To create the unstructured polypeptide XTEN, pairs of randomised 36 nucleotide DNA fragments encoding only for the amino acids A,E,G,P,S,T must be designed to form AGGT overhangs and subjected to ligation. Other amino acids avoided due to undesirable properties such as chemical instability, hydrophobicity, disulfide cross-linking ability.
The resulting 108 nucleotide XTEN fragments (each encoding 36aa) can be isolated be electrophoresis and cloned into a pET30 derived expression vector.eg.pCW0359, that consists of a promoter region(typically T7 promoter), a terminator region, a Clostridium thermocellum Cellulose Binding Domain, stop codons and the gene encoding GFP, such that the GFP gene fuses in frame with the C terminus of the XTEN fragment.
The insertion site for the XTEN fragment in the pCW0359 vector can be flanked with adaptors/linkers containing sites for the restriction enzymes Bsa1 and Bbs1, so that digestion of the vector with these two enzymes would give back the XTEN fragments with the AGGT overhangs.
Bsa1 and Bbs1 are unique restriction enzymes since they cut at sites downstream of their respective recognition sequences. This generates overhangs that can be accordingly engineered.
The DNA fragments encoding the 36aa XTENs must be screened for highest fluorescence, sequenced and selected for further use.
The 108 nucleotide XTEN fragments must be recovered form their expression vectors and then ligated into expression vectors from the same XTEN pool using restriction enzyme digestion. This would result in random dimerisation of XTEN fragments .e.g. 108+108=216 nucleotide fragments.
This dimerisation procedure must then be repeated 3 more times to result in 1278 nucleotide fragments encoding 576aa segments.
The highly expressed XTEN fragments encoding 576aa segments are fused to fragments encoding 288aa segments(produced in a similar way) to generate the final gene encoding an XTEN with 864aa.
The XTEN polypeptide must then be purified, assessed for stability, aggregation properties, solubility and contamination from host cell DNA or protein.
Sequencing of Exenatide and XTEN DNA fragments (dideoxy chain-termation method).
The DNA sequence for Exenatide is fused to a preceding sequence encoding a TEV protease cleavage site with the help of adaptors/linkers or overlap PCR.
The resulting sequence is fused to the C terminal of the gene encoding the CBD using Overlap PCR, in which the forward primer for TEV/Exenatide must possess a 5'-extension complementary to the 3' end of the CBD sequence to enable fusion of the CBD and TEV/Exenatide sequences.
The TEV protease site functions to separate the cloning sequence from the CBD gene by acting as a spacer to avoid alteration of the properties and functions of the recombinant protein by the CBD gene.
CBD serves as a high affinity tag that enables rapid and efficient purification of fusion proteins. It also holds the potential to increase the thermal stability of the fusion protein.
GFP sequence must be removed from the XTEN expression vector by replacement with a stop codon followed by ligation of the CBD-TEV/Exenatide fusion into the vector, resulting in a CBD-TEV/Exenatide-XTEN fusion sequence.
Restriction enzyme digestion of the fusions to analyse and verify constructs.
Transformation of highly competent high-level protein expression host cells. e.g. BL21(DE3) E.coli cells, by the recombinant expression vector.
Alternatively, E.coli cells can be made competent by means of CaCl2 transformation or Electroporation.
Plating of cell culture onto LB Kanamycin agar plates.
Transformed cells will exhibit antibiotic resistance and hence appear as colonies on the agar plate, since the expression vector also carries the gene for kanamycin resistance.
Selection of positive clones for verification by colony PCR and agarose gel electrophoresis.
Transformed cells(positive clones) cultured in nutrient media containing kanamycin in a shaking incubator to ensure growth of transformed cells only.
Determination of growth of culture by measuring its Optical Density(O.D) at 600nm. At this wavelength, 1 O.D would represent 0.8*109 cells/ml.
Induction of cells to over express the fusion protein using IPTG, which would initiate transcription of the T7 promoter by binding to the repressor protein or displacing it from the operator site of the DNA.
Harvest cells by centrifugation and lyse them to release intracellular components including fusion protein of interest. Cells can be ruptured either mechanically (Homogenisation, Micro-fluidisation, Sonication) or lysed chemically ( EDTA + Lysosyme + SDS).
Removal of cell debris by differential centrifugation to yield a clear lysate that would contain the soluble protein.
Treatment of lysate with Ethidium Bromide followed by subjection to CsCl density gradient centrifugation.
This would result in proteins floating on top of the gradient due to lowest buoyant density.
Isolation of protein particles by puncturing the tube with a syringe and withdrawing them manually.
Purification of fusion protein by means of chromatography. To obtain a protein of high purity, the best approach is to use multiple chromatography steps, whereby the protein sample is passed through several different chromatography columns in succession.
The target protein can be purified on the basis of its affinity, charge, hydrophobicity and size.
Protein sample applied onto a cellulose matrix where the CBD would bind specifically and effectively to the matrix and facilitate protein purification. The CBD fusion protein can be eluted by using ethylene glycol.
Addition of TEV protease to the sample so that CBD and TEV sequences are cleaved off from the fusion protein.
Protein sample applied onto an Ion-exchange chromatography column which allows for separation of proteins based on their net charge.
The fusion protein must be eluted and subsequently applied onto a Hydrophobic Interaction chromatography column, which allows for separation of proteins based on their hydrophobic characteristics.
The target protein can be eluted by lowering the salt concentration in the matrix stepwise, and then concentrated by centrifugation and filtration.
N-Terminal Sequencing - Determination of the amino acid sequence of the fusion protein based on the principle of Edman Degradation with the help of a device called Sequenator.
SDS-PAGE - Non-reducing SDS-PAGE analysis to preserve native structure of the protein and to estimate its molecular weight by comparing its position on the gel to the positions of proteins of known molecular weights. The fusion protein may not necessarily migrate to to a position corresponding to its predicted molecular weight due to the fact that its conformation is unstructured/unfolded.
Detection of the protein by staining with Coomassie dye.
Mass Spectrometry - to determine the precise mass of the fusion protein, based on the principle of separation of ions according to their mass(m) to charge(z) ratio(m/z).
This requires cleavage of the fusion protein into short peptides followed by their ionisation and detection based on the above mentioned principle.
Circular Dichroism (CD) - Far ultra-violet CD spectrum of the fusion protein to be determined to check for the absence or presence of secondary structural features such as α helices, β sheets and β turns.
This technique is based on the principle that biological molecules(such as proteins) exhibit a difference in the absorption of left circularly polarised light and right circularly polarised light.
Ideally, the CD spectrum of the unstructured fusion protein should possess a single deep minimum around 200nm with a CD of approximately 0 in the 210-220nm region.
Size Exclusion Chromatography (Gel Filtration Chromatography) - The fusion protein must be applied onto a gel filtration column which allows for fractionation of proteins based on their size.
Larger proteins travel faster in the column since smaller proteins get trapped in porous beads and are thereby delayed.
Mass of fusion protein estimated by calibrating the column with proteins of known masses.
It is also generally the final step of purification.
Multi-angle Light Scattering - The fusion protein must be eluted from the liquid chromatography column and by using a light scattering detector, its absolute molar mass and native state can be determined.
Here, the amount of light scattered by a solution of the fusion protein at at-least 3 different angles is measured and by using the value of the intensity of light scattered, the molecular weight of the solute (fusion protein) can be determined.
