Impact of modfications on TEV cleavage sequence?

Ester cleavage conditions?

Hi all, I need to cleave the ester in Ethyl 2-amino-1H-imidazole-5-carboxylate to make the carboxylic acid and retrieve the product from solution to use in downstream applications of linking to...

01 March 2021 8,766 3 View

Can we remove the fmoc group after peptide cleavage?

I have synthesized the peptide using Fmoc based solid phase peptide synthesis. Also I have cleaved the peptide from the resin and lyophilized it, but later realized that, I forgot to deprotect the...

21 February 2021 4,537 3 View

Will SARS-CoV-2 Spike be broken into S1 and S2 by denaturing conditions?

Hello, I am performing a western blot of my SARS-CoV-2 Spike-pseudotyped lentiviral particles. I denature my virus by adding concentrated virus to 2X Laemmli buffer at 1:1, then boiling for...

17 February 2021 4,844 4 View

The peptide synthesized with CTC resin have mass more (+68 Da) than the desired mass of the synthesized peptide. What could be the reason?

I am synthesizing 7 amino acid long peptide sequence with CTC resin, 50oC coupling for 10 min. On cleavage (TFA/TIPS/Water 95:2.5:2.5) I got peptide with good yield. However, It has 68 Da more...

30 January 2021 8,483 3 View

How long does the peptide cleavage cocktail last before it gets expired?

The cleavage cocktail is composed of Phenol/thioanisole/water/TFA/triisopropylsilane. I only prepare 10 mL cocktail per peptide but can the activity of the cocktail prepared at 100 mL last for a week?

10 January 2021 7,424 3 View

What buffer to use for biotinylation as alternative to PBS?

I'm looking to biotinylate a protein that precipitates in PBS. The recommended reconstitution buffer for the protein is urea/Tris buffer, pH 7,5 (7 M urea, 5 mM EDTA, 15 mM mercaptoethanol, 20 mM...

04 January 2021 7,681 5 View

Can TEV protease cleave between glutamine and methionine efficiently?

Based on this paper The P1 ' specificity of tobacco etch virus protease ,TEV protease can cleave between the Gln and several amino-acids (besides Gly/Ser) with acceptable efficiency in its...

26 November 2020 5,987 4 View

I would like to ask if anyone among you knows what is the ideal concentration of TFA to use at a chemical synthesis of peptides?

During a chemical synthesis of peptides by the F-moc method, the following happened: all the initial steps of the synthesis occurred as planned, but in the cleavage, TFA is used. In the lab, there...

02 November 2020 2,997 8 View

Why the expressed protein band Molecular Weight (MW) is lower than expectation?

I tried to express my protein in BL21(DE3) bacteria. The desired protein MW is 65.5 KDa and I know my protein will be in an insoluble form (inclusion body). I separate soluble and insoluble...

20 October 2020 3,975 8 View

How to cut the thrombin cleavage site if it is hindered by hydrophobic region?

I'm currently doing protein purification. my protein size is around 42kDa, however, my protein is attached with fusion tag which are Trx-tag and His-tag at the N terminal. I did optimize my buffer...

12 October 2020 2,083 3 View

Robert Mallis

This is permissable. See this very nice paper for details. I have verified with a subset myself and it does work.

Kapust, R.B., Tözsér, J., Copeland, T.D., Waugh, D.S. (2002) The P1' specificity of tobacco etch virus protease. Biochem Biophys Res Commun 294, 949-955.

Giambattista Guaitoli

Hello Robert,

really thank you for you incredibly quick answer! I am working with a construct exhibiting the sequence I mentioned, and I can achieve only 60% of cleavage. Do you maybe have any suggestion regarding how to boost its activity?

Many many thanks again and have a nice weekend!

Giambattista

Just off the top of my head, I know there are papers that test detergent addition, TEV seems quite tolerant. Otherwise I have found that reliability comes with using higher substrate concentrations, if your protein is too dilute, then the reaction won't go closer to completion. I will check back and see if anyone chimes in. This is not a problem that I myself have encountered.