["Collaborating Expert | Jie Huang, M.S.", "Biology University of Science and Technology of China"], ["Reviewed by | Dr. Nie Yifeng", "Nanobiomedicine University of Chinese Academy of Sciences"]
Summary
A protein expression system is a system consisting of a host, an exogenous gene, a vector, and auxiliary components. The vector contains an exogenous gene fragment, which is mediated by the vector, with the aim of realizing the expression of the exogenous gene in the host. In this paper, we present the eukaryotic expression of a universal secondary PA/G-HRP fusion protein used for mouse- and rabbit-derived IgG as an example.
Principle
The fusion gene fragments of Staphylococcus aureus protein A (SPA), Streptococcus protein G (SPG) and horseradish peroxidase (HRP) were synthesized from the whole gene, and then cloned into the pcDNATM3.1 backbone of the eukaryotic expression vector, and then transiently transfected into the HEK293F cells of the human embryonic kidney, and then identified the expression of the fusion proteins by SDS-PAGE and Western blot. The fusion protein expression was characterized by SDS-PAGE and Western blot.
Appliance
Stable and efficient preparation of multifunctional proteins was realized, and products with high purity and good homogeneity were obtained.
Operation method
Eukaryotic expression of proteins - mammalian system
Principle
The fusion gene fragments of Staphylococcus aureus protein A (SPA), Streptococcus protein G (SPG) and horseradish peroxidase (HRP) were synthesized from the whole gene, and then cloned into the pcDNATM3.1 backbone of the eukaryotic expression vector, and then transiently transfected into the HEK293F cells of the human embryonic kidney, and then identified the expression of the fusion proteins by SDS-PAGE and Western blot. The fusion protein expression was characterized by SDS-PAGE and Western blot.
Materials and Instruments
Instruments:
Constant temperature shaker, centrifuge, electrophoresis apparatus
Protein gel developer, vortexer
Reagents:
HEK293F cells, Restriction endonuclease EcoRⅠ
BamHⅠ
pcDNATM3.1 (-) Eukaryotic expression vector
HyCloneTM CDM4HEK293TM serum-free culture medium
HyCloneTM HyCell TransFx-HTM Culture medium
Bovine Serum Protein BSA, FectoPRO Transfection Kit
Kaomas Brilliant Blue (Cauliflower)
Move
1. Construction of pPA-HRP, pPG-HRP and pPA/G-HRP plasmids:
The amino acid sequences of known PA, PG and HRP were fitted according to codon preference and gene synthesized by the company. The synthesized sequences were inserted into pcDNATM3.1(-) eukaryotic expression vector with EcoRⅠ and BamHⅠ cleavage sites, and the correctly sequenced plasmids were named as pPA-HRP, pPG-HRP, and pPA/G-HRP, respectively.
2. Expression of pPA-HRP, pPG-HRP and pPA/G-HRP in HEK293F cells:
HEK293F cells were cultured using HyCloneTM CDM4HEK293TM serum-free culture medium in 80 mL/L CO2, 37 ℃, 120 r/min cell culture shaker. When the cell number was adjusted to (1~1.5) × 106 cells/mL, the cells were transferred to HyCloneTM HyCell TransFx-HTM culture medium.
Referring to the instruction manual of FectoPRO transfection reagent, the constructed eukaryotic expression plasmids pPA-HRP, pPG-HRP and pPA/G-HRP were transiently transfected into HEK293F cells, which were placed on a cell culture shaker for further incubation. 6 d later, the cell culture supernatant was collected by centrifugation, and then subjected to SDS-PAGE and Western blot analysis.
3. Protein expression identification:
After cell culture, centrifuge the cells at 2000 r/min for 5 min and collect the supernatant. Add 5× SDS-PAGE buffer, boil for 10 min, vortex and centrifuge, and then perform SDS-PAGE. after electrophoresis, the gel was stained with Cauloblue for 3 h. Then, add decolorant until the bands were clearer and the background was cleaner, and then analyze protein expression according to the staining condition.
The gels were transferred to nitrocellulose membranes and closed with TBS containing 30 g/L BSA for 1 h at room temperature, and the HA-tagged antibody was used to detect the expression of PA-HRP, PG-HRP and PA/G-HRP fusion proteins.
Caveat
The E. coli E.coli expression system, which is commonly used in vector selection, is economical, but due to the lack of important lipids and molecular stings, its use for the expression of eukaryotic proteins may affect some of the functions of the proteins themselves.
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