Basic Information:
Gene name: KIM1
Gene size: about 1500 bp
Protein molecular weight: about 39 kDa
Subcellular localization: membrane protein, predominantly located on epithelial cells near the convoluted tubule.
Catalog No. PA1000-5533
Overview: In this experiment, we will successfully prepare a recombinant form of kidney injury molecule 1 (KIM1) protein using a bacterial expression system, and perform preliminary functional studies in the obtained recombinant protein. KIM1 is a membrane protein that is highly expressed during damage and is thought to play an important role in regeneration and damage repair.
Experimental design:1Gene cloning:
The full-length sequence of the Kim1 gene was amplified from human kidney cDNA by PCR and cloned into an appropriate expression vector.
2.Bacterial expression:
The constructed KIM1 expression vector was transformed into Escherichia coli (E.).coli) expression host strains, and monoclonal strains with normal expression of kim1 protein were screened by using the selection of montatin.
3.Protein Expression and Purification:
The Kim1-expressing strain was cultured in an appropriate medium, and after induction of expression, bacterial cells were harvested, and the Kim1 protein was extracted by ultrasonic lysis and other means, and the protein was purified by affinity column chromatography and other technologies.
4.Identification and quantification:
The molecular weight determination and purity identification of the purified Kim1 protein were carried out by SDS-PAGE and Western blot. Determine the concentration of Kim1 protein using BCA or other appropriate protein quantification methods.
5.Biological Function Studies:
a) Determine the antigenicity: Verify the antigenicity of KIM1 protein by ELISA and other methods, and apply it to animal experiments or in vitro cell experiments.
b) Structural and functional studies: through crystallography and other methods, the molecular structure of KIM1 protein and its possible functional regions are analyzed.
c) Research related to kidney injury: to detect the potential application of KIM1 protein in the early diagnosis and monitoring of kidney injury through urine samples, etc.
Summary: By successfully preparing the recombinant KIM1 protein by using the bacterial expression system and conducting preliminary functional studies, we can further understand the mechanism of KIM1 protein in the process of kidney injury and repair, and provide new clues for the early diagnosis and improvement of kidney diseases.