Re: [ccp4bb] His Purification
Have you tried dialyzing the "purified" protein after elution into a no imidazole buffer while doing TEV cleavage, and then reloading on to a His column to remove the tag and TEV? I've found in my experience that this often recaptures a lot of the background proteins. If you're getting a 70kDa contaminant, may be a chaperone. Try washing during your purification with a buffer supplemented with 5-10mM ATP + 5-10mM MgCl2, incubate for about 5-10 min on ice the resin with the buffer, and then wash it off. Last option is to do ion exchange. On a really fine gradient, you may be able to separate your contaminants from each other.. Best of luck
Re: [ccp4bb] His Purification
my comment on expression of protein being low and nonspecific binding. Amount of protein of expressed protein less = less resin = less nonspecific binding? (one have to do experiments to find right amount of resin to get least non-specific binding still pull out most of your protein of interest is a good idea?) the original question was how can one do oncolumn digestion and will TEV bind to resin. Yes it will if it have tag. alternate source for TEV see www.mobitec.com MoBiTec GmbH, sell a TEV with additional GST tag. But if your protein is 87 and TEV should be possible to separate on GFC and you should add a GFC right after your TEV digestion which is always a good thing to do. earlier suggested NEB NiCo21(DE3) was promised to be useful but the benefit of this host system is still not clear to my experience. Hope this helps Padayatti On Thu, Jan 19, 2012 at 7:21 AM, Artem Evdokimov wrote: > Your mileage will vary: prset is an almost always quasi constituitive vector > due to leakage, with additional induction upon expression of T7 pol. This is > not always bad since some proteins do not fold well upon avalanche inducton > but do in fact fold when expressed at a trickle (even good old lac promoter > sometimes works better than T7). Due to leaky expression toxic proteins > usually have issues with prset, and often result in microcolonies, variable > colonies, loss of expression etc etc. It is not my first, nor my second > choice for an expression vehicle but it is something that is in my opinion > worth trying if one finds oneself in a tight corner. Either that, or call > Ghostbusters! > > Artem > > On Jan 19, 2012 5:19 AM, "Mark J van Raaij" wrote: >> >> avoid pRSET would be my recommendation - my impression from a couple of >> examples is pRSET gives badly folded protein. >> Can you easily subclone it in a better expression vector? >> (personally, even if it were not so easy, I would reclone it in another >> expression vector). >> >> Mark J van Raaij >> Laboratorio M-4 >> Dpto de Estructura de Macromoleculas >> Centro Nacional de Biotecnologia - CSIC >> c/Darwin 3 >> E-28049 Madrid, Spain >> tel. (+34) 91 585 4616 >> http://www.cnb.csic.es/~mjvanraaij >> >> >> >> On 18 Jan 2012, at 13:56, PULSARSTRIAN wrote: >> >> > Hello Every one, >> > I am trying to purify a human protein in a >> > bacterial expression system of around 82 kDa (with a 5 kDa His tag, so >> > fusion protein is 87 kDa) which was cloned in pRSET-A vector. The Problem >> > is >> > I am not able to get rid of the infamous contamination proteins of arnA >> > gene >> > (72 kDa) and glmS gene (67 kDa). >> > I am using TEV protease to cleave my protein (82 kDa) from the tag (5 >> > kDa). This TEV protease has N- terminal His tag. So I first elute my fusion >> > protein with higher imidazole concentration and then do TEV cleavage by >> > adding TEV protease,, but sadly It co-elutes other contamination proteins >> > such as 72 kDa and 67 kDa, as mentioned above.. >> > >> > Now, I wanted to know,,, can I do "On beads cleavage" by directly >> > adding TEV enzyme when the fusion protein is still bound to Ni-NTA beads?? >> > But I am worreid TEV protease which has N- terminal His tag also try to >> > bind on Ni-NTA beads.. >> > >> > Please help me.. >> > >> > Thanks!! >> > >> > >> > >> > -- >> > B4U -- Pius S Padayatti,PhD, Phone: 216-658-4528
Re: [ccp4bb] His Purification
Your mileage will vary: prset is an almost always quasi constituitive vector due to leakage, with additional induction upon expression of T7 pol. This is not always bad since some proteins do not fold well upon avalanche inducton but do in fact fold when expressed at a trickle (even good old lac promoter sometimes works better than T7). Due to leaky expression toxic proteins usually have issues with prset, and often result in microcolonies, variable colonies, loss of expression etc etc. It is not my first, nor my second choice for an expression vehicle but it is something that is in my opinion worth trying if one finds oneself in a tight corner. Either that, or call Ghostbusters! Artem On Jan 19, 2012 5:19 AM, "Mark J van Raaij" wrote: > avoid pRSET would be my recommendation - my impression from a couple of > examples is pRSET gives badly folded protein. > Can you easily subclone it in a better expression vector? > (personally, even if it were not so easy, I would reclone it in another > expression vector). > > Mark J van Raaij > Laboratorio M-4 > Dpto de Estructura de Macromoleculas > Centro Nacional de Biotecnologia - CSIC > c/Darwin 3 > E-28049 Madrid, Spain > tel. (+34) 91 585 4616 > http://www.cnb.csic.es/~mjvanraaij > > > > On 18 Jan 2012, at 13:56, PULSARSTRIAN wrote: > > > Hello Every one, > > I am trying to purify a human protein in a > bacterial expression system of around 82 kDa (with a 5 kDa His tag, so > fusion protein is 87 kDa) which was cloned in pRSET-A vector. The Problem > is I am not able to get rid of the infamous contamination proteins of arnA > gene (72 kDa) and glmS gene (67 kDa). > > I am using TEV protease to cleave my protein (82 kDa) from the tag (5 > kDa). This TEV protease has N- terminal His tag. So I first elute my fusion > protein with higher imidazole concentration and then do TEV cleavage by > adding TEV protease,, but sadly It co-elutes other contamination proteins > such as 72 kDa and 67 kDa, as mentioned above.. > > > > Now, I wanted to know,,, can I do "On beads cleavage" by directly > adding TEV enzyme when the fusion protein is still bound to Ni-NTA beads?? > > But I am worreid TEV protease which has N- terminal His tag also try to > bind on Ni-NTA beads.. > > > > Please help me.. > > > > Thanks!! > > > > > > > > -- > > B4U >
Re: [ccp4bb] His Purification
avoid pRSET would be my recommendation - my impression from a couple of examples is pRSET gives badly folded protein. Can you easily subclone it in a better expression vector? (personally, even if it were not so easy, I would reclone it in another expression vector). Mark J van Raaij Laboratorio M-4 Dpto de Estructura de Macromoleculas Centro Nacional de Biotecnologia - CSIC c/Darwin 3 E-28049 Madrid, Spain tel. (+34) 91 585 4616 http://www.cnb.csic.es/~mjvanraaij On 18 Jan 2012, at 13:56, PULSARSTRIAN wrote: > Hello Every one, > I am trying to purify a human protein in a bacterial > expression system of around 82 kDa (with a 5 kDa His tag, so fusion protein > is 87 kDa) which was cloned in pRSET-A vector. The Problem is I am not able > to get rid of the infamous contamination proteins of arnA gene (72 kDa) and > glmS gene (67 kDa). > I am using TEV protease to cleave my protein (82 kDa) from the tag (5 kDa). > This TEV protease has N- terminal His tag. So I first elute my fusion protein > with higher imidazole concentration and then do TEV cleavage by adding TEV > protease,, but sadly It co-elutes other contamination proteins such as 72 > kDa and 67 kDa, as mentioned above.. > > Now, I wanted to know,,, can I do "On beads cleavage" by directly adding TEV > enzyme when the fusion protein is still bound to Ni-NTA beads?? > But I am worreid TEV protease which has N- terminal His tag also try to bind > on Ni-NTA beads.. > > Please help me.. > > Thanks!! > > > > -- > B4U
Re: [ccp4bb] His Purification
Make sure the imidazole is removed before you apply the TEV digested sample back to the Ni column for the reverse Ni step. Sounds like the expression level of your protein is low. You many consider to improve the expression. Those contamination proteins disappear from Ni elution when the target protein is abundant. As mentioned by Greg Costakes, on beads/column cleavage is less efficient comparing to solution digestion. However on column digestion is useful for some difficult-to-purify proteins. We have used our product TurboTEV (http://www.accelagen.com/TurboTEV.htm) for on column digestions. We found that keeping the TurboTEV to target protein ratio at 1:20 to 1:100 (same ratio for digestion in solution) gives good digestion, although never complete. That means you need to use >0.2 mg/ml TurboTEV for a resin with binding capacity of 20 mg/ml. We made TurboTEV storage buffer compatible with Ni resins. TurboTEV has dual GST- and His-tags. The geometry could be different from His-tagged TEV products. TurboTEV also has very high specific activity due to a novel stabilizing mechanism that is independent of S219 mutations. These properties probably make TurboTEV more efficient for on column digestion than other TEV products. You need to experiment a little bit with His-tagged TEV products. Chun Accelagen From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of PULSARSTRIAN Sent: Wednesday, January 18, 2012 4:57 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] His Purification Hello Every one, I am trying to purify a human protein in a bacterial expression system of around 82 kDa (with a 5 kDa His tag, so fusion protein is 87 kDa) which was cloned in pRSET-A vector. The Problem is I am not able to get rid of the infamous contamination proteins of arnA gene (72 kDa) and glmS gene (67 kDa). I am using TEV protease to cleave my protein (82 kDa) from the tag (5 kDa). This TEV protease has N- terminal His tag. So I first elute my fusion protein with higher imidazole concentration and then do TEV cleavage by adding TEV protease,, but sadly It co-elutes other contamination proteins such as 72 kDa and 67 kDa, as mentioned above.. Now, I wanted to know,,, can I do "On beads cleavage" by directly adding TEV enzyme when the fusion protein is still bound to Ni-NTA beads?? But I am worreid TEV protease which has N- terminal His tag also try to bind on Ni-NTA beads.. Please help me.. Thanks!! -- B4U
Re: [ccp4bb] His Purification
Another option is to try NEB NiCo21(DE3) cells. http://www.neb.com/nebecomm/products/productC2529.asp I have no relation to NEB beyond being a customer. They've mutated GlmS to eliminate binding to IMAC resins and have added chitin affinity tags to SlyD, ArnA and Can to allow simple post- IMAC removal of those common contaminants. I've just started testing them and would be interested in feedback from others. Best, Cynthia On Jan 18, 2012, at 10:21 AM, Ed Pozharski wrote: On Wed, 2012-01-18 at 18:26 +0530, PULSARSTRIAN wrote: The Problem is I am not able to get rid of the infamous contamination proteins of arnA gene (72 kDa) and glmS gene (67 kDa). This is only a problem if you plan to have imac purification as your only step. If the goal is crystallization, such products can definitely be used in initial trials (in fact, impurities may provide a benefit of nucleation), but you would have to introduce a second step eventually, which is often the ion-exchange. The co-purified proteins will be definitely removed at that step, so perhaps your time is better spent optimizing some high-res ion-exchange gradient. Two ways I can think of if you'd rather stick with IMAC is to try cobalt instead of nickel (might have different non-specific binding profile) or overload the resin with your protein (the presumption here is that it has higher affinity than impurities and you will get rid of them by simple competition). Or maybe the imidazole gradient could help. Cheers, Ed. -- "I'd jump in myself, if I weren't so good at whistling." Julian, King of Lemurs
Re: [ccp4bb] His Purification
May be this one helps " The current study presents the design, engineering, and characterization of two E. coli BL21(DE3) derivatives, NiCo21(DE3) and NiCo22(DE3), which express the endogenous proteins SlyD, Can, ArnA, and (optionally) AceE fused at their C terminus to a chitin binding domain (CBD) and the protein GlmS, with six surface histidines replaced by alanines. " http://www.ncbi.nlm.nih.gov/pubmed/21602383 Regards,Raj Date: Wed, 18 Jan 2012 18:26:39 +0530 From: bhanu.hydpri...@gmail.com Subject: [ccp4bb] His Purification To: CCP4BB@JISCMAIL.AC.UK Hello Every one, I am trying to purify a human protein in a bacterial expression system of around 82 kDa (with a 5 kDa His tag, so fusion protein is 87 kDa) which was cloned in pRSET-A vector. The Problem is I am not able to get rid of the infamous contamination proteins of arnA gene (72 kDa) and glmS gene (67 kDa). I am using TEV protease to cleave my protein (82 kDa) from the tag (5 kDa). This TEV protease has N- terminal His tag. So I first elute my fusion protein with higher imidazole concentration and then do TEV cleavage by adding TEV protease,, but sadly It co-elutes other contamination proteins such as 72 kDa and 67 kDa, as mentioned above.. Now, I wanted to know,,, can I do "On beads cleavage" by directly adding TEV enzyme when the fusion protein is still bound to Ni-NTA beads?? But I am worreid TEV protease which has N- terminal His tag also try to bind on Ni-NTA beads.. Please help me.. Thanks!! -- B4U
Re: [ccp4bb] His Purification
On Wed, 2012-01-18 at 18:26 +0530, PULSARSTRIAN wrote: > The Problem is I am not able to get rid of the infamous contamination > proteins of arnA gene (72 kDa) and glmS gene (67 kDa). This is only a problem if you plan to have imac purification as your only step. If the goal is crystallization, such products can definitely be used in initial trials (in fact, impurities may provide a benefit of nucleation), but you would have to introduce a second step eventually, which is often the ion-exchange. The co-purified proteins will be definitely removed at that step, so perhaps your time is better spent optimizing some high-res ion-exchange gradient. Two ways I can think of if you'd rather stick with IMAC is to try cobalt instead of nickel (might have different non-specific binding profile) or overload the resin with your protein (the presumption here is that it has higher affinity than impurities and you will get rid of them by simple competition). Or maybe the imidazole gradient could help. Cheers, Ed. -- "I'd jump in myself, if I weren't so good at whistling." Julian, King of Lemurs
Re: [ccp4bb] His Purification
Have you tried reverse purification over the Ni-NTA column? That is the typical next step in purification after His-tag cleavage. Or did you mean to say that the impurities elute off with your cleaved protein during the reverse purification? If this is the case, you could try adding a reducing agent to help prevent unwanted interactions. You could also try a subsequent purification using ion exchange. As for on-column digestion... it is possible but comparatively inefficient. Hope this helps. --- Greg Costakes PhD Candidate Department of Structural Biology Purdue University Hockmeyer Hall, Room 320 240 S. Martin Jischke Drive, West Lafayette, IN 47907 ** Hard work often pays of in time, but Procrastination always pays off now ** - Original Message - From: "PULSARSTRIAN" To: CCP4BB@JISCMAIL.AC.UK Sent: Wednesday, January 18, 2012 7:56:39 AM Subject: [ccp4bb] His Purification Hello Every one, I am trying to purify a human protein in a bacterial expression system of around 82 kDa (with a 5 kDa His tag, so fusion protein is 87 kDa) which was cloned in pRSET-A vector. The Problem is I am not able to get rid of the infamous contamination proteins of arnA gene (72 kDa) and glmS gene (67 kDa). I am using TEV protease to cleave my protein (82 kDa) from the tag (5 kDa). This TEV protease has N- terminal His tag. So I first elute my fusion protein with higher imidazole concentration and then do TEV cleavage by adding TEV protease,, but sadly It co-elutes other contamination proteins such as 72 kDa and 67 kDa, as mentioned above.. Now, I wanted to know,,, can I do "On beads cleavage" by directly adding TEV enzyme when the fusion protein is still bound to Ni-NTA beads?? But I am worreid TEV protease which has N- terminal His tag also try to bind on Ni-NTA beads.. Please help me.. Thanks!! -- B4U
[ccp4bb] His Purification
Hello Every one, I am trying to purify a human protein in a bacterial expression system of around 82 kDa (with a 5 kDa His tag, so fusion protein is 87 kDa) which was cloned in pRSET-A vector. The Problem is I am not able to get rid of the infamous contamination proteins of arnA gene (72 kDa) and glmS gene (67 kDa). I am using TEV protease to cleave my protein (82 kDa) from the tag (5 kDa). This TEV protease has N- terminal His tag. So I first elute my fusion protein with higher imidazole concentration and then do TEV cleavage by adding TEV protease,, but sadly It co-elutes other contamination proteins such as 72 kDa and 67 kDa, as mentioned above.. Now, I wanted to know,,, can I do "On beads cleavage" by directly adding TEV enzyme when the fusion protein is still bound to Ni-NTA beads?? But I am worreid TEV protease which has N- terminal His tag also try to bind on Ni-NTA beads.. Please help me.. Thanks!! -- B4U
