Model Process for Removal of Caseins from Milk of Transgenic Animals

Abstract

We describe a method for selective removal of caseins from milk. The method was developed as a model for transgenic milk processing. Raw cow milk spiked with nonmilk proteins was chosen as the model to resemble transgenic animal milk containing recombinant proteins. The most important elements of the process are (1) "deconstruction" of casein micelles in milk by destroying their Ca(2+) core using a chelating agent (EDTA), thus freeing any protein that might be entrapped in casein aggregates, and (2) "reconstruction" of micelles by providing them with a new Ca(2+) core, thus precipitating them away from the whey proteins, and the protein of interest. Calcium phosphate particles (CAP) were used to reform the disrupted casein micelles. The crystal clear supernatant fraction generated by this method provided >90% recovery and 6- to 13-fold concentration of the desired protein. Product-rich supernatant contained no detectable casein residues, as silver-stained SDS-PAGE and Western blot analyses demonstrated.

Oral protein delivery: Current status and future prospect

“…The preclinical studies demonstrated that CAP nanoparticles are non-toxic, and cause no adverse reaction at the site of administration following oral and intramuscular administration as vaccine carriers [61,62]. BioSante Pharmaceuticals discovered that CAP nanoparticles have the ability to deliver protein drugs in non-invasive and non-injectable ways [63,64]. The oral insulin formulation, called CaP-PEGInsulin-Casein (CAPIC), was constructed by aggregating casein (the principal protein of milk) around a proprietary formulation of PEG and insulin (Fig.…”

On-line casein micelle disruption for downstream purification of recombinant human myelin basic protein produced in the milk of transgenic cows

“…Several approaches employing casein micelle disruption were trialed: (i) isoelectric point precipitation of caseins using 1.0 M acetic acid to pH 4.6 and solubilization of the casein pellet using 8.0 M urea, (ii) disruption of the casein micelles by addition of either urea or arginine to the milk samples to a final concentration of 8.0 and 2.0 M respectively, and (iii) selective precipitation of caseins using CAP nanoparticles as previously described [26]. The rhMBP was then recovered from the clarified fractions obtained in each case using Ni 2+ Sepharose FF followed by SP Sepharose FF in the presence of 8.0 M urea (PD-10 disposable columns, cv 5 ml each).…”

“…It has been reported that more than 90% removal of caseins was achieved by deconstruction of casein micelle calcium core using EDTA then precipitation of caseins using insoluble calcium phosphate nanoparticles (CAP). This approach was claimed to be successful in the liberation of recombinant proteins that could be associated with the casein micelles [25,26]. Other approaches using additives such as arginine [27] or increasing the milk ionic strength by adding salt [28] have been reported in order to disrupt the interaction between casein micelles and valuable proteins in milk.…”

Optimized recovery of monoclonal antibodies from transgenic goat milk by microfiltration

“…Traditional methods used by the dairy industry to isolate proteins from milk involving pasteurization followed by enzymatic coagulation or acid precipitation at pH 4.6 (pI of casein) are unsuitable for the recovery of heterologous proteins because they can be temperature and pH sensitive. Additionally, the coagulation process can trap a large amount of the target protein within the casein pellets resulting in poor yields (Morcol et al, 2001).…”