신경 조직의 재생을위한 가용성 및 불용성 바이오틴 단백질의 발현, 분리 및 정제

The overall goal of this procedure is to successfully design, express, and isolate recombinant biotinylated proteins that can be incorporated into a variety of applications such as probes, sensors, drug delivery, and tissue engineering. This is accomplished by first transforming the custom designed plasmid into a bacterial host cell line using small scale cultures, and then inducing expression of the target protein. Next, the presence and solubility of the target protein is determined in order to formulate proper purification procedures.

Then the procedure is scaled up to produce larger yields of the target protein, which are then isolated using affinity chromatography techniques. Finally, the proteins are purified and then concentrated for downstream applications and analyses. Ultimately, SDS page and FPLC results verify that recombinant proteins have been successfully isolated and purified, and the proteins are further tested for functionality.

The main advantage of recombinant protein engineering is it allows us to create large scale or milligram scale quantities of custom designed proteins to meet our exact scientific specifications. For example, we can add new functionalities to portions of these proteins to enable new properties. We'll be showing you today how you can biotin the ends of specific proteins, and we use this to specifically immobilize or tether these proteins to surfaces to guide cellular functions such as neuronal outgrowth or neuronal differentiation.

Individuals new to this technique should not have much difficulty when following the procedures for either native or non-native protein isolation. Many steps are the same, but require specific buffer formulated for either native or non-native conditions. Careful attention to details during the beginning.

Transformation and test expression stages will ensure high protein yield during the later final scale up process. Visual demonstration of this method is important because it shows how recombinant proteins can be made using simple laboratory equipment and supplies. Utilizing the simple batch reaction, e equalize and growth media are grown up in leader scale, producing 10 milligrams of protein per main culture.

Additionally, chromatography techniques can be used and demonstrated to show how proteins are further purified for downstream application Today, Alicia, graduate Cinema Lab will be demonstrating the procedure After cloning and testing for expression at target protein of interest, and growing a 20 milliliter culture overnight. According to the text protocol, pour the overnight culture into 1.8 liters of sterile growth medium with ampicillin and use a paster pipette to add six to eight drops of sterile anti foam 2 0 4 place cultures into a 37 degrees Celsius water bath through a 0.2 micron filter, bubble compressed air into the cultures through aeration stones. When the culture reaches an OD 600 of 0.7 to 0.8 at one millimolar IPTG and induce it for four hours at 37 degrees Celsius or overnight at 18 degrees Celsius, depending on whether the protein was found in the soluble or insoluble fraction in the test expression.

Experiments to collect the cells, transfer the culture into one liter centrifuge bottles, and spin it down for 15 minutes at 14, 000 times gravity and four degrees Celsius. Pour off the super natin and using a thin spatula, scoop the bacterial pellet into two 50 milliliter tubes. Pellet the cells again by centrifugation for a few minutes before storing the pellets at minus 80 degrees Celsius.

To perform non-native protein. Isolation for an insoluble protein to each frozen e coli pellet. Add 20 milliliters of lysis, wash buffer, and vortex and shake to resuspend to eliminate any large chunks.

After incubating on a mutator overnight, the solution will look like a viscous slurry centrifuge. The slurry at 20, 500 times gravity and room temperature for 30 minutes. Transfer the supernatant to a fresh tube and discard the pellet for native protein isolation.

Resus suspend the pellets in lysis buffer at a final volume of 30 milliliters with the resuspended pellet on ice. Set the sonicate at a 30%amplitude with a pulse of 30 seconds on 30 seconds off, and sonicate for five minutes. Bobbing the tube up and down during sonication to completely break up the cell pellet.

Centrifuge the slurry at 20, 500 times gravity and four degrees Celsius for 30 minutes. Then transfer the supernatant to a fresh tube and discard the pellet. To perform affinity chromatography on a non-native isolated protein, add one milliliter of nickel NTA resin solution to each centrifuge tube and incubate on a mutator or shaker at room temperature for at least one hour.

Pour the slurry into an affinity column and allow the solution to drip through the stop cock valve completely into a waste beaker. Add 10 milliliters of wash buffer to the centrifuge tube to remove residual resin and add the solution to the column after the solution has dripped through. Use a glass stirring rod to stir the resin and allow it to finish dripping before adding another wash.

Next, use 10 milliliters of wash buffer to repeat the rinse of the centrifuge tube and transfer it to the column. Then perform eight more washes of the column with 10 milliliters of lysis wash buffer for each wash. After the washes are complete, close the stop cock and replace the waste beaker with the 50 milliliter centrifuge tube.

Add 15 milliliters of elucian buffer to the column, stir the resin and allow the solution to sit for five minutes. Then open the stop cock and collect the EIT before repeating with an additional 15 milliliters of elution buffer to purify a native protein. After adding the nickel NTA resin to the protein solution, incubated on a mutator at four degrees Celsius for at least one hour.

After using wash buffer to wash the column 10 times, add five milliliters of elution buffer and incubate the resin for five minutes before using a fresh tube to collect most of the EIT while the EIT is dripping from the column, add 90 microliters per well of Bradford reagent to a clear 96 well plate. After each five milliliter elucian with buffer, allow 10 microliters of solution to drip from the column into a well containing 90 microliters of Bradford reagent. Continue with the EEU until protein is no longer detected to dialyze and or Rena.

The proteins transfer each EIT to dialysis tubing and dialyze against the appropriate buffer, one for four hours at four degrees Celsius before replacing with the respective buffer. Two overnight at four degrees Celsius. Use spin concentrators to concentrate the dialyzed proteins to less than five milliliters and further purify using size exclusion chromatography if desired by atin eight.

According to the text protocol during test expression, NGF and SE three A were first induced for four hours at 37 degrees Celsius and SDS page analysis determined both proteins were located in this soluble fraction. Protein expression seemed fair and therefore another test expression with overnight induction at 18 degrees Celsius was examined. NGF expression improved in the soluble fraction, whereas there was no noticeable difference with SEMA three.

A NGF was isolated under native and non-native conditions and run through the FPLC column for purification. Although NGF was in the soluble fraction during test expression, native isolation produced a low yield at both 37 degrees Celsius and 18 degrees Celsius inductions. However, a higher yield of NGF was obtained through non-native isolation with re naturation.

As a result, NGF was isolated under non-native conditions. After overnight induction at 18 degrees Celsius non-native isolation resulted in no SEMA three a production with 8.61 plus or minus 3.1 milligrams per main culture of two liters for native isolation. Therefore, SEMA three A was induced for four hours at 37 degrees Celsius and isolated using native isolation parameters.

FPLC peaks were collected and NGF and SEMA three A samples were analyzed with SDS page. Additional protein peaks found in the FPLC output for SEMA three A were collected and analyzed with SDS page and were not located in the SEMA three a molecular weight region. These fractions are most likely degradation products because they did not behave functionally in cell-based assays, as did the SEMA three.

A peak indicated here. Once established and mastered recombinant protein, isolation and purification should only take a few days if performed properly. However, initial bacteria transformation and test test expression stages take longer in order to properly identify the protein solubility.

Once the solubility is determined, a library of the transformed bacteria can be stored in the minus 80. Additionally, after protein expression, e coli pellets can be stored in the minus 80 as well until further isolation and experimentation. When attempting this procedure, it is important to make sure that buffers are sterilized and made fresh in order to prevent proteases from denaturing and breaking down your proteins.

Isolation buffers should be made and sterilized every four to six weeks, and dialysis buffers should be made the day before isolation. N-I-N-T-A and FPLC columns should be clean according to the manufacturer's protocols in order to prevent cross-contamination from multiple biotinylated proteins. After watching this video, you should have a good understanding of how to express, isolate, and purify custom designed biotinylated proteins using a bacterial host expression system.

This is similar to what is done both scientifically and commercially at small and large scale. Thus, this has broad application.