The Multifaceted Benefits of Protein Co-expression in Escherichia coli
February 5th, 2015
Protein co-expression is a powerful alternative to the reconstitution in vitro of protein complexes, and is of help in performing biochemical and genetic tests in vivo. Here we report on the use of protein co-expression in Escherichia coli to obtain protein complexes, and to tune the mutation frequency of cells.
The overall goal of the following experiment is to coex express different proteins in esia coli and to assess their assembly in vivo. This is achieved by using the two compatible plasmids, P bad, and P good to co transform e coli top 10. As a second step, the co transformed strain is cultured at 30 degrees Celsius and then induced to overexpress the proteins.
Next soluble proteins are extracted and subjected to gel filtration in order to assay the association of the overexpressed proteins. The results show that co-expression in e coli is an efficient tool for the production of protein complexes assembled in vivo and is convenient to bypass the poor solubility of free subunits Protein. Coex expression is a powerful tool in biochemistry, letting the overexpression of in single cells of different proteins going backwards.
One can recognize that the overexpression of single genes was essential for the study of Lopi number proteins. However, when the challenge was the study of protein complexes, one had to I overexpress, isolate and reconstitute in vitro single individual proteins. This is a lab intensive approach, which also implies that the yield of the protein complex of interest is lower than those of the individual proteins assembled into the protein complex.
In addition, one can face the problem that one of the protein interactors features low solubility. This method can help answer key questions in the biochemical field, such as the characterization of protein complexes composed of a multitude of different subunits. To begin the experiment, prepare electro competent cells of the top 10 e coli strain.
Transfer a single colony to one milliliter of LB media. Incubate the tube at 37 degrees Celsius while shaking at 180 RPM. Dilute the pre culture one to 500 in 25 milliliters of fresh LB media and incubate the culture at 37 degrees Celsius until it reaches log phase 0.6 OD centrifuge the cell suspension at 5, 000 RPM for 20 minutes after centrifugation.
Resus, suspend the pellet in 25 milliliters of 10%volume per volume, ice, cold, sterile, glycerol water. And repeat this step four times. Having the Resus suspension volume each time until 1.5 milliliters is reached.
Next, resuspend the pellet in 10%glycerol and divide the cell suspension in 50 microliter aliquots. Store the aliquots at negative 80 degrees Celsius for up to six months. To continue the experiment, dissolve the desired plasmid in sterile water, supplemented with 0.5 millimolar EDTA, and thaw an aliquot of electro competent cells on ice.
Add 2.5 to five nanograms of vector and mix the thawed cells. Dispense the cells into a 0.1 centimeter vet. Apply a 1.8 kilovolt pulse to the cells, and immediately transfer the electroporated cells into one milliliter of SOC media.
Incubate the cells while shaking for one hour transfer 100 microliter aliquots to petri dishes containing LB auger and antibiotic, and incubate the cells overnight at 37 degrees Celsius. Purify the T by streaking single colonies on Petri dishes with a sterile loop. Transfer a small amount of glycerol stock with co transformants onto a Petri dish containing LB media and antibiotics.
Let the cell suspension dry onto the Petri dish and streak the cell droplet. Incubate the dish at 37 degrees Celsius overnight. After incubation, using a sterile toothpick, transfer a single colony to one milliliter of LB antibiotic media.
Incubate the culture for eight hours at 37 degrees Celsius. Dilute the pre culture one to 500 in fresh media and incubated at 30 degrees Celsius for 15 hours. Next, add one millimolar.
Each of Aose and IPTG incubate the culture at 30 degrees Celsius for two and a half hours. Collect the cells and store the pellets at negative 20 degrees Celsius. Thaw the pellets on ice and resuspend them in lysis buffer.
Then gently homogenize the cel suspension with a cold glass potter. Next, sonicate the cells at 15 watts for 15 seconds, followed by a 15 second cooling interval. And repeat this process four times centrifuge.
The total protein extracts at 10, 000 Gs for 20 minutes at four degrees Celsius. To recover the soluble fraction. Transfer 20 microliters of each soluble protein extract to a micro centrifuge tube containing 80 microliters of loading buffer and boil the solution for five minutes to analyze each soluble protein extract by 12.5%Acrylamide SDS page, load 18 microliters of each sample and run the electrophoresis at 140 volts for one and a half hours.
After centrifugation, load the supernatant onto a bookner funnel equipped with three layers of paper filter and apply a gentle vacuum. Discard the pellet to avoid excessive foaming. Keep the vacuum erlenmeyer flask on ice during filtration.
After filtration, perform a Bradford assay to determine protein concentrations equilibrate a water jacketed 16 by 70 gel filtration column with 50 millimolar tris, HCL 150 millimolar, NACL one millimolar EDTA at pH eight. Using a one milliliter sample loop, load the soluble protein extract onto the column. Perform the chromatography at 0.6 milliliters per minute and keep the column temperature at four degrees Celsius.
Next, collect 0.9 milliliter fractions To analyze them by SDS page. Transfer 20 microliters of each relevant fraction to a micro centrifuge tube containing 80 microliters of loading buffer and boil the tubes for five minutes. Load 18 microliters of each sample and perform the electrophoresis at 140 volts for 1.5 hours.
Follow the SDS page by assay, the three prime five prime exonuclease activity of each fraction in 96. Well microplate using P-N-P-T-M-P substrate estimate DNA polymerase activity using the PPX enzyme coupled assay and INT as the electronic scepter transfer. A single colony of e coli top 10 containing the PBA epsilon and the P good one epsilon D 12 A 11 vectors to one milliliter of LB media treated with antibiotics.
Incubate the culture overnight at 37 degrees Celsius. The next morning dilute the pre culture one to 250 in three flasks containing 10 milliliters of fresh media. Add the inducers one millimolar each of arabinose IPTG or AOSE and IPTG and incubate the induced culture at 37 degrees Celsius for eight hours.
Prepare non induced cultures. In parallel. Collect one milliliter aliquots and dilute them one to 500 in new flasks containing 10 milliliters of fresh media, supplemented or not supplemented with inducers.
Then incubate the mixture overnight at 37 degrees Celsius. The next day. Repeat the steps beginning with the dilution of the pre culture and collect one milliliter aliquots.
The aliquots are then placed in the freezer. Next, determine the number of generations that have occurred in each culture. Transfer 100 microliters of appropriate cereal, dilution of inoculum and culture on LB plates.
Incubate the plates overnight at 37 degrees Celsius the following morning. Count the colonies on the lb plates. Calculate the log of the number of cells present in the inoculum or log I and in the culture at the end of growth or log C and determine the number of generations.
Then centrifuge the culture at 5, 000 Gs for 20 minutes and resuspend the cells in one milliliter of 50 millimolar tris, H-C-L-P-H 7.6 50 millimolar, NACL to perme. The cells add two to three drops of chloroform and vortex for 20 seconds. Finally, determine the glucose dase activity of each aliquot in a 96.
Well microplate add 100 microliters of permeable cells and 100 microliters of using p nitro phenol d glucocide substrate to each well, while avoiding the creation of air bubbles in the wells. Read the absorbance at 420 nanometers using a microplate reader and filter. And an SDS page was performed from total protein extracts, isolated from cells, not induced or induced to overexpress alpha, Dr.Epsilon or alpha Dr.And epsilon.
The gel demonstrates that the simultaneous addition of IPTG and ARABINOSE to the culture media triggers the co-expression of alpha DR.And epsilon soluble proteins were then extracted from cells. Coex expressing these proteins. Gel filtration and enzyme assays demonstrated that alpha, DR.And epsilon do not associate protein co-expression can be used to obtain a better control of mutator strains.
Beta glucosidase activity was measured in a phenotypic test to determine the effects of Epsilon. D 12. A mutagenic variant expression beta glucosidase activity was acquired in about 20 generations when the wild type epsilon subunit was induced alone, or in conjunction with the D 12.
A variant beta gluc activity was acquired by e coli at moderate levels. While attending this procedure, it is important to test the co-expression conditions. It is important to remember in particular, there are some major parameters which deserve attention.
First, the temperature of induction. Second, the concentration of inducer, and third, the induction length. Finally, it could also be important to test different host strains in parallel.
Since this could affect enormously the final yield of the protein complex of interest.
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Overview
This study explores the co-expression of proteins in Escherichia coli to facilitate the assembly of protein complexes in vivo. By utilizing compatible plasmids, the researchers demonstrate an efficient method for producing soluble protein complexes, overcoming challenges related to protein solubility.
Key Study Components
Area of Science
- Biochemistry
- Protein Engineering
- Microbiology
Background
- Protein co-expression is an alternative to in vitro reconstitution of protein complexes.
- This method aids in biochemical and genetic testing in vivo.
- Escherichia coli serves as a model organism for protein assembly studies.
- Challenges include the solubility of free protein subunits.
Purpose of Study
- To co-express different proteins in E. coli.
- To assess the assembly of these proteins in vivo.
- To evaluate the efficiency of co-expression in producing protein complexes.
Methods Used
- Co-transformation of E. coli with two compatible plasmids.
- Culturing the transformed strain at 30 degrees Celsius.
- Inducing overexpression of target proteins.
- Extracting soluble proteins and performing gel filtration to analyze protein association.
Main Results
- Co-expression in E. coli effectively produces protein complexes.
- The method allows for the assembly of proteins that are otherwise poorly soluble.
- Results indicate a successful bypass of solubility issues with free subunits.
- Co-expression is validated as a powerful tool in biochemistry.
Conclusions
- Co-expression is a viable method for studying protein interactions in vivo.
- This approach enhances the production of functional protein complexes.
- Future applications may include more complex protein assembly studies.
Frequently Asked Questions
Chapters in this video
0:05
Title
2:11
Isolation of E. coli Co-transformants
4:19
Co-expression of α and ε Subunits of E. coli DNA Polymerase III
6:20
Co-expression of α Subunit of Deinococcus radiodurans DNA Polymerase III and ε Subunit of E. coli DNA Polymerase III
6:49
Gel Filtration Chromatography
8:09
Mutation Analysis of Populations Co-expressing the Wild-type ε Subunit of E. coli DNA Polymerase III and the Mutagenic εD12A Variant
10:43
Results: Effective Protein Co-expression in E. coli
11:55
Conclusion
