Methods for purifying and isolating recombinant chondroitinases
The invention relates to rationally designed polysaccharide lyases and uses thereof. In particular, the invention relates to modified chondroitinase B. The modified chondroitinase B enzymes of the invention are useful for a variety of purposes, including cleaving and sequencing polysaccharides such as glycosaminoglycans (GAGs) as well as removing polysaccharides from a solution. The invention also includes methods of inhibiting anticoagulant activity, inhibiting angiogenesis, treating cancer, and inhibiting maternal malarial infection.
1. A method for isolating a recombinant chondroitinase, comprising:
lysing a cell culture containing a recombinant chondroitinase having a terminal histidine tag, and
passing the recombinant chondroitinase over a charged Ni 2+ column to isolate the recombinant chondroitinase,
wherein the recombinant chondroitinase is a modified chondroitinase B having an amino acid sequence of the mature peptide of SEQ ID NO: 2 or conservative substitutions thereof, wherein at least one residue at a position selected from the group consisting of 116, 184, 213, 219, 245, 250, 271, 272, 296, 298, 318, 333, 363 and 364 of SEQ ID NO: 2 has been substituted or deleted.
2. The method of claim 1 , wherein the recombinant chondroitinase is selected from the group consisting of chondroitinase AC, chondroitinase B or a modified chondroitinase B.
3. A method for purifying a recombinant chondroitinase, comprising:
inducing a culture of cells containing a recombinant chondroitinase with an inducing agent for greater than four hours, and
isolating the recombinant chondroitinase from the cells to produce a purified chondroitinase,
wherein the recombinant chondroitinase is a modified chondroitinase B having an amino acid sequence of the mature peptide of SEQ ID NO: 2 or conservative substitutions thereof, wherein at least one residue at a position selected from the group consisting of 116, 184, 213, 219, 245, 250, 271, 272, 296, 298, 318, 333, 363 and 364 of SEQ ID NO: 2 has been substituted or deleted,
and wherein the inducing agent is isopropyl-B-D-thiogalactopyranoside.
4. The method of claim 3 , wherein the cells are incubated with the inducing agent at a temperature of between 20° and 26° C.
5. The method of claim 3 , wherein the cells are incubated with the inducing agent for at least 8 hours.
6. The method of claim 4 , wherein the cells are incubated with the inducing agent for at least 8 hours.
7. The method of claim 3 , wherein the recombinant chondroitinase includes a terminal histidine tag.
8. The method of claim 7 , wherein the method further comprises passing the recombinant chondroitinase over a charged Ni 2+ column to isolate the recombinant chondroitinase.