1.Purpose of the experiment:
The purpose of this study was to prepare Ca4 (carbonic anhydrase 4) protein by genetic recombination technology and to study its function in carbonic anhydrase-catalyzed reaction.
2.Ca4 Protein Basic Information:
Ca4 belongs to the carbonic anhydrase family, which regulates the pH value by accelerating the conversion of water and carbon dioxide, and plays an important role in maintaining the acid-base balance of organisms. Ca4 protein is composed of 260 amino acids and has a molecular weight of about 30 kDa.
3.Protocol Procedure:
1) Gene cloning: RNA is extracted from human cells and cDNA of Ca4 protein is synthesized by reverse transcriptase. The CDN** segment was ligated to the expression vector and transformed into E. coli (E.).coli).
2) Bacterial culture and induction: inoculate the bacterial solution containing the recombinant vector into the medium containing antibiotics, and after the concentration of the bacterial solution reaches the specified value, isopropyl-D-thiogalactoside (IPTG) is added to induce the expression of the target protein.
3) Cell rupture and extraction: Collect the bacteria induced by IPTG and perform ultrasonic disruption or chemical rupture to extract the protein from the cell matrix.
4) Affinity chromatography purification: Affinity chromatography columns, such as nickel affinity resin columns, are used to purify according to the His-tag on the Ca4 protein.
5) Protein identification and analysis: Proteins were separated by SDS-PAGE electrophoresis and verified by Western blot or mass spectrometry.
6) Ca4 enzyme activity assay: determine the catalytic activity of Ca4 protein in water and carbon dioxide conversion reactions, and evaluate its function.
4.Experimental results and discussion:
Through the above experimental design, we successfully prepared the Ca4 recombinant protein. The resulting protein was verified by SDS-PAGE and Western blot with the correct molecular weight and specificity of the target protein. Further enzyme activity assays showed that Ca4 protein exhibited enzymatic activity similar to that of natural enzymes. This proves that we have successfully prepared a functional Ca4 protein.