1.Purpose of the experiment:
The purpose of this study was to obtain high-purity DNASE1 recombinant protein by genetic engineering technology, which can be used to further study the biological function of the protein and its potential applications in autoimmune diseases.
2.Basic information about DNase1 recombinant protein:
Protein name: Deoxyribonuclease I (DNase1).
Genetic**: Human.
Protein molecular weight: approximately 32 kDa
Proteolytic digest: consists of 267 amino acid residues.
Biological function: DNase1 is an endonuclease that functions mainly in tissues with high exosome densities in the nucleus. It is capable of degrading DNA molecules and has the effect of scavenging extracellular free DNA to maintain a stable extracellular environment.
3.Protocol Procedure:
Step 1: Obtain the DNase1 gene fragment.
DNase1 gene fragments were amplified from human chromosomal DNA and obtained by polymerase chain reaction (PCR).
Step 2: Clone the gene fragment into the expression vector.
The DNase1 gene fragment is ligated to an expression vector (e.g., PET-28A) and the gene fragment is inserted into the expression vector using restriction digestion and DNA ligase.
Step 3: Transform the recombinant plasmid into E. coli.
The recombinant plasmid is introduced into Escherichia coli (e.g., BL21(DE3) strain), and the bacteria are subjected to the recombinant plasmid ingestion by heat shock freezing or electroporation to form a transformed bacteria.
Step 4: Grow the inverted bacteria.
Incubate the invariants into a medium containing the appropriate antibiotic (e.g., kanamycin) and incubate them on a shaker or flask at an appropriate temperature (e.g., 37 °C) with regular shaking to incubate.
Step 5: Protein expression induction.
At an appropriate bacterial density (e.g., OD600 is 0.).6-0.8), an inducer (e.g., IPTG) was added to stimulate the activity of the promoter in the recombinant plasmid that promoted protein expression, so that Escherichia coli began to express DNase1 recombinant protein.
Step 6: Cell disruption and lysis.
After culturing the bacteria to the appropriate stage, the bacterial cells are disrupted by ultrasonic disruption or high-pressure disruption to release the DNase1 recombinant protein.
Step 7: Yeast protein expression purification.
Purification for DNase1 specificity and removal of other impurities is performed using techniques such as affinity chromatography columns (e.g., fusion protein affinity resins) or ion exchange columns (e.g., phosphate columns).
Step 8: Identify the purified protein.
Protein analysis methods such as SDS-PAGE and Western blot were used to detect and identify the purified DNase1 recombinant protein.
Step 9: Analyze and save the results.
DNase1 enzyme activity was determined by colorimetric or fluorometric methods, and the results were analyzed. Aliquot the purified DNase1 recombinant protein in a -20°C freezer for future experiments.
Through the above experimental design, high-purity DNase1 recombinant protein can be obtained, which provides strong support for subsequent functional research and application.