Finding the best proteins

There is a beautiful lesson to be learned from the Gossamer Condor[1]where the team of Paul MacCready succeeded where others have failed: to keep a, by man-powered aircraft in the air long enough to draw the infinity sign between two markers one half mile apart, starting and ending the course at least three meters above the ground. They did so on the 23rdof August, 1977.

Paul MacCready sought out to do “do more with less”, however the lesson I learned from this engineering achievement was: “fail fast to succeed faster”. Where other big companies, like Boeing failed, they succeeded. Paul MacCready’s team used cheap and simple materials and techniques so they could build 2 or 3 different configurations a day. They could sample what works and, more importantly, what does not work in a rapid way. Thus, when I start a lecture on making proteins, people look at first confused when I ask them if they have heard of the Kramer prize and its first winner.

So, what does it all have to with making and finding proteins? The process to produce recombinant proteins by use of living cells, currently the state-of-the art especially for the pharmaceutical industry[2], is a lengthy one. For some recombinantly produced antibodies there could be over 1000 different steps. At each of the steps the protein is exposed to different environments, which may interfere with its natural state.

In nature proteins are important, because within the cell and the body they provide structure, perform chemical reactions and pass on information. For example, collagen in connective tissue, α-amylase breaks down long-chain carbohydrates during digestion, or dopamine receptors in the brain. Within cells, many processes are put in place to make the right proteins at the right time, while folding them correctly into their native and active 3D-structures. However, by interfering with these natural processes and tricking cells to make more of the protein we would like to have, for example recombinantly produced human insulin[3], cells may not fold the protein of interest correctly. Misfolded proteins are not active, can induce adverse effects if administered as drugs, and if misfolded protein stick together they form insoluble complexes called aggregates, with no functionality and even greater adverse effects.

Therefore, when we start making small changes to proteins to see if they have an improved functionality compared to its original design, we need to test for many parameters, but functionality and protein stability are crucial. Before we make changes to our proteins, which may have the potential to become safe and functional drugs, we need to make sure our processes are designed well and that our means to evaluate the protein is sound. In the current landscape of protein preparation, the use of cells (i.e. biotechnology) is employed the most. However, one can argue that it takes long to make some protein with many different variations in cells: from weeks to months. How can we fail “faster to succeed faster” to evaluate more proteins?

One disruptive technology which has started to show its promise in an industrial setting[4]is the use of the cellular machinery that makes the proteins without its envelope. Proteins are made by other proteins, enzymes, who transcribe DNA into RNA and translate RNA into an amino acid sequence, a strand of the building blocks of a protein, that then folds into its 3D-structure. This technology is referred to as in-vitro transcription translation (IVTT) or cell free protein synthesis (CFPS)[5]. In short, a cell is cracked open like an egg, and its content is split in a soluble fraction and an in-soluble fraction. Part of the soluble fraction contains all the enzymes needed to make proteins, this part is isolated, often concentrated and then mixed with amino-acids, enzymes to provide energy, and other components needed to synthesize protein. RNA induces the process of protein synthesis, which is often transcribe from DNA within the mixture. Protein production in such an open system is short. Depending on the lysate used the expression can be between 1 – 16 hours, while the process is not designed to keep the cells alive and dividing at a set rate, but on protein synthesis.

Another powerful feature is that many different cell lysates can be used, for example rabbit reticulocyte, E. coli, mammalian cells, such as CHO or HeLa cells, L. tarentolae,tobacco plants, and many others.

The promise of recombinant protein preparation from GMO animals failed in the past; one main reason being the turn-over rate between preparation, purification and testing was very long. One can argue that the turn-over rates of cells are too long as well. In a market where the life-cycles of pharmaceutical products are getting shorter, while the overall volume is growing, more safe and well-designed protein-based drugs are needed. In this landscape CFPS has the potential to act as a valuable tool in the same way mylar foil was crucial for the Gossamer Condor’s success.

 

Marco G. Casteleijn © 2018

Publish first on BrighOwl blog: https://brightowl.pro/blogs/2018/06/finding-the-best-proteins/


[1]https://en.wikipedia.org/wiki/MacCready_Gossamer_Condor#cite_note-1

[2]Casteleijn M.G. and Richardson D. (2014) Engineering Cells and Proteins – creating pharmaceuticals. European Pharmaceutical Review, 19(4): 12-19

[3]https://www.dnalc.org/view/15255-Producing-human-insulin-using-recombinant-DNA-Walter-Gilbert.html

[4]https://en.wikipedia.org/wiki/Sutro_Biopharma

[5]Marco G. Casteleijn, Arto Urtti, Sanjay Sarkhel. 2013. Expression without boundaries: Cell-free protein synthesis in pharmaceutical research. Int J Pharmaceut, 440 (1): 39-47

 

Embrace the other tribe

eu-broken1

“I do solemnly swear (or affirm) that I will faithfully execute the office of President of the United States, and will to the best of my ability, preserve, protect and defend the Constitution of the United States.”

In our lifetime we have seen many upheavals in various countries around the world and many horrible atrocities committed against humanity. We are more and more connected to the flow of that news, yet more and more desensitised at the same time. Our sense of global humanity is an interesting construct, since we are hard-wired to live in smaller tribes and be empathic to familiar people. We can only really store a certain amount of people in our brain (1), beyond that realm we encounter “them” as another tribe.

While researching the tribe versus globalisation, I came across this opinion piece by  (2), and find truth in these lines regarding current events:

Important traumatic events have plunged people all over the globe into an identity crisis. Their response is tribalisation: going back to the tribe they know best.

and while this may give solace to ‘why is this happening?’, it does not give us a sense of security and stability.

This blog is mostly about writing and sharing our cooking project. Yet our collective identity crisis affects also me and our family. Our daughter flipping of the president elect during his press conference is her response to our insecurity about the future. I would like to stress here to be honest: firstly our family’s future, then the broader spread of humanity.

As a researcher I am depended on a favourable political landscape towards (my kind of) science to obtain funding. Without funding I will not be employed, since my salaries are depended on external funding. The EU has been a driving power in providing funding strategies beyond my current tribe in Finland. The political landscape in Finland to fund science has always been good, but resent years this is on the decline (3). This trend we also see in the USA, a frontrunner for scientific endeavours for many decades.

Now it seems we enter a new phase, something I am writing about in my very slowly progressing SciFi novel-thingy. At this rate it seems that it may even be fiction before I can get it published. A new world power is emerging, with another one waiting in the wings. Of course I am referring to China and India, the Dragon and the Elephant. Two countries that look at each other with suspicion, but with a clear understanding of the power of investments in new technologies and creating legislations that open a freedom of operation within their own tribes.

But a citizen of the World I am wary of the near future. The USA has through previous technical and scientific developments and acquisitions build up a wealth and power that influences the rest of the population with a literary unbalanced force. In the last election for example about 40% of the USA (which holds only 5% of the world population) decided to put a person in place with very limited views of the World. We can all argue about the values that that Tribe he represents and we can all argue about it as well, but for our global perspective, it is a step in the wrong direction. Scientific facts do not care about public opinion. Gravitational force is still here wether you deny it or not. Same about evolution, climate change, gender issues, GMOs, vaccinations, and the power of innovation.

Our future may be uncertain, I think our ancestors shared our concerns. They looked at the stars, they conversed with the bones of their ancestors, they invented religion to strengthen the bonds of the tribe in order to survive better. Will a collective connection directly to everybody else aid us? Do we need to enhance our brain (via science mind you) so that Dunbar’s number becomes infinite? Will an encounter with an off-Worldly tribe spark the birth of the Human Tribe? Many stories have been written about this.

So my take on it is that humanity is a collection of stories, and that the narrative of those stories is healing. Some people like poetry, others read horror stories, but at the end of the day we can tell each other stories about that one poem of inspiration or the scary bits of a horror story without forcing the other to read it. To find common ground, to revel in being human, to talk and listen and embrace the other tribe…

 


Sources:

(1) https://en.wikipedia.org/wiki/Dunbar’s_number

(2) https://euobserver.com/opinion/131413

(3) https://www.timeshighereducation.com/news/finland-funding-cuts-catastrophe-research

 

IMAGE CREDITS: CC / Flickr – Theophilos Papadopoulos

Kitchen lab – Episode #2 Yeah!

Yes I am writing. Poetry, grants, and another grant (and even a third one), and a lonely short story. I will get to the book thingy soon. But I wanted to share our second episode of Kitchen lab.

Here our family will talk about how to make a tasty bbq also healthy. Please join us on our youtube channel and give it a thumbs up if you like it, comments are more than welcome. Of course I would love some science questions as well!

Kitchen lab update

In our scientific endeavours at the University we serve three pillars:

  1. Research and pushing that little bubble of knowledge further into the unknown.
  2. Teach students.
  3. Serve society and open the dialogue with the public.

Carl Segan understood that very well and in the first rendition of the popular TV show “the COSMOS” and then the update by Neil Degrass Tyson brought some wonder to the people homes. Science, some teaching and entertainment.

Our little TV show Kitchlab is trying to do the same, and trust me I am not comparing me to the two well know scientist mentioned above, but hey we can all dream.

So after our first episode, soon our second, but just because it is so much fun here is our intro. Freshly pressed.

Our next episode is about how to do a tasty and health bbq. See you soon on our Youtube channel!

 

 

The Mondays

IRES_RNAfold
“It looks like my story line is going… where?”

It is Monday. I am writing, yes the curse of November may be lifted yet. So what is going on? In the short fiction course I am taking we are required to (finally) write a short story. I am working on it. In the course I am a week behind, it has been busy, but these is writing going on.

This week we are finally looking at editing and I hope to get some insight into that, since it is still an elusive skill to me. The hyperlink story, brought to you by me and Ville where we deliver a story in 100 word chunks (or drabbles) via a challenged title has taken time, since each story is so packed with information or needs to deliver very precise. Also it is a hyperlink story, so each drabble connects to some others, while it may not be clear. This could be strutted easily if there was a plan prior. Yet we do not have. So structure is only applied while and after writing. Indexing took some time, then side drabbles run in parallel now to support other stories. It is becoming more and more fun, but if anybody would ever get something out of it I do not know.

So if you ever have time I invite you to read it if you want (you can find it all in The Cave (see link on top); some discussion or feed-back on hyperlink stories is something I am looking for.

Did you ever write in that format? Is it a rewarding forum? Or is a classic tale with a real beginning, middle and end better?

NaNoWriALotLess

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I know it has been a while to vent ideas via the blinking cursor into the white void of blogism. It is so.

I, again, tried to believe I could finish my first draft of my novel by entering NaNoWriMo. I even had time for it, yet I wrote less than I ever did (at least for this novel).

Instead, “Wordexpanding”, a hyperlink novel written together with my dear friend and fellow poet Ville at the Cave (the blog where I hide a lot of my writing) & Chameleon’s stories has flown away and is going fast. I really enjoy these 100 word stories and poems that we are trying to forge, without any discussions, into a larger story. They may take some research and the dense messages of 100 words is reflected in the time it takes to write them. There is no NaNoWriMo robot-high speed traffic of characters spawn via the blinking cursor into the cloud-memory mind.

So that pressure was to restrictive. Now I will write again for “The Death of an Emperor”, I will try week by week, and I will deepen the characters better (thanks to a great on-line course I am taking at the same time). Also for the course I need to write, though its a different kind of writing.

Finally, then soon I will be reading again. The winter is upon us, the cozy warm bus awaits. Humming to bring me 2 hours of reading a day. Neil Stepheson latest book is lurking at me from my bookshelf for far to long.

So I unfurl myself in the dark day today, awakening to the possibilities and so I wrote once again today. NaNoWriNoMore, but #Iamwriting!

 

 

“Shizzle, Inc” by Ana Spoke on tour

Available on Amazon
Available on Amazon.com

Today is Tuesday, and instead of letting you know how my writing is going, I want to introduce you this week to a talented young writer who just published her first book. It is getting great reviews and if you love comedy (and who does not love to laugh) please go check it out on Amazon.

Ana is on tour, well not in person, like most of us she has a day job, so a virtual tour it is. I was curious how she did it, so she is letting us know how this writer thingy is working out. Please also visit her blog to get more behind the scenes of the glamorous life of an author (spoiler: it is not like the Hollywood kind-a-glamerous). I started this blog and my ‘book-thingy’ very recent, lets just say she is way ahead of me. Enjoy:


Marco: Awesome you wrote a book. Is this your first one and what is it about.

Ana: Shizzle, Inc is the first book I’ve finished. There were lots more started that died on the vine, and perhaps for a good reason.

Shizzle, Inc is a comedy novel told from the perspective of Isa Maxwell, a recent community college graduate, who is losing her grip on life in more ways than one. Isa believes that the only solution to her financial and relationship woes is to become famous, like many nobodies before her have done on the reality TV. Luckily, Isa lands on her bottom in a billionaire’s office and, as a result, lands a job as his protégé. Just as things start looking up, they get more complicated. We are talking life and death, and maybe even slow torture kind of problematical.

I’ve had a lot of fun writing this novel, and I hope readers will have fun reading it. It wasn’t easy, with a full-time job to manage, but I will always cherish the evenings spent cracking myself up over my laptop.

Marco: Why was it important for you to write this story and even more important why did you want it out there in the ‘reader verse’?

Ana: I love making people laugh, probably more than anything. I even do it in my “serious” job, using humor to engage people in boring topics, illustrate a point, or even win over difficult customers. I’ve made my CEO laugh out loud in a presentation about financial modelling – I can’t help it!

I guess writing this novel has been the ultimate test of my funny bone – do I have enough oomph for a whole story, or should I go back to cracking up my colleagues and management? It is also a test of whether I have what it takes to reinvent myself as an author. As much as I love working for a bureaucratic giant, aghm, it doesn’t compare to the dream of writing full-time. Plus, I’m sure there’s an element of approval- and attention-seeking in there, too.

Marco: I just blogged about iteration and polishing you lines and the whole story. Are you a masochistic editor, do you read to story out loud, what are you methods?

Ana: I am an obsessive-compulsive editor. I can’t stop editing even when I’m writing the first, “vomit”, draft. I’ve spent probably a year editing and re-editing the story, and, having recently discovered bits of the very first draft, can say it was worth it. I have not tried reading it out loud, but I have printed and massacred endless copies. I have sent my drafts to several beta-readers. I have hired a structural editor; a copy editor, and a proofreader. I have used Grammarly to catch some of the very last mistakes. And then, after all that, I went through it one more time. Just in case.

Writing and publishing Shizzle, Inc has been a trip. To start with, it takes balls to say to yourself, “Hey, methinks I might write something. How ‘bout a novel?” It then takes constant effort not to: get distracted; start another novel; give up altogether; hang on to your darlings; get defensive and precious about every joke; get depressed and moody; question “why do it anyway, when I could be watching TV or going outside”; resist temptation to “just post it on Amazon and get it over with”; get so scared of finishing that you keep editing it for another decade, and so on. It is now a daily head trip when I look at it on Amazon and think, there it is. It didn’t exist, and now it does, and I’m the one responsible. And people like it. All of the six reviewers so far really, really like it!

I love every aspect of this new project/passion/hobby/budding career. I’ve loved answering questions on this blog tour, and I would like to thank you, Marco, for hosting me. Now it’s your turn – here are some curve balls for you:

  1. I see that you are a postdoctoral student, working on pharmaceutical protein research. How has this “other life” affected your writing?
  2. What has been your most significant writing achievement to date? 
  3. What are your plans for the next project?

Marco: I will get back to you about that, this time is al about you. 

The Fridays – the Introduction some more…

"certain angle, certain light"
“certain angle, certain light”

I know it has been a while, sorry about that. This application had to out, and a few nights in a row writing until 3 in the morning fries your brain a bit. At least it fried mine.

I promised to continue about the introduction, and maybe a funnel. Well that is still what the introduction should do. It sets the problem, and places that problem in a larger setting from the start. So instead of saying that the coffee was so expensive in the store, we rather talk about the rain problems in Brazil, which lead to bad harvest, which drove the prices up. You get the point.

Then slowly, or if you have a limited amount of words, you zoom in to the last paragraph where you summarise what you are about the talk about in more detail.

The aim of our study was to investigate the underlying paradigm shift in coffee prices world wide, and in Scandinavian roasters in particular. We found a strong correlation between rainfall and the quality of bean used in Sweden, however in Finland lack of compromise on the use of, and availability of lesser beans drove the prices up by 12%.

You see in this made up scenario the end of the funnel.

Now you may wonder why the picture of the angle. Well I was asked how to find your own voice in a scientific text. Often there is little room for a personal opinion, so how to stress your view in a review of the literature and while mapping out the problem? It seems difficult if you want to be comprehensive over selective.

In writing about the problem and during the selection of literature to represent there is always a key paper that gives you insight or inspiration or both. For example, in my doctoral dissertation I seemed at first all over the map. I did some protein engineering, then we ran out of funding, then we worked on a different enzyme I isolated from a hyper-thermophile, and I improved some methods to make more protein. Finally, some computation modelling was done on some of our proteins as well. What as mess right?

Then I found a paper, and its message can be summarised in three concepts: you either make it, find it, or improve the process. Eureka, my patchwork was sown together: I was engineering a protein: making it. We delved in the data-bases to find the enzyme I isolated: finding it. I improved methods (processes) to improve things. I put that candy shell around the rest and it worked.

The story, the literature, the funnel, all introduction (I ended up splitting the stories up in 4 chapters) were all the same. I just found a better angle, I just found my own voice…