Purification of sarcosine oxidase SOX
Sarcosine oxidase is abbreviated as SOX. It is an important enzyme in the metabolism of muscle metabolite creatinine. It is used to detect the content of creatinine in biochemical testing. Its enzyme activity directly affects the accuracy of the test results. Purification is required in the test. After SOX.
Gene engineering preparation and purification of sarcosine oxidase
Determination of creatinine by sarcosine oxidase method:
The coupling of creatine oxidase, creatininase, and creatinase CRH catalyzes the final degradation of creatinine into glycine and hydrogen peroxide, based on which creatinine can be measured. The content of creatinine in blood and urine is an important indicator for judging whether kidney function is normal. The creatinine oxidase method is usually used to determine creatinine: the chromogen substrate TOPS is used to measure the hydrogen peroxide generated by creatinine after SOX catalysis through Trinder reaction. , The amount of color product is measured by absorbance, which is directly proportional to creatinine content.
The sarcosine oxidase used is prepared by genetic engineering and purified:
1.The sarcosine oxidase (SOX) gene derived from Bacillus sp. was cloned and transformed into E. coli for expression. After induction by IPTC reagent, the wet cells of E. coli were collected, and 20mmol/L trihydric was used. Methylaminomethane hydrochloric acid (Tris-HCl) buffer (pH 8.0) washed and centrifuged in a centrifuge;
2.After the centrifugation is completed, the supernatant is removed, and then the cells are resuspended in 20mmol/L Tris-HCl buffer (pH8.0) and ultrasonically broken. The wall breaking condition is 4min/1min (ultrasonic time/intermittent time), the wall breaking time is 20min, until the solution becomes clear, the crude enzyme solution of sarcosine oxidase is obtained.
3.The sarcosine oxidase was purified by agarose sepharose-4-aminopyrrole-2-carboxylic acid affinity chromatography, and the affinity chromatography column was equilibrated with 20 mmol/L Tris-HCl buffer (pH 8.0), and then the crude Enzyme solution was injected into the chromatography column, and the target protein was eluted with 0.03mol/L NaC1/20mmo1/L Tris-HC1 buffer (pH8.0).
The purified sarcosine oxidase was analyzed by SDS-PAGE, and there was an obvious single protein band at the relative molecular mass of 43kDa. After sepharose-4-aminopyrrole-2-carboxylic acid affinity purification, the impurity protein in the crude enzyme solution was basically removed, and the purified SOX reached electrophoresis purity, the specific enzyme activity was 30.8 U/mg, and the enzyme activity recovery rate was 91.2%. The relative molecular mass of SOX was identified by MALDI-TOF. The relative molecular mass of the recombinant protein was 43.8kDa, which was comparable to SOX from Bacillus sp.B-0618 (relatively extraordinary mass 43839Da). The purified sample was stored at 4°C for later use.
There are a variety of reagents and instruments in the purification process of sarcosine oxidase. As an ivd raw material manufacturer, Desheng can provide SOX enzyme preparations, Tris buffers and the chromogen substrate TOPS required for the determination of creatinine.
When it comes to the chemiluminescent reagent Luminol, it is not unfamiliar to everyone and is commonly used in the field of criminal investigation to detect blood stains. Researchers have found that the principle of luminol luminescence can be used to detect specific substances in saliva, such as proteins, DNA, and related indicators of microorganisms, in order to determine physical conditions.