1.Purpose of the experiment:
The purpose of this study was to construct the recombinant protein of NPCC2 using genetic recombinant technology, and to identify its function and expression to further understand its biological characteristics and potential applications.
2.Basic information about NPC2 protein:
NPC2 (Niemann-Pick C2) protein is a cholesterol transporter encoded by the human NPC2 gene, which plays an important role in cholesterol metabolism and cholesterol trafficking pathways. The NPCC2 protein contains 151 amino acid residues with a molecular weight of about 174 kda。This protein is mainly found in the endoplasmic reticulum and lysosomal cavity and is involved in the intracellular cholesterol transport process by interacting with other proteins.
Catalog No. PA1000-5741
3.Protocol Procedure:
Step 1: Gene cloning.
a.Firstly, the coding region of human NPC2 gene was amplified by PCR, and the target sequence was amplified with appropriate primers.
b.The amplification product was enzymatically digested, and the coding region of the NPCC2 gene was ligated with the expression vector to form a recombinant vector.
c.Transform the recombinant vector into Escherichia coli (e.).coli) host bacteria, screening positive clones.
Step 2: Recombinant protein expression and purification.
a.Positive recombinant strains were selected, and the recombinant proteins were obtained by large-scale fermentation culture by inducing the expression of genes.
b.The recombinant protein in the medium was collected, and the protein was precipitated and extracted by centrifugation and other steps.
c.Purification was performed using affinity chromatography columns and ion exchange chromatography columns to remove impurities and unbound proteins;
d.Purified proteins were identified using methods such as SDS-PAGE and Western blot.
Step 3: Functional Identification.
a.In vitro lysosomal membrane liposome binding assay was used to verify the binding ability of recombinant NPC2 protein to cholesterol or other lipids in lysosomes.
b.The cholesterol transport efficiency of recombinant NPCC2 protein and wild-type protein was compared and analyzed, and the cholesterol level after transport was detected by high performance liquid chromatography.
c.Other further molecular biology or biochemical experiments, such as nuclear magnetic resonance (NMR), mass spectrometry, etc., can be performed to further study the function of NPC2 protein.
4.Results and Discussion:
Through gene cloning technology, the NPCC2 recombinant protein expression vector was successfully constructed, and the recombinant protein with high expression was obtained during the expression process in Escherichia coli. After the purification steps of affinity chromatography and ion-exchange chromatography, the degree of purification of the recombinant NPC2 protein and the presence of the protein of interest were confirmed by SDS-PAGE and Western blot identification.
The ability of NPCC2 protein to bind cholesterol and other lipids was verified by in vitro experiments, and its efficiency in the process of cholesterol transport was further improved. The experimental results showed that the recombinant NPC2 protein could effectively bind cholesterol and improve its transport efficiency, which further verified its biological function in the cholesterol metabolism pathway. In addition, further NMR and mass spectrometry analyses can provide more detailed information on the structure and function of NPC2 proteins.
5.Summary:
In this experiment, the NPCC2 recombinant protein expression system was successfully constructed and functionally identified. The results of this experiment can provide a theoretical basis for further study of the relationship between NPCC2 protein and cholesterol metabolism and cholesterol transport related diseases, as well as for the development of related methods.