The overall goal of this procedure is to synthesize a highly stable protein hydrogel that can be used as a general scaffold for protein immobilization. This is accomplished by first synthesizing two protein hydrogel building blocks in a bacterial host. Each building block contains a subunit of a chimeric protein that serves as a crosslinker and one half of the NPU split ene.
These proteins are then purified by affinity chromatography in the second step. The purified protein building blocks are mixed initiating in teen trans splicing reactions that re constitutes a self assembling polypeptide flanked by cross linkers triggering hydrogel formation. This hydrogel is highly stable in solution incorporation of an appropriate binding motif into the protein block.
Copolymers enables the convenient site-specific incorporation of functional gular proteins into the hydrogel building blocks resulting in the formation of a hydrogel, which immobilizes proteins immobilized gular proteins exhibit low leaching rate over three weeks at room temperature. Finally, enzymes immobilized in this protein hydrogel are protected from denaturation by organic solvents. Thus, this hydrogel can also serve as a bioreactor for organic synthesis.
Main advantage of this technique over existing methods for stable protein immobilization, including those involving cross-linking surface LI or sing with the solid scaffolds encapsulation of enzymes in porous supports or inter enzyme Cross linking is the ability to stably immobilize target proteins in a porous and highly hydrated environment without any chemical modifications of the protein side chain. This technology also affords the assembly of multiple enzymes within a pathway in a predetermined three-dimensional arrangement that may facilitate substrate channeling between enzymes. Prior to starting this procedure, generate the two protein block copolymers N and C via plasmid construction by recombinant DNA technologies followed by protein expression and REIA coli B 21 de three to purify n resuspend the cell pellets in buffer A at 10 milliliters per gram of wet pellet.
Then immerse the pellet suspension in an ice water bath and disrupt the cells by sonication at 10 amps with a one second pulse and a six second pause for one minute when finished. Centrifuge the lysate at 6, 000 times G for 20 minutes at four degrees Celsius. After removing the tube from the centrifuge, discard the S supernatant and resuspend the pellet in buffer DA following centrifugation using the same conditions as before, passed the supernatant through a five milliliter NTA column previously equilibrated with buffer DA using a low pressure chromatography system.
Then wash the column with 30 milliliters of buffer DA supplemented with 45 millimolar ole elute, the purified protein using 20 milliliters of buffer DA supplemented with 150 millimolar ole. Transfer the purified protein to a centrifuge tube containing a 30 kilodalton ultra filtration spin column. Centrifuge the sample at 2, 800 times G and four degrees Celsius until the volume is less than one milliliter.
When finished, add 14 milliliters of DPBS buffer to the column to dilute the protein sample. Repeat the centrifugation and dilution steps three more times after buffer exchange, add DTT to the purified protein to give a final concentration of two millimolar. Next, concentrate the protein to about 100 milligrams per milliliter by centrifugation through a 30 kilodalton ultrafiltration spin column at 2, 800 times G at four degrees Celsius for approximately 45 minutes to one hour.
To make a 100 microliter hydrogel mix, 42 microliters of a 100 milligram per milliliter stock solution of C with 10 microliters of 5%sodium azide five microliters of 100 millimolar DTT and 42 microliters of a 100 milligram per milliliter stock solution of N.In a two milliliter glass vial, add one microliter of DPBS buffer to the vial to achieve a final volume of 100 microliters and manually mix all the components via a swirling motion using a pipette tip. After centrifusion the mixture for two minutes at 8, 000 times G, incubate it at room temperature overnight to allow the ENE trans splicing reaction to reach completion. Following this confirm hydrogel formation by turning the tube upside down to make 50 microliters of a 1.2 millimolar GFP functionalized hydrogel.
Combine 17 microliters from a stock solution containing 100 milligram per milliliter of CSH three ligand and nine microliters of a 230 milligram per milliliter solution of SH 3G FP in a one-to-one molar ratio in a 1.7 milliliter centrifuge tube, and incubate the mixture at room temperature for 30 minutes. Next, add five microliters of 5%sodium azide 2.5 microliters of 100 millimolar DTT and 0.5 microliters of DPBS to the same tube. Then add 16 microliters of N to achieve a one-to-one molar ratio of N and CSH three ligand and mix the sample via a swirling motion using a pipette tip centrifuge the mixture at 8, 000 times G for two minutes.
When finished, incubate the mixture at room temperature overnight in the dark to form a hydrogel and encapsulating SH 3G FP.For an additional application, prepare a 0.63 millimolar stock solution of horse radish peroxidase HRP in DPBS to make 30 microliters of a 1.6 millimolar hydrogel and trapping HRP combined 12 microliters of a 100 milligram per milliliter stock solution of C with two microliters of HRP three microliters of 5%sodium azide and 0.5 microliters of 300 millimolar DTT and a 1.7 milliliter centrifuge tube. Add 12 microliters of a 100 milligram per milliliter solution of N and 0.5 microliters of DPBS and mix the solution via a swirling motion using a IPE tip. After centrifusion the mixture at 8, 000 times G for two minutes, incubate it at room temperature overnight.
For the enzymatic reaction, submerge the hydrogel in one milliliter of a reaction cocktail containing 5.8 millimolar of NN dimethyl PPH phenol diamine 5.8 millimolar of phenol and 2.9 millimolar of turt butyl hydro peroxide in n heptane manually disrupt the gel using a pipette tip to increase the contact surface area of the hydrogel and the solvent. Detect the HRP product and indel phenol type dye by taking samples of the solvent at different times and measuring the optical absorbance at 546 nanometers in a plate reader mixing of purified N and C in the presence of the reducing agent, DTT induces the formation of a third protein, the ligated product J individually. The hydrogel building blocks n NC exists as viscous fluids mixing of n NC yields a transparent semi-solid material that is retained on the bottom of a glass vial after inversion indicative of the formation of a hydrogel.
This ene mediated protein hydrogel exhibits high solution stability. There is little to no loss of cross-linked hydrogel scaffold after 21 days at 22 degrees Celsius in DPBS buffer. As the total amount of protein released into the DPBS buffer only slightly exceeds the theoretical amount of the spliced ene from the hydrogel dense cytometry revealed that during hydrogel formation, trans splicing reactions were about 80%efficient.
SDS page gel analysis showed that only trace amounts of the trans spliced product were present in the hydrogel surrounding buffer. Confirming that loss of the cross-linked hydrogel scaffold to erosion is minimal. The main protein present in the hydrogel surrounding buffer is the spliced out intune.
No visible signs of erosion were observed in an undisturbed hydrogel, submerged in aqueous solution at room temperature for over three months. The hydrogel is also highly stable at 37 degrees Celsius and in both acidic and basic buffers. To facilitate protein immobilization, a protein and its peptide ligand were used to dock proteins of interest into the hydrogel scaffold.
We chose the SH three protein, a SAR homology three domain from the adapter protein CRK as the docking protein for fusion to a protein of interest and its ligand as the docking station peptide for incorporation into the hydrogel scaffold. This interaction pair was chosen because of the relatively small molecular size and high affinity finity. S SH three ligand was inserted between NPUC and cut A to form CSH three ligand.
The S SH three protein was fused to the end terminus of a model target globular protein GFP. The process described in the protocol yields a hydrogel containing 1.2 millimolar trans spliced hydrogel backbone building blocks and 1.2 millimolar GFP. The GFP containing hydrogel exhibited a similar stability to the hydrogel lacking GFP with about 35%total protein loss after 21 days.
In DPBS buffer, most of the proteins present in the erosion buffer were the cleaved in teens. The leaching of SH 3G FP from a hydrogel containing the S SH three ligand is about 30%after three weeks, significantly smaller than that from a hydrogel lacking the s sh three ligand as seen here hydrogel containing the docking station peptide. S SH three ligand retains most of the GFP fluorescence.
After three weeks while the hydrogel lacking SSH three ligand becomes essentially non fluorescent. The density of immobilized GFP in this work is about 33 mole percent of the hydrogel. A higher immobilization density can potentially be achieved when multiple docking station peptides are incorporated into the hydrogel building block.
Next, the HRP enzyme was incorporated into the protein hydrogel to demonstrate its ability to support biocatalysts in organic solvents. Enzyme activity was measured by monitoring the oxidative coupling of NN dimethyl pph, Lene diamine, and phenol with turt butyl hydro peroxide over time. The hypothesis was that the hydrated environment of the hydrogel would protect the attached enzyme from the denaturing effect of the organic solvent.
After immersing an organic solvent, the HRP containing hydrogel was manually disrupted into small clusters to increase the hydrophilic hydrophobic interface area. Hydrogel incorporated HRP effectively catalyzed the rapid oxidation reaction giving rise to a colormetric product. The product accumulation follows a linear slope indicating little to no enzyme in activation.
During the experiment, HRP dissolved A-D-P-B-S first followed by addition to the organic solvent, was able to catalyze the conversion, but at a much reduced reaction rate. The low conversion rate of enzymes dissolved in DPBS is likely due to the small interfacial area between the DPBS and the organic solvent, which limits the rate of substrate product diffusion. Incorporation of the highly hydrophilic s fragment in the hydrogel backbone, which effectively locks the water inside the hydrogel and prevents the organic solvent from accessing the hydrogel interior, enables the hydrogel to withstand the denaturing effect of the organic solvent.
These results indicate that the ene mediated protein hydrogel can be an effective scaffold for enzymatic reactions in organic solvent. The structural flexibility and high stability of this self assembling hydrogel allows it to be used not only in protein and mobilization, but also in other applications including biofuel cells, injectable drug delivery carriers, and tissue engineering scaffolds.
