Part:BBa_K1694004
Single-chain variable fragment (Anti-EGFR)
Introduction: Single-Chain Variable Fragment of Cetuximab
ScFv (Single-Chain Variable Fragment)
ScFv (single-chain variable fragment) is a fusion protein containing light (VL) and heavy (VH) variable domains connected by a short peptide linker. The peptide linker (GGSSRSSSSGGGGSGGGG) is rich in glycine and serine which makes it flexible.
Features of scFv:
1. Specificity：Though remove of the constant regions , scFv still maintain the specificity of the original immunoglobulin.
2. Efficient：ScFv is smaller than the entire antibody, so that the loading of production to E.coli is lower.
Cetuximab
(International Nonproprietary Name (INN) : epidermal growth factor receptor (EGFR) inhibitor) is a chimeric (mouse/human) monoclonal antibody and it specific binds to target antigen epidermal growth factor receptor (EGFR).With high affinity it can prevent ligand binding and activation
We selected the single chain variable fragments (scFv) of monoclonal antibodies Cetuximab and named it Anti-EGFR
Epidermal growth factor receptor
EGFR is a transmembrane tyrosine kinase receptor that regulates the cell division and cell apoptosis.
EGFR is characterized by an extracellular ligand-binding domain, a transmembrane domain, and a cytoplasmic domain containing the tyrosine kinase region followed by a carboxyl-terminal tail with tyrosine autophosphorylation sites.
EGFR is overexpressed on the cell surfaces of various solid tumors.
Mutations in the gene encoding EGFR that lead to overexpression of this protein will lead to uncontrollable cells proliferate.
Mechanism:
1. Cetuximab inhibition
When Cetuximab binds to the extracellular domain of the EGFR, it prevents the activation and subsequent dimerization of the receptor, inhibition in signal transduction and anti-proliferative effects. Moreover, this agent may inhibit EGFR-dependent primary tumor growth and metastasis.
They may occur by the decreasing in receptor activation and dimerization.
2.EGFR activation
Firstly, the ligand binding at the extracellular domain of EGFR will lead to the occurance of active homo- or hetero-dimers.
Dimerization induces the activation of the tyrosine kinase(TK) domain, leading to autophosphorylation of the receptors on multiple tyrosine residues.
This phosphorylation triggers recruitment of a range of adaptor proteins, , followed by a series of intracellular signaling cascades that finally will affect the cell proliferation, apoptosis, invasion, metastasis, and angiogenesis.
1.http://www.sciencedirect.com/science/article/pii/S0959804901002301
2.https://en.wikipedia.org/wiki/Cetuximab
3.[ ]M. Whirl-Carrillo, E.M. McDonagh, J. M. Hebert, L. Gong, K. Sangkuhl, C.F. Thorn, R.B. Altman and T.E. Klein. "Pharmacogenomics Knowledge for Personalized Medicine" Clinical Pharmacology & Therapeutics (2012) 92(4): 414-417
Experiment:
After receiving the DNA sequences from the gene synthesis company, we recombined each scFv gene to pSB1C3 backbones and conducted a PCR experiment to check the size of each of the scFvs. The DNA sequence length of the scFvs are around 600~800 bp. In this PCR experiment, the scFv products size should be near at 850~1050 bp. The Fig. showed the correct size of the scFv, and proved that we successful ligated the scFv sequence onto an ideal backbone.
Application of the part:
1.
Cell staining experiment:
After creating the part of anti-EGFR，we are able to co-transform them with different fluorescent parts into our E. Cotector.
The next step is to prove that our co-transformed product have successfully displayed scFv of anti-EGFR and expressed fluorescent part.
To prove this, we have decided to undergo the cell staining experiment by using our E. Cotector to detect the EGFR in the cell lines.
Each type of E. Cotector has been co-transformed with two different fluorescent colors ---RFP and GFP
Procedure：
First of all, the main materials that we needed are red and green fluorescent of co-transform E.coli with scFv of anti-EGFR, red green blue fluorescent E.coli without scFv and the cancer cell line – SKOV-3 that expressed EGFR, for staining used.
SKOV-3 is a kind of epithelial cell that expressed markers such as EGFR.
After injecting E.coli into the wells, we had to shake the plate in darkness for 45minutes. After staining for 45 minutes, we will wash away the unbind E.coli with PBS solution for a few times before observing the staining result under fluorescent microscope.
Below are our staining result：
Negative control:
There are red and green fluorescent anti-EGFR E.Cotectors stick on the cell’s surfaces as the anti-EGFR probes on E.Cotectors successfully detect and bind with EGFR.
2.
cell staining experiment:
After creating the part of scFv and transforming them into our E.Cotector, we were going to prove that our detectors have successfully displayed scFv of anti-EGFR. To prove this, we have decided to undergo the cell staining experiment by using our E.Cotector to detect the EGFR in the cell lines.
Procedure：
First of all, the main materials that we needed are red green blue fluorescent E.coli with scFv of anti-EGFR, red and green fluorescent E. coli without scFv and the cancer cell line – SKOV-3 that expressed EGFR,for staining used. SKOV-3 is a kind of epithelial cell that expressed markers such as EGFR.
After injecting E.coli into the wells, we had to shake the plate in darkness for 45 minutes. After staining for 45 minutes, we will wash away the unbind E.coli with PBS solution for a few times before observing the staining result under fluorescent microscope.
Below are our staining result：
Negative control:
Modeling:
In the modeling part, we discover optimum protein production time by using the genetic algorithm in Matlab.
We want to characterize the actual kinetics of this Hill-function based model that accurately reflects protein production time.
When we have the simulated protein production rate, the graph of protein production versus time can be drawn (Fig.1) (Fig.2) (Fig.3). Thus, we get the optimum protein production time
Compared with the simulated protein production rate of time, our experiment data quite fit the simulation.
Sequence and Features
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- 1000COMPATIBLE WITH RFC[1000]
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