US2014273152A1PendingUtilityA1

Recombinant manganese oxidase

41
Individually held — no corporate assignee on recordPriority: Mar 13, 2013Filed: Mar 13, 2014Published: Sep 18, 2014
Est. expiryMar 13, 2033(~6.6 yrs left)· nominal 20-yr term from priority
Inventors:Bradley M. TeboCristina Butterfield
C12N 9/0004C12N 9/0065C12N 9/0091C12Y 111/01013C12N 2009/02
41
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Claims

Abstract

Disclosed herein is a recombinant Bacillus manganese oxidase complex, including a bacterial expression vector for the expression of the complex and a method of synthesizing the complex.

Claims

exact text as granted — not AI-modified
1 . A recombinant bacterial expression vector comprising:
 a first polynucleotide that encodes SEQ ID NO: 5 or a homolog thereof;   a second polynucleotide that encodes SEQ ID NO: 6 or a homolog thereof;   a third polynucleotide that encodes SEQ ID NO: 7 or a homolog thereof;   and a promoter sequence operably linked to the first polynucleotide, second polynucleotide and third polynucleotide.   
     
     
         2 . The bacterial expression vector of  claim 1  further comprising a fourth polynucleotide that encodes SEQ ID NO: 4 or a homolog thereof. 
     
     
         3 . The bacterial expression vector of  claim 1  wherein the promoter sequence comprises an inducible promoter. 
     
     
         4 . The bacterial expression vector of  claim 3  wherein the promoter comprises an IPTG inducible promoter. 
     
     
         5 . The bacterial expression vector of  claim 1  within an  E. coli  bacterium. 
     
     
         6 . The bacterial expression vector of  claim 5  wherein the  E. coli  bacterium is of the BL21 DE3 strain. 
     
     
         7 . A recombinantly produced manganese oxidase complex comprising:
 a first polypeptide comprising SEQ ID NO: 5 or a homolog thereof   a second polypeptide comprising SEQ ID NO: 6 or a homolog thereof and   a third polypeptide comprising SEQ ID NO: 7 or a homolog thereof.   
     
     
         8 . The manganese oxidase complex of  claim 7  wherein the manganese oxidase complex comprises one polypeptide of SEQ ID NO: 7; 0-8 polypeptides of SEQ ID NO: 5 and 0-8 polypeptides of SEQ ID NO: 6, wherein the total number of polypeptides of SEQ ID NO: 5 and SEQ ID NO: 6 totals between 6 and 8. 
     
     
         9 . The manganese oxidase complex of  claim 8  wherein the manganese oxidase complex comprises exactly three polypeptides of SEQ ID NO: 5 and exactly three polypeptides of SEQ ID NO: 6. 
     
     
         10 . The manganese oxidase complex of  claim 7  further comprising SEQ ID NO: 4 or a homolog thereof. 
     
     
         11 . The manganese oxidase complex of  claim 7  further comprising a label. 
     
     
         12 . The manganese oxidase complex of  claim 11  wherein the label is a protein tag. 
     
     
         13 . A method of synthesizing a manganese oxidase complex, the method comprising:
 inoculating culture media with an  E. coli  bacterium comprising the bacterial expression vector of  claim 1 , wherein the culture media comprises a first concentration of copper ion;   adding IPTG to the culture media;   adding a copper salt to the culture media to reach a second concentration of copper ion;   wherein the second concentration of copper ion is at least 10-fold higher than the first concentration of copper ion; and wherein the culture media is not agitated after the second concentration of copper ion is reached.   
     
     
         14 . The method of  claim 13  wherein the first concentration of copper ion is 0.2 mM and wherein the second concentration of copper ion is 2 mM. 
     
     
         15 . The method of  claim 13  wherein the copper salt is CuSO 4 . 
     
     
         16 . The method of  claim 13  further comprising purifying the manganese oxidase complex. 
     
     
         17 . The method of  claim 16  wherein the complex is purified by FPLC. 
     
     
         18 . The method of  claim 16  wherein the manganese oxidase complex comprises a label covalently attached to a component of the complex. 
     
     
         19 . The method of  claim 18  wherein the label is a protein tag. 
     
     
         20 . The method of  claim 19  wherein the complex is purified using the protein tag.

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