM each). Reassociation of mixed KLH subunits present in the commercially available product Immucothel® was performed using a standardized buffer solution containing 50 mM Tris–HCl, 150 mM NaCl, 10 mM CaCl2 and 10 mM MgCl2 (pH 7.4) over a minimum period of one week, at 4°C. This solution was selected as being close to our KLH stabilizing buffer (used routinely for the 4°C storage of native KLH), but with a slightly elevated concentration of both divalent cations (i.e. 10 mM instead of the usual 5 mM) to potentiate KLH reassociation. The reformation of the higher molecular mass oligomeric and polymeric forms of KLH was monitored throughout from air-dried negatively stained specimens prepared on continuous carbon support films and across the small holes of holey carbon support films, and also by cryo-negative staining. Compared to our previous studies, KLH reassociation has been found to be much slower, but a high percent recovery of total KLH (ca. 87%, produced by centrifugal pelleting), was achieved after a period of seven–ten days reassociation at 4°C. In vitro production of naturally occurring oligomeric forms of KLH1 and KLH2 now predominates, and the proportion of smaller diameter tubular polymeric forms is much less. After reassociation of the mixed KLH1 and KLH2 subunits present in Immucothel®, separation of intact KLH1 from dissociated KLH2 was achieved by gel filtration chromatography after dialysis against 1% ammonium molybdate–0.2% PEG (Mr 1000) at pH 5.7 and monitored by native PAGE. This purification enabled the reassociation characteristics of the two purified KLH subunits (from a 130 mM glycine–NaOH buffer solution at pH 9.6) to be investigated in our 10 mM divalent cation-supplemented Tris–saline buffer. In both cases, the second reassociation, performed over a period of one–two weeks at 4°C, led to the production of the characteristic decameric oligomeric forms; for KLH1 the didecamer predominates, and for KLH2 didecamers and short multidecamers predominate, but in both cases a small quantity of the previously described helical/tubular polymers is also present. Subsequent centrifugal pelleting of this reassociated KLH1 and KLH2, with resuspension and storage at −70°C in stabilizing buffer containing 10% (w/v) trehalose, has been found to provide a biochemically and structurally characterized stock of purified KLH oligomers. The prolonged time-periods required by the overall reassociation and purification procedure do not appear to detract from the quality or stability of the high molecular mass forms of KLH1 and KLH2 so produced.