Method of producing and purifying soluble recombinant COQ5 protein and soluble recombinant COQ5 protein thereof
AssigneeChang Gung University
InventorsHsiu-Chuan Yen, Shih-Wei Chen
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Abstract
The present invention relates to a producing and purifying method of soluble recombinant coenzyme Q5 homolog (COQ5) protein, which is expressed in soluble form by Escherichia coli (E. coli), under native conditions. The method is characterized by pre-treating bacterial lysate with low concentration of ionic detergent, such as sodium dodecyl sulfate (SDS), before purification; and the purifying method is performed under native condition without using urea to avoid the problems of requiring lengthy processes to remove urea in purified protein solution or re-aggregation and precipitation of protein after removal of urea.
Claims7
A method of producing and purifying recombinant mitochondrial targeting signal (MTS)-truncated coenzyme Q5 homolog (COQ5) protein in non-denaturing conditions, comprising the steps of:
- providing an Escherichia coli (E. coli) host cell transformed with an expression vector containing a DNA fragment that encodes the recombinant COQ5 protein;
- inducing the expression of the recombinant COQ5 protein in the E. coli host cell;
- lysing the bacterial cells with a lysis buffer comprising lysozyme and deoxyribonuclease I (DNase I);
- pre-treating the bacterial cell lysate with less than 0.2% of an ionic detergent;
- centrifuging the cell lysate for removing insoluble proteins or debris and collecting the supernatant containing the recombinant COQ5 protein;
- and
- applying the supernatant to nickel-nitrilotriacetic acid (Ni-NTA) beads under native conditions that lack exposure to urea to obtain purified recombinant COQ5 protein.
A method of producing and purifying recombinant mitochondrial targeting signal (MTS)-truncated coenzyme Q5 homolog (COQ5) protein in non-denaturing conditions, comprising the steps of:
- providing an Escherichia coli (E. coli) host cell transformed with an expression vector containing a DNA fragment that encodes the recombinant COQ5 protein;
- inducing the expression of the recombinant COQ5 protein in the E. coli host cell;
- lysing the bacterial cells with a lysis buffer comprising lysozyme and deoxyribonuclease I (DNase I);
- pre-treating the bacterial cell lysate with less than 0.2% of an ionic detergent;
- centrifuging the cell lysate for removing insoluble proteins or debris and collecting the supernatant containing the recombinant COQ5 protein;
- and
- applying the supernatant to nickel-nitrilotriacetic acid (Ni-NTA) beads under native conditions that lack exposure to urea to obtain purified recombinant COQ5 protein, wherein the recombinant COQ5 protein is a MTS-truncated human COQ5 protein with 6× histidine tag (His tag) at the N-terminus, comprising the amino acid sequence of SEQ ID NO:
- 2.
Dependencies are inferred from the claim text.