Archive for February 16th, 2017

Purifying TMV: a blast from the archives

16 February, 2017

We have had an in-house method for purifying Tobacco mosaic virus (TMV) and its various relatives ever since I got to Cape Town – and it was propagated by copying and re-copying of what was effectively an abstract for a talk given at our local Experimental Biology Group quarterly meeting in early 1970, published in the South African Medical Journal.

Marc van Regenmortel was Professor of Microbiology at the time, and had a long history of physicochemical and serological work on TMV and strains and mutants of TMV. He also had Barbara von Wechmar, later to become my PhD supervisor, working for him as a Scientific Officer – and together they came up with an ingeniously simple, easy, high-yielding method to purify TMV out of infected tobacco.

So why do we care now? Well, we’re trying to purify some derivatised TMV [details redacted while patent is sought], and Sue Dennis in my lab could only find techniques that involved extraction with chloroform, PEG/salt precipitation x 2, high-speed centrifugation – all of which sounded unnecessarily laborious, given I knew we had a better method.

Trouble is – I cleaned up my office a while back, and seeing as “we’ll never work with TMV again, will we??”, I’d thrown out all of the old practical manuals that included it.

So I go to the old papers I could find online, and they all referred to “von Wechmar and van Regenmortel, 1970”, with no methodological details. And of course, there was no record of this paper anywhere I could find, not even using [obscure Russian language site details redacted].

Then I chanced upon the very bare bones online archive of the SAMJ, married that up with the much snazzier-looking-but-devoid-of-desired pdfs official site to find issue numbers – and there we were! Via some fascinating side trips through a history of the plague in Cape Town, among other things, but finally, a PDF of the original EBG abstract.

tmv-method

In fact, I have a big section of our coldroom with myriad bottles of purified TMV, all at 5 mg/ml concentration or higher, still infectious, and up to 40 years old – all made by this technique.

tmv sedim

So Sue is about to apply it right now, as she conveniently has a freshly mashed extract of N benthamiana ready waiting, and we have PEG and NaCl…we’ll give the charcoal/Celite a miss this time, because it can get a bit messy, but it is THE way to get pigments out of your virus preps – or even nanoparticles, @FrankBioNano & @Lomonossoff_Lab?