BUG SPLATS!

February 28, 2014

A quick technical note/tip for folks doing recombinant protein expression in E.coli


When doing large-ish scale expression of proteins in E.coli, it is common to freeze the cell pellet post-harvesting and prior to cell lysis and protein purification. Typically this is achieved by re-suspending the cell pellet in a small about of Luria Broth or PBS and then re-pelleting the cells in a 50ml falcon tube, and freezing them at -80ºC.

Whilst this is convenient, the down side of this is that it introduces another centrifugation step into your harvesting protocol, and a solid lump of cell paste can take a while to thaw out fully, and can be difficult to get completely homogenous and lump-free prior to whatever lysis technique you choose to use.

A colleague of mine who recently joined the lab from AstraZeneca (after they closed much of the Alderley Park research facility) has brought with him an ingenious way of speeding both harvesting and thawing out considerably: BUG SPLATS.

Poo in a bag?

Revolutionary bacteria pellet freezing protocol!

Rather than re-suspend your pellet in LB/PBS and re-pellet it, just scoop it out with a spatula (or similar) and place the bug pellet into a small (~10x20cm) press-lock bag.

Collect all the pellet into the bottom of the bag and then smooth it out so that your bug splat is nice and thin (<5mm is ideal), and stick it in the freezer.

The thin bug splat will freeze faster than a pellet in the bottom of a falcon tube.

It will also thaw out much faster. For thawing – snap the brittle, frozen bug splat into small chunks, and empty them into a suitable container (50ml falcon or similar), and add the required amount of lysis/sonication buffer. It should thaw out rapidly, and it should be easier to obtain a smooth, homogenous solution before lysis than a large ~25ml lump of frozen paste in the bottom of a falcon tube.

As the pellet is brittle, you can also snap smaller piece off if you don’t want to thaw the whole thing out.

So there you go – a ludicrously simple way of saving time in the lab, and making the freeze/thaw process faster, which is (anecdotally) better for your protein.


The week in Structures – 8th May 2013 #PDB

May 8, 2013

Week #2: Trying to jumpstart our immune system to fight off HIV. 


Fighting the good fight against HIV.

In this weeks PDB releases, we see a couple of HIV-antibody structures, continuing the work of the Kwong and Mascola labs in the NIH. In 2011, these labs released a paper (published in Science) studying various antibodies that recognises and neutralise HIV-1. If you can recall your high school biology classes, antibody recognition of an invading pathogen is the first step in activating the immune system that hopefully results in successfully fighting off the infection. This work has been further developed with papers this year in Cell and Nature - Prolific and important work!

The structure shown below contains the “FAb fragment” (gold & bronze cartoon) of the antibody bound to gp120 (the big blue blob).

Antibody recognition of HIV gp120 - a surface marker of the HIV virus. After PDB # 4JB9

Antibody recognition of HIV gp120 – a surface marker of the HIV virus. After PDB # 4JB9

Glycoprotein 120 (gp120) is a component of the HIV surface spikes – which recognise a range of different molecules on the surface of our cells and initiate infection. It is because of the this that gp120 is an attractive vaccine candidate – however, recombinant gp120 has failed deliver results in clinical trials, and it is hoped that by studying antibody-gp120 interactions and perhaps by designing a novel gp120-dervied antigen, an effective HIV vaccine can be developed.

Presumably the authors discuss the relevance of their findings in the paper (not released at the time of writing here) in the context of HIV treatment and rational vaccine design. (PDB codes 4JB9 and 4J6R). (EDIT: Other work in the same edition of Science discuss similar topics, here and an article “Rational HIV Immunogen design to targets specific germline B-cell receptors.”)

Other structures of note this week at the excellently named “star domain of quaking protein in complex with RNA” and some work looking at the mechanism by which angiopoietin 1 and 2 interact with the Tie2 receptor tyrosine kinase ([paper] | [PDB] ), which might lead to therapeutics that prevent tumours growing their own blood supplies (angiogenesis blockers).


On branded drugs.

May 5, 2013

I went shopping yesterday…


Anti-allergy meds

On the left, a box of 14 generic allergy relief tablets, each containing 10mg Cetirizine HCl and Lactose – costing £1.

On the right, a box of 14 branded allergy relief tablets, each containing 10mg Cetirizine HCl and Lactose – costing £5.67. In a sale. Down from £7.57.

The active ingredients are  identical. The evidence for the efficacy of the active ingredients is identical. So how can companies justify charging 7.5 times more? I understand and acknowledge that effective marketing and other psychological factors might lead to a more effective placebo component of any clinical effect [1][2] – but a 7.5 fold increase in effectiveness?

The chemical structure of cetirizine.

The chemical structure of cetirizine. (Photo credit: Wikipedia)

If we assume that the sellers make some sort of profit on the generic, then someone must be making a huge profit on the branded anti-allergy meds. Which seems a little immoral. If you can offer people relief for 7.1p per day, why charge as much as 54p per day?

Whether or not this pricing is down to the pharmaceutical industry I cannot be sure – but big pharma don’t have the greatest public image at the moment – and examples like this sat on the shelf of your local supermarket perhaps serve as another example of why.


The week in Structures – 1st May 2013 #PDB

May 1, 2013

The first in a (hopefully) weekly series of reviews of interesting structures in latest PDB release**.


High-resolution* Cryo-EM models of Human and Drosophilia Ribosomes!

Proteins are the major molecular players in life as we know it – they are the chemists, the engineers, the messengers and the defenders of life. They make new chemical compounds that we need, they break old compounds down so that we might re-use them – they transmit messages between different cells and tissues and help identify invading pathogens. In short – proteins do pretty much everything, and as such are the subject of intense study and scrutiny.

What better subject for the first of my PDB release highlight posts, than the Ribosome – a vast (at least at a molecular level) and ancient molecular machines that are responsible for the synthesis of proteins in our cells. The (perhaps outdated) “central dogma” of biology is that DNA is transcribed to mRNA, which is translated into protein, and the proteins then go and do everything. Anger et al have released 5Å resolution Cryo-EM-derived models of human and drosophila (Fruit Fly) ribosomes – they are so vast that they have to be split across several PDB files each…

Human (3J3A, 3J3D, 3J3B, 3J3F)

Drosophila (3J38, 3J3C, 3J39, 3J3E)

We're gonna need a bigger boat.

… and so complex as to defy creation of a clear and crisp picture.

The Paper concerning these is due to be published in Nature, but is not available at the time of writing (EDIT – Now it is – here (£)). Presumably, along with the methods  employed in what must be a massive modelling task, there will be some discussion of the differences between these two eukaryotic ribosomes, and the prokaryotic ribosome structures that were the subject of the 2009 Nobel Prize for Chemistry.

Other highlights in this weeks crop of 287 PDB releases include yet more BACE-inhibitor complexes (potential lead compounds for Altzheimer‘s treatments) and CDK2 and CDK8 -inhibitor complexes, which may be lead to novel anti-cancer therapies.


*ok – 5Å might not be great for crystallography, but for our Cryo-EM-based friends, this is pushing the limits and is clearly the result of a great deal of hard work and a massive amount of particle picking.

** The PDB is the “Protein Data Bank” – all structures of proteins/DNA and related molecules are deposited into the PDB and then made available to everyone (for free) prior to or at the time of publication of the paper that describes the work pertaining to the structure. The PDB issues new releases every Wednesday morning (UK time).


Like science?

April 30, 2013

Like science and the progress and prosperity it brings the UK? Then sign the pants off this bad boy. http://ow.ly/kyyQ7 #sivpoint8


POP QUIZ – What’s this?

April 16, 2013
Erm...

What the hell is this thing?

A little game – What the hell is this thing? Take a guess in the comments below – the red blobs are me deleting labels that might have given it away.

The prize is kudos, the admiration of your peers, and a life time subscription to this blog. ;-)


#QEDcon 2013 ISS passes

April 9, 2013

Information shamelessly lifted from meteorwatch.org and metcheck.com.

Friday Night (12th)

  • 21:43-21:48 – “Incredibly Bright” – cloud cover 23%

Saturday Night (13th)

  • 20:52-20:59 – “Incredibly Bright” – cloud cover 99% :-(
  • 22:29-22:32 – “Incredibly Bright / Short pass” – cloud cover 100% :-(

Sunday Night (14th) (for the stragglers)

  • 21:39-21:44 – “Incredibly Bright” – cloud cover 94% :-(

So… Friday night it is then, caveats about weather forecasting notwithstanding. If we can drag ourselves out of the bar for 5 minutes.


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