Application
Core protein solutions that drive interdisciplinary research and industrial breakthroughs
CFPS is invincible; the challenge of membrane proteins has been effortlessly overcome.
Release date:
2024-09-09
Membrane proteins play crucial roles in numerous cellular processes, including transport, signal transduction, and proliferation and differentiation, and they constitute important drug targets. However, due to low expression levels, conformational instability, and difficulties in isolation and purification, many studies are stalled by the challenge of expressing membrane proteins efficiently.
A research group at Shandong Agricultural University encountered a challenging problem: a certain membrane protein proved difficult to express in a prokaryotic system, and despite two years of repeated attempts, they were still unable to overcome this hurdle.
Perotin Bio, leveraging its proprietary cell-free protein synthesis (CFPS) system, has successfully expressed multiple full-length transmembrane proteins. The research group, having learned of Perotin Bio’s expertise, decided to entrust the company with an attempt to express this membrane protein using the CFPS platform, after gaining a deeper understanding of CFPS technology and Perotin Bio’s technological strengths in the cell-free field.
Test Method
Upon receiving the customer’s plasmid, Perotin Bio’s R&D team promptly began constructing a linear expression template. To directly monitor the expression of the target membrane protein and enhance its solubility, they also engineered an expression construct incorporating GFP. Subsequently, they employed a specialized CFPS system tailored for membrane proteins—derived from the PLDK002 kit—and supplemented it with a detergent. [1] , the cell-free reaction system was assembled on the day the plasmid was received.
Experimental Design:
| Group |
NC |
A1 |
A2 |
B1 |
B2 |
| Whether to integrate GFP |
- |
- |
- |
+ |
+ |
| Add detergent? |
- |
- |
+ |
- |
+ |
| Purification tag |
- |
His tag |
|||
Note: [1] This detergent was selected based on experimental data from the Perrotin biofilm protein expression database.
Test Results
Figure 1: The target membrane protein fused with GFP emits green fluorescence under the gel imaging system.
The next day, the supernatant from the reaction was collected and subjected to affinity purification and expression analysis. As shown in Figure 1, both groups B1 and B2 exhibited green fluorescence under the gel imaging system, indicating that the GFP‑fused target membrane protein can be stably expressed in soluble form in the CFPS supernatant, regardless of whether a detergent is added.
Figure 2. SDS-PAGE analysis of the target membrane protein.
After inclusion and decolorization, as shown in Figure 2, the target membrane protein that did not undergo fusion (groups A1 and A2) was also expressed in a soluble form in the supernatant. Furthermore, by comparing groups A1 and A2 with groups B1 and B2, it was observed that the addition of a detergent promotes the soluble expression of this membrane protein.
Summary
The Perrotin CFPS system lived up to expectations, successfully expressing a membrane protein that had eluded the project leader for two years within just two days—truly deserving its reputation as a “savior for hard-to-express proteins”! This successful experiment once again underscores the exceptional prowess of Perrotin CFPS technology in the expression of challenging proteins, particularly membrane proteins.
A surprise at the end!
To enable more membrane protein research teams to benefit from CFPS technology and to meet the eager expectations of new customers, Perotin Bio is extending its “Membrane Proteins in the Cloud—Custom Protein Expression Service: No Charge if No Protein Is Expressed” promotion through December 31, 2024!
Link: Cloud-Based Membrane Protein – Custom Protein Expression Services; No Charge if the Protein Is Not Expressed.
Contact Us:
Phone: 0512-67900128
Official website: www.cellfreeprotein.cn
Address: Room 302, Building 1, Weili Medical Science and Technology Park, No. 69 Jiepu Road, Suzhou Industrial Park
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