Difference between revisions of "Part:BBa K5522005"
Line 32: Line 32:
 
</head>
 
</head>
 
<body>
 
<body>
−
 
+
Existing Part: BBa_K2817003
 
     <!-- Introduction Section -->
 
     <!-- Introduction Section -->
 
     <h2>Summary</h2>
 
     <h2>Summary</h2>

Revision as of 09:51, 30 September 2024


pET28a-IL10


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 4402
    Illegal BglII site found at 4546
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 2622
    Illegal NgoMIV site found at 2782
    Illegal NgoMIV site found at 4370
  • 1000
    COMPATIBLE WITH RFC[1000]


pET28a-IL10 Gene Documentation Existing Part: BBa_K2817003

Summary

In order to increase protein expression and solubility, we designed to add different tags and applied codon optimization techniques.

  1. The sequences were compiled in this fashion: SUMO-IL-10-Fc(BBa_K5522004). A 6×His tag was added to the C-terminal of the protein. The SUMO modification enhances the stability of the protein. The Fc modification slows the degradation of proteins in vivo. The His tag is used for protein purification.
  2. The codon optimization improves gene expression and enhances translational efficiency within E. coli. We constructed a new plasmid BBa_K5522005 (pET28a-IL10), induced protein expression, and tested inhibition of IFN-gamma, indicating its anti-inflammatory effect. We also compared our new part with IL18.
  3. This project provides a potential new treatment for IBD and compares the effects of several different proteins.

Documentation

Usage and Biology

The protein encoded by this gene is a cytokine primarily produced by monocytes and, to a lesser extent, lymphocytes. It has pleiotropic effects in immunoregulation and inflammation, down-regulating the expression of Th1 cytokines, MHC class II Ags, and costimulatory molecules on macrophages. It also enhances B cell survival, proliferation, and antibody production. This cytokine can block NF-kappa B activity and is involved in regulating the JAK-STAT signaling pathway. Knockout studies in mice suggested its function as an essential immunoregulator in the intestinal tract[1].

Construction design

Figure 1:Plasmid map of pET28a-IL10
Fig 1. The plasmid map of pET28a-IL10

The pET28a vector is a widely used vector in synthetic biology, containing a T7 promoter and lac operator, allowing for fine-tuned control over the plasmid's recombinant protein expression (induced by IPTG). It includes a poly-histidine sequence meant to simplify purification. After combining the human SUMO and Fc gene fragments (obtained from NCBI), going through codon optimization, and adding NheI and XhoI restriction sites, we plan to ligate the SUMO-IL-10-Fc fragment onto the plasmid for testing in BL21 cells to verify protein expression effectiveness.

Experimental Approach

We transformed the pET28a-Sumo-IL10-Fc synthesized by the company into E. coli BL21 to promote protein expression as our positive control. In Fig 2A, the agarose gel electrophoresis results showed that we obtained the expected length of sumo-IL10-Fc, indicating the successful completion of the construction. The target gene sequence was consistent with the sequencing results (Fig 2B).

Gene sequencing and colony PCR amplification of sumo-IL10-Fc
Fig 2. Gene sequencing and colony PCR amplification of sumo-IL10-Fc

Characterization/Measurement

The purified IL10 protein was 62.1kDa. SDS-PAGE successfully verified the IL10 proteins extracted and purified from E. coli BL21 (Fig 3).

SDS-PAGE verification of extracted proteins
Fig 3. SDS-PAGE verification of extracted proteins

Compared to coomassie Brilliant Blue staining, the principle of Western detection involves the antibody-antigen specific reaction, allowing for high detection specificity. As shown in Fig 4, the protein size we obtained is consistent with the expected size, demonstrating successful protein expression.

Detection of recombination protein expression by western blot
Fig 4. Detection of recombination protein expression by western blot

We stored the Sumo-IL-10 proteins at different temperatures (-80, -20, 4, 37 ℃) for 24 hours and incubated them with mouse primary T lymphocytes. After stimulating the cells with IL-18, we detected the IFN-γ levels in the cell supernatant.

In Fig 5, the concentration of IFN-γ gradually decreased with increased Sumo-IL-10 protein concentrations, illustrating the inhibitory effect of recombinant Sumo-IL-10 proteins on IFN-γ production. The results showed that the activity of the protein is affected by temperature. Storing at -80°C preserves the stability of recombinant proteins. Moreover, the anti-inflammatory effect of recombinant proteins is dose-dependent.

Influence of dose and storage temperature on protein activity
Fig 5. The influence of dose and storage temperature on protein activity

References

  • [1] Piersiala K, Hjalmarsson E, da Silva PFN, Lagebro V, Kolev A, Starkhammar M, Elliot A, Marklund L, Munck-Wikland E, Margolin G, Georén SK, Cardell LO. Regulatory B cells producing IL-10 are increased in human tumor draining lymph nodes. Int J Cancer. 2023 Aug 15;153(4):854-866. doi: 10.1002/ijc.34555. Epub 2023 May 5. PMID: 37144812.