Expression and purification of HtpX-like small heat shock integral membrane protease of an unknown organism related to Methylobacillus flagellatus.
The M48 conserved family of peptidases contains a single catalytic zinc ion tetrahedrally co-ordinated by two histidines within an HEXXH motif. The proteases of this class are generally toxic to the cell and thus difficult to express and purify. Here, we report the expression and purification of the small HtpX-like heat shock metalloprotease from an unknown organism related to the obligate methylotrophic anaerobic bacterium, Methylobacillus flagellatus. The protease was expressed in the Escherichia coli vector - pT7. Optimization of expression was done to increase the yield and solubility of the expressed protein. Improved refolding procedures from inclusion bodies of pT7 E. coli system were devised to get the protease in an active and stable form. The protease was purified to near homogeneity in its active form from the refolded proteins of the inclusion bodies by a two-step (cation exchange followed by gel filtration) high performance liquid chromatography (HPLC). The purified protease was active on zymography and casein hydrolysis assays. The activity of the protease was found to be optimum at pH 7.4 and at a temperature of 37 degrees C but significant activity was also retained at higher temperatures of 45-50 degrees C. Centrifugal fractionation showed that it is a membrane localized endopeptidase. The methods described here can serve as guidelines to express and purify other homologues of M48 family of proteases for functional and structural studies.
Siddiqui
Asim A
AA
Department of Biotechnology, All India Institute of Medical Sciences, Ansari Nagar, New Delhi-110029, India.
Jalah
Rashmi
R
Sharma
Yagya D
YD
D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D017931:DNA Primers; D006360:Heat-Shock Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D002479:Inclusion Bodies / Q000378:metabolism / Q000648:ultrastructure; D007700:Kinetics; D020631:Methylobacillus / Q000235:genetics*; D017510:Protein Folding; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism
D001426:Bacterial Proteins; D017931:DNA Primers; D006360:Heat-Shock Proteins; D011994:Recombinant Proteins