A linear EGFR-binding peptide using 6xHis tag for purification.

Enhanced Green Fluorescent Protein (eGFP) as a positive control to confirm Ssp DnaB and Mxe GyrA intein system is working for expressing a short EGFR-binding peptide in a plasmid with CBD (Chitin Binding Domain) for purification.

One-step circularization of EGFR-binding peptide upon Ssp DnaB and Mxe GyrA intein splicing with CBD (Chitin Binding Domain) for purification.

One-step circularization of scrambled EGFR-binding peptide upon Ssp DnaB and Mxe GyrA intein splicing with CBD (Chitin Binding Domain) for purification. Act as a negative control model.

Enhanced Green Fluorescent Protein (eGFP) as a positive control to confirm Npu intein system is working for expressing a short EGFR-binding peptide in a plasmid with CBD (Chitin Binding Domain) for purification.

One-step circularization of EGFR-binding peptide upon Npu intein splicing with higher efficiency and using CBD (Chitin Binding Domain) for purification.

One-step circularization of scrambled EGFR-binding peptide upon Npu intein splicing with CBD (Chitin Binding Domain) for purification. Act as a negative control model.

The EGFR binding peptide is a 12 amino acid short peptide that can bind specifically to the epidermal growth factor receptor (EGFR) on the surface of lung cancer cells.

A scramble EGRR peptide is a short amino acid sequence that has been randomly generated and does not have any specific biological activity or binding affinity to epidermal growth factor receptor (EGFR). Its function is to serve as a negative control in experiments to assess the specificity and activity of EGFR binding peptide allowing for the discrimination of true binding or functional effects from non-specific interactions.

The Ssp DnaB intein is a self-splicing protein domain that originates from the bacterium Synechocystis sp. PCC6803 and is capable of excising itself from its host protein through a series of chemical reactions.

Ref: Mathys, S., Evans, T.C., Chute, I.C., Wu, H., Chong, S., Benner, J., Liu, X.-Q., and Xu, M.-Q. (1999). Characterization of a self-splicing mini-intein and its conversion into autocatalytic N- and C-terminal cleavage elements: facile production of protein building blocks for protein ligation. Gene 231, 1-13. https://doi.org/10.1016/S0378-1119(99)00103-1.

The Mxe GyrA intein is a self-splicing protein domain that originates from the bacterium Mycobacterium xenopi and is capable of excising itself from its host protein through a series of chemical reactions.

Ref: Mills, K.V., Johnson, M.A., and Perler, F.B. (2014). Protein Splicing: How Inteins Escape from Precursor Proteins*. Journal of Biological Chemistry 289, 14498-14505. https://doi.org/10.1074/jbc.R113.540310.

The Npu intein is a self-splicing protein domain that originates from the bacterium Nostoc punctiforme and is capable of excising itself from its host protein through a series of chemical reactions. It is notable for its high splicing efficiency and has been widely used in protein engineering and biotechnology applications.

A protein degradation tag that direct the tagged protein to the ClpXP machinery for degradation. It reduces the toxicity of Npu intein.

The chitin binding domain is a protein module that can be fused to other proteins to enable their purification using chitin chromatography. N-terminus linked CBD and Its function is to bind specifically to chitin, allowing for the selective isolation of chitin-tagged proteins from complex mixtures.

The Npu intein is a self-splicing protein domain that originates from the bacterium Nostoc punctiforme and is capable of excising itself from its host protein through a series of chemical reactions. It is notable for its high splicing efficiency and has been widely used in protein engineering and biotechnology applications.

The T7 promoter is a DNA sequence derived from the T7 bacteriophage that is recognized by the T7 RNA polymerase enzyme for highly efficient transcription of DNA into RNA. Its function is to drive the expression of genes and production of recombinant proteins in molecular biology and biotechnology applications.

The Hexahistidine affinity tag is a short amino acid sequence that can be added to a recombinant protein to facilitate its purification using immobilized metal affinity chromatography (IMAC). It can bind to metal ions such as nickel or cobalt that are attached to a solid support, allowing for selective purification of the tagged protein from a complex mixture.

Ribosome binding site facilitates the binding of the ribosome to initiate translation of the mRNA into protein.

T7 promoter is good for protein expression in e. coli. Requires the T7 RNA polymerase. Also contains a lac operator (inducable with lactose/ lactose substitute), and a His tag for protein purification.

The T7 terminator is a short DNA sequence that signals the end of transcription by the T7 RNA polymerase enzyme. Its function is to ensure the accurate and efficient termination of transcription, preventing the synthesis of unwanted RNA transcripts downstream of the intended gene or sequence.

The lacI promoter controls the expression of the lac repressor protein in bacteria.

The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-β-D-thiogalactopyranoside (IPTG).

The pUC origin of replication facilitates the replication of plasmids in E. coli.

The Kanamycin resistance gene confers resistance to the antibiotic kanamycin in bacteria.

f1 bacteriophage origin of replication.

The chitin binding domain is a protein module that can be fused to other proteins to enable their purification using chitin chromatography. C-terminus linked CBD 2 Its function is to bind specifically to chitin, allowing for the selective isolation of chitin-tagged proteins from complex mixtures.

Enhanced Green Fluorescent Protein (eGFP) is a protein that fluoresces green. It can be fused to other proteins of interest, allowing to track the expression of the fused protein in real-time. eGFP as a positive control to confirm that an intein system is working for expressing a short EGFR-binding peptide in a plasmid.