Patent ID: 8916691

Claim:
A method of producing a histidine-tagged LIM mineralization protein in a recombinant human host cell, which comprises: transfecting human host cell with an isolated nucleic acid molecule encoding the histidine-tagged LIM mineralization protein, culturing said cell under conditions promoting the expression of the LIM mineralization protein in said cell; and, purifying the LIM mineralization protein comprising the steps of fractionation by gel filtration prior to metal affinity chromatography, wherein said fractionalization includes: suspending the LIM mineralization protein in a 20 mM phosphate lysis buffer, the phosphate lysis buffer comprising pH 7.0 containing 50 mM Tris-HCl, pH 7.5 and 5 M NaCL, spinning the LIM mineralization protein in a centrifuge at 10,000 g at 4° C. to produce a supernatant, applying the supernatant onto a gel filtration column pre-loaded with low and high molecular weight protein markers at a low flow rate, the flow rate comprising 1 ml/minute, and wherein said metal affinity chromatography includes: applying the at least one fraction onto a metal affinity column, washing non-specific and low affinity proteins from the metal affinity column with a first phosphate buffer, eluting affinity-bound proteins from the metal affinity column with a second phosphate buffer, concentrating the affinity-bound proteins, and dialyzing the concentrated affinity-bound proteins to produce a LIM mineralization protein that is substantially free of any carbohydrate moiety, wherein the method is performed in vitro.