Project hVpr/hKRS : Part II

Introduction

The purpose of these experiments was to test whether the hVpr protein and the hKRS bind with each other so as to prevent the hKRS from functioning. The hVpr, or human immunodeficiency virus type one Vpr protein, is a viron-associated protein that localizes in the nucleus of infected cells [Forget]. It was shown to bind hKRS and inhibit its ability for aminoacylation of tRNA^Lys [Stark].

The hVpr protein is 15kDa in size, and its gene, 283bp, is cloned into the pET11 plasmid under expression of the T7 promoter. The protein has three short sequences attached to the C terminus: the protein kinase A domain, the flag epitope, and a block of 6 histidine residues [Zhao]. These resulting attachments to Vpr make the protein run at just above 17kDa on an SDS PAGE.

The hKRS, or human lysyl-tRNA synthetase, is cloned into the pUC19 plasmid under the expression of the camv35s promoter and contains the Flag epitope at the N terminus, this plasmid is labeled pUC19 hKRS. The KRS protein is ~80kDa in size, and its gene is ~2000bp long.

To test the interaction of the two proteins, an in-vivo bioassay was done using temperature sensitive E.coli cells which lose the gene for the E.coli KRS at high temperature [Shiba]. The mutant cells transformed with the pUC19 hKRS plasmid rescue the temperature sensitive E.coli cells [Luttrull]. Therefore, these temperature sensitive cells were chosen to host both the hVpr and hKRS protein to test for possible inhibition of the hKRS protein by the hVpr protein. To clone the hKRS gene into the pET11 hVpr plasmid, AatII site linked primers flanking the hKRS gene were used for PCR [Byung-Eun]. The resulting PCR fragment was cloned into pET11 hVpr using the AatII restriction sites, yielding clones with two orientations of the hKRS vector within the pET11 hVpr plasmid. The resulting clones were named pET11hVpr/hKRS. To determine the expression orientations a restriction digestion was done using SspI and StuI which yields one of two fragments. A fragment around ~947bp indicates expression towards each other, and a fragment around or ~2224bp indicates expression in the same direction. Both inserts were found in the cells screened for the pET11 hVpr/KRS clones, labeled VK1 and VK2 for short.

The resulting pET11hVpr/hKRS plasmids, labeled VK1 and VK2, were then tested in temperature sensitive cells. The mutant cells are rescued at 42˚c by the pUC19 hKRS plasmid, but not by the pET11 hVpr/KRS plasmids or the pET-11 hVpr plasmid. Growth curves done show that the mutant cells containing either of the pET11 hVpr/KRS plasmids have a much longer lag phase than the other plasmids tested. However, the lag was not observed when growth curves were done in DH5α cells. After growing the mutant cells containing pET11 hVpr/KRS at 30˚c for five hours, the cells were then raised to 42˚c, where their growth stops. This indicated that even after lag phase, cells containing the pET11 hVpr/KRS plasmid will stop growing.

Next, tests for expression of hKRS and hVpr were done with BL21 DE3 LysS cells. The hKRS protein has the FLAG tag, and the hVpr protein has both the FLAG and His tag.

Materials and Methods

Protein Expression of pET11 hVpr/KRS

Purpose: Check for protein expression of pUC19, pET11 hVpr, pUC19 hKRS, and pET11 hVpr/KRS in BL21 DE3 LysS cells using Flag tag for visualization of both proteins by Western blot analysis.

Procedure: Plasmids pET11 hVpr, pUC19 hKRS, pET11 hVpr/KRS were transformed into BL21 DE3 LysS cells and plated on LBamp agar. Overnight cultures were prepared using single cell colonies from plates and incubated 10 hours at 37˚c with 250rpm agitation. Then the cells were induced with 1mM IPTG for an hour. 1ml pellets were harvested of induced and un-induced cells and used for 12% gel SDS PAGE. 70ul of 1X loading buffer were added to each pellet and heated at 100˚c for 15 minutes, and then 25ul of each sample was loaded onto the SDS gel. Western blot was conducted and then treated with 1˚ monoclonal flag antibody and 2˚ anti-monoclonal HRP antibody. Blot visualized using chemoluminescence.

Results: Negative control un-transformed BL21 DE3 LysS cells showed no background, and positive control pET11 hVpr showed expression both induced and un-induced. The pET11 hVpr/KRS cells showed only hVpr both induced and un-induced, however, the pUC19 hKRS cells showed only a very faint band when induced. Blot left side: ladder, BL21 cells, un-induced pET11 hVpr and pET11 hVpr/KRS; Blot right side: ladder and induced BL21 cells, pET11 hVpr, pET11 hVpr/KRS, and pUC19 hKRS.

Conclusion: The pET11 hVpr is expressed both induced and un-induced. The pET11 hVpr/KRS cells express hVpr, not hKRS, visibly when induced and un-induced. The pUC19 hKRS showed only a very faint band when induced. Therefore, the hKRS under the control of the 35s promoter is not a good candidate for co-expression with hVpr, which is under the control of the T7 promoter, using BL21 DE3 LysS cells.

Construction of Plasmid pET21d hKRS

Purpose: Insert Flag tagged hKRS from pUC19 hKRS into pET21d so that it is under control of T7 promoter using the unique NcoI and NotI restriction sites. Test for protein expression after confirmation of clone.

Procedure: The pUC19 hKRS and pET21d plasmids were each digested using NcoI and NotI in single step reactions using NEB buffer #3. The Flag tagged hKRS fragment, ~1.9Kb, was separated from pUC19 hKRS by 0.8% agarose gel purification with GFX column. The pET21d fragment, ~5.4Kb, was isolated by 0.7% agarose gel purification with GFX column. Fragments were ligated together using T4 DNA Ligase. The ligation mixture was used to transform DH5α cells, and the resulting colonies screened using PCR and the universal T7 vector primers. The clones were confirmed by growing up plasmid DNA and cutting out the Flag tagged hKRS fragment using the same enzymes, NcoI and NotI. Once the pET21d hKRS plasmid was confirmed, it was checked for hKRS expression using BL21 DE3 LysS cells. 1ml pellets were harvested of 2 hour 2mM IPTG induced and un-induced cells grown at 30˚c and 37˚c. 50ul of 1X loading buffer were added to each pellet and heated at 100˚c for 15 minutes, and then each sample was loaded onto a 12% SDS PAGE. Western blot was conducted and then treated with 1˚ monoclonal Flag antibody and 2˚ anti-monoclonal HRP antibody. Blot visualized using chemoluminescence.

Results: Plasmids pUC19 hKRS and pET21d plasmids cut with NcoI and NotI to yield linear plasmid and hKRS gene for ligation.

PCR screened colonies from transformation of the ligation mixture test positive for clones when showing product at ~2Kb. Colonies #1 and #4 selected for plasmid isolation. Lanes from left to right: 1 kb Ladder, PCR pET11 hVpr (+ control), - control, colonies 1 thru 12, PCR pET21d.

The pET21d hKRS plasmid DNA from the two positive colonies was confirmed by NcoI and NotI digestion yielding ~2Kb fragment of hKRS gene. The hKRS protein is only expressed when induced, and the inductions of hKRS at 30˚c and 37˚c both had protein degradation. Blot from left to right: Ladder, un-induced hKRS at 37˚c and 30˚c, induced hKRS at 37˚c and 30˚c, and induced overnight Flag tagged hVpr at 30˚c as positive (X labels blank lanes).

Conclusion: pET21d hKRS strongly expresses Flag tagged hKRS only when induced, but degradation of the protein is observed.

Construction of Plasmid pET21 T7VK

Purpose: Isolate T7 controlled hVpr vector from pET11 hVpr using PCR, and clone it into pET21d hKRS. PCR hVpr vector to include BglII cut site flanking T7 promoter and add SphI cut site flanking terminator. Then insert into pET21d hKRS using unique restriction sites BglII and SphI. The two vectors are expressed away from each other with ~30bp separating the start of each T7 promoter. Test for protein expression after confirmation of clone.

Procedure: Digested pET21d hKRS in two step reaction with BglII and SphI using GFX column in between to purify. Reverse PCR primer designed to insert SphI site flanking T7 terminator of hVpr vector in pET11 hVpr. Forward primer with BglII cut site flanking T7 promoter, labeled FwrT7hVprBglII: 5’-CGG CGT AGA GGA TCG AGA TCT CGA TCC CGC-3’. Reverse primer with engineered SphI cut site flanking T7 terminator, labeled RevT7hVprSphI: 5’-TAT CAA GGA CGT ACG TCG TTT TTT GGG GAG-3’. PCR reaction carried out using PWO polymerase. Agarose gel run to confirm PCR fragment of ~0.3-0.4Kb, then PCR reaction mixture was purified by GFX column. PCR fragment digested in two-step reaction with BglII and SphI using GFX column in between to purify. Digested PCR fragment then isolated by 2% agarose gel purification with GFX column. PCR fragment ligated into open pET21d hKRS using T4 DNA Ligase. Transformed DH5α cells with ligation mixture, and PCR screened colonies for clones using primers FwrT7hVprBglII and RevT7hVprSphI. The clones were confirmed by growing up the plasmid DNAs and cutting them with the enzyme XhoI. Protein expression checked for using BL21 DE3 LysS cells with 1mM IPTG induction for one hour, 12% SDS PAGE, and Western blot with chemoluminescence for Flag tag visualization of proteins.

Results: Plasmid pET21 T7VK was cloned from pET11 hVpr PCR fragment and pET21d hKRS using unique BglII and SphI restriction sites, and the purified DNA was run on gel for concentration comparison.

Transformation colonies from ligation were PCR screened with the same primers used in cloning and results run on 1.5% agar gel (lane 12 and 13 switched due to loading error). Colonies #13, 15, 16 tested positive.

Plasmid clones confirmed by digestion using XhoI, originally a unique enzyme site in pET21d hKRS and PCR fragment of hVpr, but now yields ~2.6Kb fragment from new construct pET21 T7VK. Results run on 0.8% agar gel. . Clone #16 may have multiple inserts as indicated by a faint third XhoI digest band running between 0.5Kb and 1.0Kb. Lanes from left to right: 1 kb Ladder, pET11 hVpr, XhoI cut pET11 hVpr, pET21 hKRS, XhoI cut pET21 hKRS, colony #13, XhoI cut colony #13, colony #15, XhoI cut colony #15, colony #16, XhoI cut colony #16.

There was no expression observed of either protein from clones #13 and #15. Clone #16 expressed hVpr only. Lanes from left to right: clone #16, clone #15, clone #13, ladder, pET21d hKRS (positive)

DNA sequencing of clones was done, with the reverse sequence of clone #15 returning the best results. The hVpr gene is highlighted in yellow, and the pET21d sequence highlighted in red with the terminator highlighted in green. The sequence was then compared to the pET21d sequence using blast, and also the hVpr gene sequence obtained from NCBI GenBank file reference # NC_001802. The non-matching base pairs are marked with an asterisk (*). These non-matches were ruled out by protein translation sequence comparison of both genes.

Conclusion: Plasmids pET21 T7VK from clones #13 and #15 did not express either hVpr or hKRS. Clone #16 may have multiple inserts of hVpr gene. Insert spacer between T7 promoters, possibility that T7 promoters are too close together.

Insert Spacer DNA into pET21 T7VK to create plasmid pET21 T7VKS

Purpose: Space out the T7 promoters by inserting small stretch of DNA in between the two T7 promoters at the BglII site in pET21 T7VK using spacer fragment isolated from pUC19. The spacer separates the two genes T7 promoter sequences by at least 170bp.

Procedure: Digested pUC19 with BamHI and NdeI in single step reaction to create 233bp DNA fragment from middle of Lac Z gene. Isolated fragment with agarose gel purification on GFX column. The fragment is digested again with Sau3AI to yield a 141bp fragment and is isolated by agarose gel purification on GFX column. Plasmid pET21 T7VK is then cut with BglII and purified by GFX column. The opened plasmid is then treated with CIP, and isolated by agarose gel purification on GFX column. BglII, BamHI, and Sau3AI all have compatible overhangs, and the two fragments are ligated together using T4 DNA ligase. After transformation of the ligation mixture to DH5α cells, colonies are PCR screened with p19SpcFwr (GAT CGG TGC GGG CCT CTT) and p19SpcRev (GAT CCC CGG GTA CCG AGC). Positive colonies are selected for plasmid DNA prep and then screened by digestion with EcoRI and XhoI. The resulting positive clones are labeled pET21 T7VKS. Protein expression checked for using BL21 DE3 LysS cells using pET11 hVpr and pET21 hKRS as positives. 100ul of overnight culture are used to inoculate 10 ml LBamp media, and incubated at 37˚c with shaking until 0.4-0.5 OD₆₀₀ is reached. Then 1mM IPTG is added and 1ml cell pellets are harvested at one and three hour intervals. 40ul of 1X SDS loading buffer are added to each pellet, vortexed, heated at 100˚c for 15 minutes, and then run on 12% SDS PAGE. Western blot is conducted with Flag antibodies. Blot is incubated for one hour with 1˚ monoclonal flag antibody, given 3 ten minute PBS washes, and then incubated for one hour with 2˚ anti-monoclonal HRP antibody followed by another round of PBS washes as before. Blot visualized using chemoluminescence.

Results: Agarose gels of DNA fragments for ligation. Left gel (left to right) shows the pUC 19 plasmid, 123 ladder and the BamHI/NdeI cut lacZ fragment. The right gel (left to right) shows the purified BglII cut pET21d, Sau3AI cut lac Z fragment, and 123 ladder.

There was no expression of either protein from pET21 T7VKS clones generated from pET21 T7VK #13 and pET21 T7VK #15. The pET21 T7VKS clone generated from pET21 T7VK#16 expressed only hVpr. Expression was done in duplicate with cells harvested at one and three hour intervals. Lanes from left to right: Ladder, colony #1 and colony #2 at one and three hour intervals, positive hVpr, positive hKRS.

Conclusion: No co-expression of proteins. Utilize new plasmid developed by Novagen called pACYC Duet. It has two MCS for co-expression of 2 proteins using the T7 expression vector with only one promoter and terminator. The plasmid also contains chloramphenicol resistance and the P15A replication origin, this allows for co-transformation into cells with other plasmids that contain a different replication origin and antibiotic resistance.

Construction of Plasmid pACYC hKRS

Purpose: Create plasmid with hKRS to co-transform with pET11 hVpr for protein expression. Insert Flag tagged hKRS from pUC19 hKRS into pACYC Duet so it is under control of T7 promoter using the unique NcoI and NotI restriction sites. Test for protein expression after confirmation of clone using BL21 DE3 cells, which do not contain chloramphenicol resistance.

Procedure: The pUC19 hKRS and pACYC Duet plasmids were each digested using NcoI and NotI in single a step reaction. The Flag tagged hKRS fragment and the pACYC Duet fragment were isolated by agarose gel purification with GFX columns. The resulting two purified fragment samples were compared for concentration on a 0.7% agarose gel. Then the fragments were ligated together using T4 DNA Ligase. The ligation mixture was used to transform DH5α cells and plated on LB agar with chloramphenicol. Then colonies were selected for plasmid DNA isolation. The new plasmid construct was confirmed by cutting out the Flag tagged hKRS fragment using same enzymes used for cloning, NcoI and NotI. Protein expression checked for using BL21 DE3 cells, non chloramphenicol resistant strain, with 1mM IPTG induction, SDS Page, and then a Western blot with chemoluminescence for visualization of Flag tagged proteins was conducted.

Results: Plasmid pACYC hKRS clones confirmed by digestion. Colonies PCR screened by NcoI and NotI double digest to check for hKRS gene insert into pACYC Duet plasmid.

The hKRS flag tagged protein is expressed with IPTG induction, but with degradation. Blot left to right: hKRS (positive), and pACYC hKRS with overflow in side lanes.

Conclusion: pACYC hKRS strongly expresses Flag tagged hKRS when induced by IPTG, but degradation of the protein is observed. Use for co-transformation with plasmid pET11 hVpr into BL21 DE3 cells that are not chloramphenicol or ampicillian resistant.

Co-transformation and Induction of pACYC hKRS and pET11 hVpr

Purpose: Co-transform plasmids pACYC hKRS and pET11 hVpr into BL21 DE3. Confirm co-transformation by restriction digestion, and check for co-expression of both proteins in BL21 DE3 cells.

Procedure: Both plasmids were transformed into BL21 DE3 using ampicillian and chloramphenicol LB media agar plates for selection. The reaction yielded two colonies. The colonies were then tested for the presence of both plasmids by growing overnight cultures in LB media with ampicillian and chloramphenicol, purifying the plasmid DNA, and then performing a restriction digest. The restriction digest was done with NcoI and NotI to yield 3 expected DNA fragments at 2Kb, 4Kb, and 6Kb along with the two larger parent plasmid fragments. Next, overnight cultures of the cells were used for IPTG induction. 150ul of overnight culture was used to inoculate15ml LB media containing ampicillian and chloramphenicol. After 2 hours incubation at 37˚c with shaking, the cells reached an OD₆₀₀ of approximately 0.5. At this time the cells were induced with IPTG for 2 hours and 45 minutes, and then 1ml samples were taken and frozen in N₂. Western blot was done using Flag antibody to visualize both the hVpr and hKRS.

Results: pET11 hVpr is ~6Kb when linearized. pACYC hKRS cut by NotI and NcoI yields two fragments at ~2Kb and ~4Kb.

Both hKRS and hVpr proteins are visible on Western blot with Flag antibody after induction.

Conclusion: The two colonies were confirmed to have both plasmids transformed in each. Glycerol stocks were made of these cells for use in induction and purification of both hKRS and hVpr proteins. The transformed cells co-expressed both hVpr and hKRS proteins only when induced.

Ni+ His tag Affinity Batch Purification of hVpr and hKRS to Test for Protein Binding

Purpose: Induce hKRS and hVpr together in-vivo and then purify hVpr via His tag to see if it pulls out hKRS along with it. Check results using Flag tag Western Blot

Procedure: The buffers were prepared as follows:

Equilibrium Buffer (0.1M Na Phosphate pH8) - 950ml Na₂HPO₄ + 50ml NaH₂PO₄
Wash Buffer - same as equilibrium buffer, but with IMDZ added to a 20mM concentration
Elution Buffer - 250ml equilibrium buffer + 8.51g IMDZ + 8.751g NaCl; yielding a final solution containing 0.1M Na Phosphate, 250mM IMDZ, and 0.3M NaCl
Sonication Buffer - 250ml equilibrium buffer + 347.2ul 14.4M β-mercapto EtOH + 50ml glycerol, then add H₂0 to a final volume of 500ml; yielding a final solution containing 50mM Na Phosphate, 10uM β-mercapto EtOH, and 10% glycerol

Fresh transformations of pET11 hVpr, pACYC hKRS, and the two plasmids together were used to make overnight cultures. 1ml of overnight culture was used to inoculate 100ml of LB media and incubated at 37˚c with shaking. When the cells reached 0.4-0.5 OD₆₀₀, ~2 hours, they were induced for one hour by adding 1mM IPTG. Cells were harvested in 10ml and 50ml aliquots. The 10ml pellets were re-suspended in 1ml sonication buffer with 10ul of 100mM PMFS added. They were then sonicated for 120 seconds at 50% pulse on ice. The final sample was spun down at 12500G for 5 minutes in a 1.5ml Epp tube and kept on ice. 100ul of the supernatant was also put to the side for SDS PAGE.

The Ni+ batch was prepared by spinning down 50ul Ni+ resin in an EPP tube at 5000G and the supernatant removed. Equilibrium of the resin was then done with re-suspensions of it in a ½ ml equilibrium buffer three times, by spinning it down each time in between at 5000G and removing the supernatant. After the final removal, the resin was spun down again to remove any left over equilibrium buffer. The main sample supernatant was added to the resin, and the remaining sample pellet put to the side for SDS PAGE. The batch sample was then placed into an orbital shaker at 4˚c for one hour.

After shaking, the batch sample was spun down at 5000G for 5 minutes, and the supernatant was placed to the side as the flow through sample. The resin was then washed three times for ten minutes in the orbital with 500ul wash buffer each. After each wash the resin was spun down at 5000G for 5 minutes, and the supernatant was placed to the side as the wash sample.

Elution of the protein from the resin was done by adding 250ul elution buffer to the resin, and then placing it in the orbital for one hour. Then the resin was spun down at 5000G for 5 minutes, and the supernatant was placed to the side as the extraction sample. Next, the resin was washed again three times as before, and the supernatant obtained was saved as wash #2. The final eluted and washed resin was also saved for SDS PAGE analysis.

The original supernatant, pellet, flow through, wash, extract, and resin were then run on a 12% SDS PAGE, and a Western blot was conducted for detection of the Flag tag.

Results: Both pET11 hVpr and pACYC hKRS samples have Flag tagged protein present in all samples. The co-transformed cells batch sample had both proteins visible in the pellet, supernatant, and flow through; but only hVpr present in the wash, extract, and resin. Bottom blot hKRS, middle blot hVpr, and top blot hKRS-hVpr co-expression sample. Lanes from left to right on all three: blank, positive, ladder, original supernatant, blank, flow through, wash, extract, resin, blank.

Conclusion: Many trial runs were done to try to optimize the batch reaction solutions. In the early trial runs IMDZ was added to the equilibrium buffer at 20mM and 10mM, but both proteins were retained by the beads, even though hKRS is not His tagged. In the final run no IMDZ was added to the equilibrium buffer. For this run the hVpr was purified out, but the hKRS was not from the co-transformed cells. However, even during that run, the negative control again showed that the hKRS was binding to the beads possibly due to an excess of hKRS protein loaded into the batch. Ni+ affinity batch reaction conditions could disrupt binding interactions between both proteins, and is also unreliable due to the ability of beads to bind hKRS. Therefore, gel permeation chromatography is next course of action.

Timed Induction of pET11 hVpr, pACYA hKRS , and Co-transformed BL21 DE3 Cells

Purpose: Check expression of proteins at different time intervals to view degradation pattern.

Procedure: Grew overnight culture from glycerol stocks of BL21 DE3 cells containing pET11 hVpr, pACYC hKRS, and the two plasmids together. Next, 1ml of overnight culture was used to inoculate 100ml LB media, and then incubated at 30˚c with shaking. When the cell cultures reached 0.3-0.5 OD₆₀₀, they were induced with 1mM IPTG. Then 10ml aliquots of cells were harvested at 30 minutes, 45 minutes, 1 hour, 1 ½ hours, and 2 hours. The samples were then sonicated on ice with 1ml sonication buffer containing 1mM PMFS for two minutes at 50% pulse. The resulting samples were run on a 12% SDS PAGE, and a Western blot was conducted for detection of the Flag tag.

Results: Western blot with Flag tag detection using previous hVpr sample as positive. Bottom blot hKRS-hVpr co-expression sample, middle blot hKRS, and top blot hVpr. Lanes from left to right on all three: flag positive (hVpr), ladder, blank, 30 minutes, 45 minutes, 1 hour, 1 ½ hours, 2 hours, blank.

Conclusion: Degradation is not very visible with the hVpr protein at any time after induction. The hKRS protein has the least amount of degradation at 30 minutes, and increasing signs of degradation after 45 minutes.

Purification of hVpr and hKRS Using Gel Permeation Chromatography

Purpose: Isolate hVpr and hKRS complex using gel permeation column. If the two proteins form a complex then they should elute faster than each protein individually

Procedure: Overnight cultures of BL21 DE3 cells containing pET11 hVpr, pACYC hKRS, and the two plasmids together were prepared. Next, 1ml of overnight culture was used to inoculate 100ml LB media, and then incubated at 30˚c with shaking. When the cell cultures reached 0.4-0.45 OD₆₀₀, they were induced with 1mM IPTG. Then 10ml aliquots of cells were harvested at 30 minutes. Binding buffer and column slurry was prepared as follows:

Binding Buffer (25mM HEPES pH 7.9, 50mM KCl, 0.5mM DTT, 5% glycerol, 0.1% NP-40) = 5.95g HEPES + 3.7275g KCl + 0.077125g DTT + 50ml glycerol + 1ml NP-40 and water is added to a final volume of 1 liter; then just before use PMFS is added for a final concentration of 0.4mM
Column Slurry - 1g of Sephadex G200 absorbs ~30-40ml, prepared with excess binding buffer, without PMFS added, for 72 hours

A 10ml slurry column was prepared having a void volume of ~ 5ml, which was determined by running blue dextran through it. The samples were then sonicated on ice for two minutes at 50% pulse with 1ml binding buffer and PMFS added. The samples were then spun down at 5000G for 5 minutes. The 250ul aliquots of the resulting supernatants were run on a 12% SDS PAGE, and a Western blot was conducted for detection of the Flag tag.

Results: Both hVpr and hKRS eluted in the void volume fraction when using Sephadex G200. The experiment was repeated using Bio Gel A-5, to accommodate for the proteins running heavy, but with the same results. However, hKRS was not visible on the blot of the co-expressed protiens for unknown reasons, it may have run off heavier than void volume with the possibility that it is bound with the hVpr.

A test was also done to check for protein precipitation due to freezing and thawing. Fresh supernatant from sonicated 10ml 30 minute induced hVpr and hKRS induced cells was obtained, some was frozen and thawed, and from the frozen/thawed fraction a sample was spun down for 15 minutes at 13200RPM in Epp tube centrifuge to pellet out any precipitate. Those samples, 32ul of each, were then run on SDS PAGE/Western with Flag tag detection. They did not indicate the occurrence of precipitation due to freezing. They did not indicate the occurrence of precipitation due to freezing. Blot left to right: hKRS thawed/spun (TS), hKRS thawed (T), hKRS supernantent (S), blank (*), blank (*), hVpr thawed/spun (TS), hVpr thawed (T), hVpr supernantent (S), ladder.

Conclusion: Both proteins ran heavy and eluted with the void volume, possibly due to formation of inclusion bodies. In original document of hVpr forming complex with hKRS, the hVpr used was expressed as a GST fused protein. The original pGEX hVpr sample from Stark et al, did not yield expected restriction digestion map. Paper states that 186bp fragment is obtained from digestion of plasmid with BamHI and EcoRI, but an ~800bp fragment is obtained instead. Therefore, re-create GST fused hVpr protein and co-express with hKRS to check for complex. (gel)

Construction of Plasmid pGEX-3x hVpr

Purpose: PCR hVpr from pET11 hVpr with out Flag or His tag. Create BamHI sites on both ends of full length hVpr gene, and insert digested PCR fragment of hVpr gene into BamHI site of pGEX-3x. Confirm clone by sequencing and digestion to check for multiple inserts.

Procedure: Two primers were designed, VprT and VprP. Primer VprT is at the tail end of the hVpr gene with a BamHI site insert followed by a two base overhang homologous to pGEX-3x: 5’-TAG GAT CCT ATG TCG ACA CCC AAT TCT GAA-3’. Primer VprP is at the start end of the hVpr gene with a BamHI site insert preceded by a two base overhang homologous to pGEX-3x: 5’-TGG GAT CCC CAT GGA ACA AGC CCC AGA AGA-3’. PCR reaction was done using PFU polymerase and pET11 hVpr as template. The resulting PCR fragment, ~254bp, was run on a 2% agarose gel and then purified from the gel using a GFX column. The purified PCR fragment was digested by BamHI, and the resulting digested fragment was gel purified by a GFX column from a 2% agarose gel. Plasmid pGEX-3x was digested with BamHI, and the DNA purified using a GFX column. Next, the cut pGEX-3x DNA was treated with CIP. The digested CIP treated fragment was then gel purified by a GFX column from a 1% agarose gel. The ligation of the hVpr into pGEX-3x was done with T4 DNA ligase, and the resulting reaction mixture was transformed into DH5α cells. Colonies were PCR screened using primers VprP and pGEXrev (5’-GCA GAT CGT CAG TCA GTC ACG ATG-3’) for detection of clones with proper orientation of insert. Positive colonies were selected and plasmid DNA isolated for restriction mapping and sequencing. The cloned plasmid was cut with AvrII, which cuts hVpr once but does not cut pGEX-3x, thus determining presence of hVpr and occurrence of multiple inserts. Then the clone was sequenced using the pGEXrev primer. Cells were grown and induced with IPTG, then run on 12% SDS PAGE, and a Western blot was conducted using GST anti-bodies for detection of GST fused hVpr.

Results: The purified digested pGEX-3x and PCR hVpr fragments run side by side on a 1.5% agarose gel to check each DNA sample’s concentration. Gel from left to right: purified BamHI digested and CIP treated pGEX-3x, purified BamHI digested PCR fragment of hVpr gene from pET11 hVpr, and 1 Kb Ladder. PCR screen of transformed colonies for presence of hVpr gene in pGEX-3x, expected fragment size of ~283 bases, lanes 2, 6, 8, and 9 show positive test for clone.

Restriction digestion used to confirm clone and check for multiple inserts of hVpr gene. AvrII cuts hVpr once but does not cut pGEX-3x. (gel)

Sequence results using the pGEXrev primer also confirm insertion of hVpr

Conclusion: The pGEX3x hVpr clones were confirmed. When induced, there was no visible expression, however it was suggested that the protein may be expressed at low levels, and GST purification would give better results.

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Purification of hVpr and hKRS Using Glutathione Beads

Purpose: Purify GST fused hVpr using glutathione beads, and check for protein using SDS PAGE and Western blot with GST anti-bodies

Procedure: Plasmids pGEX hVpr (Stark), pGEX3x, pGEX3x hVpr, and GST Vp16 (+ control) transformed into BL21 DE3 cells, overnight cultures prepared, and 150ul of overnight culture are used to inoculate 15ml LB media. Cultures are grown to OD ~1.0, ~2 hours, and then induced for 1hr with 1mM IPTG. Harvest 1ml and 10ml pellets. 133ul glutathione beads are spun down for 5 minutes at 5000g, and then the EtOH supernatant is removed. The beads are re-suspended in 1 ml PBS, inverted to wash, then spun down for 5 minutes at 5000g, and the supernatant is removed. Next 100ul PBS is added to create 50% glutathione slurry.

The 10ml pellets were re-suspended in 1ml PBS and sonicated, then spun down and a 10ul sample of supernatant was put to the side for SDS PAGE. The remaining supernatant was transferred to a fresh 1.5ml Epp tube, and the pellet was saved for SDS PAGE analysis. Next 20ul of 50% glutathione slurry is added to the main supernatant, and mixed by inverting gently for 5minutes. The sample is spun down and the supernatant removed and saved as the, then the beads are washed with 100ul PBS by brief vortex mix. Then the sample is spun down again for 5 seconds to sediment the beads and remove the supernatant. The beads are then eluted with 10ul elution buffer for 5 minutes at room temperature. The sample is spun down for 5 minutes and the supernatant removed for SDS PAGE analysis.

Results: Sample loads:

Supernatant: 10ul + 3ul 5x SDS buffer

Pellet: 10ul of 1ml suspension + 3ul 5x SDS buffer

Elution: 10ul + 3ul 5x SDS buffer

Beads: 10ul H₂O + 3ul 5x SDS buffer, spun down to remove beads before loading

Conclusion: GST fused hVpr was purified. Repeat experiment with cells co-transformed with pGEX3x hVpr and pACYC hKRS

--------------

Construction of Plasmid pFastBac hKRS

Purpose: Prepare plasmid with hKRS for use with the baculovirus system.

Procedure: Plasmid pM386 from Hiromi Motegi, contains N-terminal His tagged hKRS, clone into pFastBac using XbaI and XhoI sites. Ligation with T4 DNA ligase, and colonies selected for plasmid prep. Clones confirmed by cutting out hKRS gene, and sequence analysis.

Results: The forward and reverse sequencing results were blasted against the pFastBac1 sequence. The hKRS gene is highlighted in yellow, pFastBac is highlighted in geen, and the XhoI/XbaI resitriction sites are highlighted in red. They were also blasted against the full legth hKRS from NCBI GenBank reference # BC004132:

Forward:

NGCCCGTCTCCGGTCCGANCGCGCGGAATTCAAAGGCCTACGTCGACGAGCTCACTAGTCGCGGCCGCTTTCGAATCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACATATGAGAGGATCGCATCATCATCATCATCACAGCAGCGGCTGGGTCGATATGGCGGCCGTGCAGGCGGCCGAGGTGAAAGTGGATGGCAGCGAGCCGAAACTGAGCAAGAATGAGCTGAAGAGACGCCTGAAAGCTGAGAAGAAAGTAGCAGAGAAGGAGGCCAAACAGAAAGAGCTCAGTGAGAAACAGCTAAGCCAAGCCACTGCTGCTGCCACCAACCACACCACTGATAATGGTGTGGGTCCTGAGGAAGAGAGCGTGGACCCAAATCAATACTACAAAATCCGCAGTCAAGCAATTCATCAGCTGAAGGTCAATGGGGAAGACCCATACCCACACAAGTTCCATGTAGACATCTCACTCACTGACTTCATCCAAAAATATAGTCACCTGCAGCCTGGGGATCACCTGACTGACATCACCTTAAAGGTGGCAGGTAGGATCCATGCCAAAAGAGCTTCTGGGGGAAAGCTCATCTTCTATGATCTTCGAGGAGAGGGGGGTGAAGTTGCAAGTCATGGCCAATTCCNGAAATTATAAATCAGAAGAAGAATTTTTTCATATTATAACAAATGCGTCGGGGGAGACATAATTGGAGTTCAGGGGAATCCTGGTAAAACCCAGAANGGTGGAGCTTGANCATCATTCCGTATGANGATCACACTGGCTGTCTNCCTGGTTTGCATATGTTACCTCATCTTCNCTTTGGNCTCAAAGACAAGGAAAACAAGNATN

Reverse:

ATTGGTTTTGACGCNCATCCTNATTTCNCCCACCTGTGAGTTTTTACATGGCCTATGCAGANTTCCACGGATNTCATGGAAATCACGGAGAAGATGGTTTCAGGGATGGTGAAGCATATTACAGGCAGTTACAAGGTCCACCTACCACCCCAGATGGCCCAGAGGGCCAAGCTTACGATGTTGACTTCACCCCACCCTTCCGGCGAATCAACATGGTAGAAGAGCTTGAGAAAGCCCTGGGGATGAAGCTGCCAGAAACGAACCTCTTTGAAACTGAAGAAACTCGCAAAATTCTTGATGATATCTGTGTGGCAAAAGCTGTTGAATGCCCTCCACCTCGGACCACAGCCAGGCTCCTTGACAAGCTTGTTGGGGAGTTCCTGGAAGTGACTTGCATCAATCCTACATTCATCTGTGATCACCCACAGATAATGAGCCCTTTGGCTAAATGGCACCGCTCTAAAGAGGGTCTGACTGAGCGCTTTGAGCTGTTTGTCATGAAGAAAGAGATATGCAATGCGTATACTGAGCTGAATGATCCCATGCGGCAGCGGCAGCTTTTTGAAGAACAGGCCAAGGCCAAGGCTGCAGGTGATGATGAGGCCATGTTCATAGATGAAAACTTCTGTACTGCCCTGGAATATGGGCTGCCCCCCACAGCTGGCTGGGGCATGGGCATTGATCGAGTCGCCATGTTTCTCACGGACTCCAACAACATCAAGGAAGTACTTCTGTTTCCTGCCATGAAACCCGAAGACAAGAAGGAGAATGTAGCAACCACTGATACACTGGAAAGCACAACAGTTGGCACTTTCTGTCTAGTCTCGAGGCATGCGGNCCAAGTTGTCG

Forward and reverse sequence blast against hKRS BC004132:

atggcggccgtgcaggcggccgaggtgaaagtggatggcagcgagccgaaactgagcaag

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

atggcggccgtgcaggcggccgaggtgaaagtggatggcagcgagccgaaactgagcaag

aatgagctgaagagacgcctgaaagctgagaagaaagtagcagagaaggaggccaaacag

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

aatgagctgaagagacgcctgaaagctgagaagaaagtagcagagaaggaggccaaacag

aaagagctcagtgagaaacagctaagccaagccactgctgctgccaccaaccacaccact

|| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||

aaggagctcagtgagaaacagctaagccaagccactgctgctgccaccaaccacaccact

gataatggtgtgggtcctgaggaagagagcgtggacccaaatcaatactacaaaatccgc

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

gataatggtgtgggtcctgaggaagagagcgtggacccaaatcaatactacaaaatccgc

agtcaagcaattcatcagctgaaggtcaatggggaagacccatacccacacaagttccat

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

agtcaagcaattcatcagctgaaggtcaatggggaagacccatacccacacaagttccat

gtagacatctcactcactgacttcatccaaaaatatagtcacctgcagcctggggatcac

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

gtagacatctcactcactgacttcatccaaaaatatagtcacctgcagcctggggatcac

ctgactgacatcaccttaaaggtggcaggtaggatccatgccaaaagagcttctggggga

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

ctgactgacatcaccttaaaggtggcaggtaggatccatgccaaaagagcttctggggga

aagctcatcttctatgatcttcgaggagaggggggtgaagttgcaagtcatggccaattc

||||||||||||||||||||||||||||| ||||||||||||||||||||||||||||||

aagctcatcttctatgatcttcgaggaga-gggggtgaagttgcaagtcatggccaattc

cngaaattataaatcagaagaagaattttttcatatt-ataacaaa-tgcgtcgggggag

| |||||||||||||||||||||||||| |||||||| |||||||| |||||| ||||||

cagaaattataaatcagaagaagaatttattcatattaataacaaactgcgtc-ggggag

acataattggagttcaggggaatcctggtaaaacccagaanggtggagcttgancatcat

||||||||||||||||||||||||||||||||||| |||| ||| |||| ||| ||||||

acataattggagttcaggggaatcctggtaaaaccaagaagggt-gagc-tgagcatcat

tccgtatgangatcacactggctgtctncctggtttgcatatgttacctcatcttcnctt

||||||||| ||||||||| ||||||| ||| |||||||||||||||||||||||| |||

tccgtatga-gatcacact-gctgtctccct-gtttgcatatgttacctcatcttcactt

tggnctcaaagacaaggaaa Forward

||| ||||||||||||||||

Tggcctcaaagacaaggaaa BC004132

ccacctgtgagtttttacatggcctatgcaganttccacggatntcatggaaatcacgga

||||||||||||| | |||||||||||||||| |  |||| || ||||||||||||||||

ccacctgtgagttct-acatggcctatgcagactatcacg-atctcatggaaatcacgga

gaagatggtttcagggatggtgaagcatattacaggcagttacaaggtccacctaccacc

||||||||||||||||||||||||||||||||||||||||||||||||| ||||||||||

gaagatggtttcagggatggtgaagcatattacaggcagttacaaggtc-acctaccacc

ccagatggcccagagggccaagcttacgatgttgacttcaccccacccttccggcgaatc

| ||||||||||||||||||||| ||||||||||||||||||||||||||||||||||||

c-agatggcccagagggccaagcctacgatgttgacttcaccccacccttccggcgaatc

aacatggtagaagagcttgagaaagccctggggatgaagctgccagaaacgaacctcttt

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

aacatggtagaagagcttgagaaagccctggggatgaagctgccagaaacgaacctcttt

gaaactgaagaaactcgcaaaattcttgatgatatctgtgtggcaaaagctgttgaatgc

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

gaaactgaagaaactcgcaaaattcttgatgatatctgtgtggcaaaagctgttgaatgc

cctccacctcggaccacagccaggctccttgacaagcttgttggggagttcctggaagtg

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

cctccacctcggaccacagccaggctccttgacaagcttgttggggagttcctggaagtg

acttgcatcaatcctacattcatctgtgatcacccacagataatgagccctttggctaaa

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

acttgcatcaatcctacattcatctgtgatcacccacagataatgagccctttggctaaa

tggcaccgctctaaagagggtctgactgagcgctttgagctgtttgtcatgaagaaagag

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

tggcaccgctctaaagagggtctgactgagcgctttgagctgtttgtcatgaagaaagag

atatgcaatgcgtatactgagctgaatgatcccatgcggcagcggcagctttttgaagaa

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

atatgcaatgcgtatactgagctgaatgatcccatgcggcagcggcagctttttgaagaa

caggccaaggccaaggctgcaggtgatgatgaggccatgttcatagatgaaaacttctgt

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

caggccaaggccaaggctgcaggtgatgatgaggccatgttcatagatgaaaacttctgt

actgccctggaatatgggctgccccccacagctggctggggcatgggcattgatcgagtc

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

actgccctggaatatgggctgccccccacagctggctggggcatgggcattgatcgagtc

gccatgtttctcacggactccaacaacatcaaggaagtacttctgtttcctgccatgaaa

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

gccatgtttctcacggactccaacaacatcaaggaagtacttctgtttcctgccatgaaa

cccgaagacaagaaggagaatgtagcaaccactgatacactggaaagcacaacagttggc

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

cccgaagacaagaaggagaatgtagcaaccactgatacactggaaagcacaacagttggc

actttctgtctag Reverse

|| ||||||||||

ac-ttctgtctag BC004132

Conclusion: Plasmid clone confirmed and labeled pFastBac hKRS. Plasmid was then used for transposition into bacmid DNA. Bacmid hKRS clone comfirmed by PCR analysis.

Discussion

Plasmid constructs containing both hVpr and hKRS genes did not co-express the proteins. pET11 hVpr/hKRS may have had co-expression of proteins, but it was not detectable by Western blot for the Flag tag. Only the hVpr was detected from the pET11 hVpr/hKRS plasmid both induced and uninduced. The pUC19 hKRS plasmid, from which the hKRS gene expression vector was obtained, only showed a faint detectable amount of Flag tag hKRS. It was determined that the hKRS under the 35s promoter was not suitable for co-expression with hVpr, which is under the control of the T7 promoter. Therefore, the hKRS was cloned into a T7 vector contained with in the pET21d plasmid for increased expression. Now both proteins are expressed using the T7 vector.

A second plasmid construct, pET T7VK, was developed containing both genes. The T7 vector containing hVpr was cloned into the pET21 hKRS plasmid at the BglII site, such that the transcription directions of each protein were away from each other on the plasmid. No expression was obtained, and it is believed that the two promoters were to close together for proper expression. Therefore, a DNA spacer was put in between the two promoters to separate them further apart by ~174bp, versus their current separation of about ~30bp. These clones labeled pET T7VKS also did not co-express the proteins.

A new method was used for co-expression of the proteins using a two plasmid system. The plasmids are co-transformed into cells by means of separate replication origins and antibiotic resistance. The pET11 hVpr plasmid has ampicillin resistance and the pBR322 replication origin, and the pACYC hKRS plasmid has chloramphenicol resistance and the P15A replication. This allowed for co-transformation of the plasmids into BL21 DE3 cells, which did show co-expression of both proteins.

The co-transformed BL21 DE3 cells were used for an in vivo assay to see if hVpr binds with hKRS. The proteins were co-expressed using these cells, and then run in a N+ resin batch reaction. The two proteins have the Flag tag, but only the hVpr contains the His tag. The N+ resin was used to purify the hVpr via the His tag, and then the purified sample was analyzed on a Western blot using the Flag tag for detection. If the hVpr binds the hKRS in vivo by co-expression, then the hKRS should also show up on the blot with the purified hVpr from the N+ resin. The hKRS did not show up, and it was determined that the N+ resin batch reaction conditions could disrupt the binding of the two proteins. Therefore, gel permeation chromatography was used.

The hVpr and hKRS vary greatly in size, hVpr runs at ~18kDA and hKRS runs at ~80kDA. If the two proteins bind with each other, then they should elute from the chromatography column together. A column was setup with 10ml slurry of Sephadex G200. The proteins were co-expressed using BL21 DE3 and then loaded onto the column. Both proteins were found to run heavy and eluted with the void volume fraction. Next, Bio Gel A-5 was used as the column slurry, but with the same results. It is believed that the hVpr protein is forming inclusion bodies.

The hVpr was originally found to bind hKRS when it was expressed as a GST fused protein [Stark]. Analysis of the hVpr from Stark et al was found to have four amino acid changes in its N-terminal domain when compared to the pET11 hVpr obtained from Zhao et al.

The following is the protein sequence of the first 72 amino acid residues of hVpr from NC_001802, Stark et al, and Zhao et al:

MEQAPEDQGPQREPHNEWTLELLEELKNEAVRHFPRIWLHGLGQHIYETYGDTWAGVEAI

MEQAPGNQGPQREPHNEWTLELLEELKNEAVRHFPRIWLHGLGQHIYETYGDTWAGVEAI

MEQAPEDQGPQREPYNDWTLELLEELKNEAVRHFPRIWLHGLGQHIYETYGDTWTGVEAL

*****-:*******:*:*************************************:****:

IRILQQLLFIHF hVpr NC_001802

IRILQQLLFIHF pGEX hVpr Stark_et_al

IRILQQLLFIHF pET11 hVpr Zhao_et_al

************

* single, fully conserved residue

- no consensus

: conservation of strong groups

(Sequence alignment analysis via http://workbench.sdsc.edu/)

hKRS binds directly to the N-terminal domain of hVpr [Stark]. Since there is four amino acid substitutions in the N-terminal domain of the pET11 hVpr from Zhao et al compared to that of the pGEX hVpr from Stark et al, then this might account for loss of activity of the pET11 hVpr to bind with hKRS.

Reference Page

Byung-Eun, Kim. Reasearch Paper. Dr. Folk Lab. Dec. 2000.

Forget, Janique. Human Immunodeficiency Virus Type 1 Vpr Protein Transactivation Function. (1998) J. Mol. Biol. 284, 915-923.

Lutrull, Michael. Reasearch Paper. Dr. Folk Lab. May-Nov. 2000