Serratia marcescens DNA/RNA Nonspecific Nuclease produced recombinately in E. coli.
Serratia marcescens nuclease is a well known non-specific nuclease that digests single and double stranded RNA and DNA. It is widely used in the purification of recombinant proteins because it reduces or eliminates nucleic acids from the disrupted cells, and greatly decreases the viscosity of the resultant solution. This makes subsequent purification procedures easier. Commercial versions of this enzyme are available but expensive.
From the laboratory of Darrell L. Peterson, PhD, Virginia Commonwealth University.
|Name:||Serratia marcescens nuclease|
|Host:||Recombinant E coli|
|Molecular Weight:||27,444 Da|
|Amino Acid Sequence:||DTLESIDNCA VGCPTGGSSN VSIVRHAYTL NNNSTTKFAN WVAYHITKDT PASGKTRNWK TDPALNPADT LAPADYTGAN AALKVDRGHQ APLASLAGVS DWESLNYLSN ITPQKSDLNQ GAWARLEDQE RKLIDRADIS SVYTVTGPLY ERDMGKLPGT QKAHTIPSAY WKVIFINNSP AVNHYAAFLF DQNTPKGADF CQFRVTVDEI EKRTGLIIWA GLPDDVQASL KSKPGVLPEL MGCKNENLYF Q|
|Fusion Tag(s):||C terminal NLYFQ vector sequence|
|Buffer:||25 mM Tris/HCl, pH 8 + 10% glycerol|
Using the following assay, the purified nuclease has a specific activity of about 40,000 units per milligram.
The activity of the enzyme is determined by measuring the amount of high molecular weight DNA is converted to acid soluble fragments. This assay is somewhat arbitrary, however for practical purposes the following procedure is used.
The assay is performed at 0 C (on ice), since the typical use of this enzyme is during the purification of recombinant proteins from bacterial lysates, and the usual procedures are performed at 0- 4C. Calf thymus DNA (Alfa Aesar ) is dissolved in 25 mM Tris, pH8 containing 10 mM magnesium chloride, such that the solution as an A260 of around 5. 0.2 ml of this is used for each assay, and 10 ul of nuclease diluted into the same buffer are added. The mixture is incubated on ice for various time intervals, then the reaction is stopped by the addition of 1.0 ml of 0.3 N sulfuric acid and centrifuged for 10 min at 15K rpm in a microfuge. The absorbance is read at 260 nM. An identical aliquot of DNA but without added nuclease is used as the blank. 1 unit is defined as the amount of enzyme that solubilizes 1 A260 of DNA in 15 min.
|Comments:||Requires Mg for activity|
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