Preparing recombinant protein: Tips to improve growth rate and yield

I have been in the distinctive business of making proteins, looking at proteins, or finding proteins that have special properties. I mostly try to make them myself, and over the years I’ve had lots of success—and many failures. It’s part of research. We all know, from DNA we get transcribed RNA, which is translated into a strand of amino acids, which by forces of physics fold into a functional protein—sometimes with some help from chaperones. However, it does not always work. Even in a living organism with high adaptability, success isn’t guaranteed. Aggregation is a waste of energy for the organism, and now it needs time and energy to recycle the materials.

In industry, about 40 percent of all pharmaceutical proteins are made with recombinant protein production in bacterial strains, while the remainder is mostly by use of mammalian cells. In the lab, we use E. coli the most, simply because many tools are available—from cloning vectors to E. coli strains from DNA plasmid production, to cloning kits, simple transformation procedures, and plasmids designed for high yields of recombinant protein production. Many different media and fermentation procedures are designed by scientists and manufactures to increase yields of correctly folded protein. It all seems so simple, but it’s not.

Even a condensed, incomplete list of tools illustrates that we need to make many choices to find the right parameters. For now, I will skip most tools and focus only on protein yield. In the lab, we need just a little bit of protein, but as a bioprocess engineer, I always need to consider the choice of the expression system on the available means of the laboratory or production facility, modification needs of the protein produced, and compatibility of the gene control system with the bioprocess for production later on.

In terms of protein yield, either as yield per cell or total yield, it is important to remember that the product formation must be determined experimentally, and may either be growth associated or non-growth associated. The specific product rate formation, qp (kg kg h-1), is given by qp = qs Yp/s, where qs (kg kg-1 h-1) is the specific substrate rate and Yp/s (kg kg-1) is the yield coefficient. The specific product rate formation can also be expressed in terms of growth association or non-growth association by means of the Luedeking-Piret model: qp = αµ + β. Here, µ is the specific growth rate, β = 0 gives a complete growth association, and α = 0 gives a complete non-growth association. Protein product formation is mainly growth associated, at least in wild type cells. Low growth rate, for example in E. coli, may hinder product formation due to the maintenance cost of the cell, while fast product formations may hinder correct folding of proteins. These problems are related to protein production in living cells, but less so in cell-free expression systems.

Important parameters to consider are again plenty. Are we using a simple shake flask or do we have access to a fermenter? In either case, we still have to choose if we grow the cells in a batch phase or use a fed-batch culture, i.e. if we grow the cells following sigmoidal growth curve or a linear growth curve with a fixed number for µ. Batch phase in shake flasks is very common because it is cheap, however it has its own unique problems. For example, using LB medium would be a poor choice for long expressions. Cell densities are often low (OD600 < 6) due to acidification of the medium, while the pH in Terrific Broth (TB) can go up above a pH of 8.5, indicating high ammonia production due to utilization of amino acids as carbon source. The growth rate of E. coli is dramatically reduced under a pH of 5, and thus will not produce recombinant protein—it will start recycling it for its own maintenance. The utilization of amino acids as a carbon source will also lower the amount of recombinant protein.

The design of the shake flask (i.e. baffled or round), the size of the opening, and the choice of the cover are equally important, since E. coli can only grow and make recombinant protein if the oxygen transfer rate is as high as possible. Switching from an aluminium cover to very porous paper can increase the final biomass manyfold.

In conclusion, aside from the choice of DNA vector, E. coli expression strain, growth temperature, inducing agent and amount, and co-expression of chaperones, parameters to control the growth rate are equally important. Growth media, a good oxygen transfer, and a good buffering system have proven in my work to be important elements to obtain high yields of correctly folded protein.

(c) Marco Casteleijn. 2017

 

“Preparing recombinant protein: Tips to improve growth rate and yield” was published originally on the 23rd of May, 2017, in the ‘The Q’ . Republished with permission.

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Embrace the other tribe

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“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

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“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!

 

 

NaNoWriMo distractions

We all have them. Sunday evening. Perfect evening to write. Epic words: “I will do 5000 words tonight, get a good start.” Then one of your favourite books is moulded into a concept short movie by the author himself. No biggie, it is a short move. Oh wait, a Q & A with the author on Facebook, Oh what? Hour 2.1, hour 3? Two in the morning, work the next day. Word count: 0. Gain: awesome sauce Q&A and short movie.

See it here, get distracted!

Post-Human from Post-Human Media on Vimeo.

The Mondays – NaNoWriMo bugs

crestNo, do not worry! No need to send out some search dogs through the rubble of my busy life to find me. I am still alive and writing. If you had time to check my blog “The Cave”, link above, you would have seen that. The drabble war is in full swing, some haikus fell out of my head, and work as been full with writing.

Also I started a writing course, here: Fiction writing course and I am doing some pedagogy studies for my work. So loads of writing, now I have time to catch up.

Oh wait, is it November 2nd? Dang, sorry NaNoWriMo is happening again. Last time I just made it halfway, writing was too hard and it was to busy to get about 3000 words a day in before work started. Now I have a bit more time in the evening this week, so lets see if I can make a head start so <ahum> coast through the final week. Solid plan! But we will see.

How many of you have no time to respond here because you have the NaNoWriMo bug? I will try this month to have sporadic and most likely frantic, peer-pressured, “need to write more words”-panic like paragraphs thrown at this blog, more for my moral support than your entertainment. I apologise beforehand.

With that I dive into my coffee, go to the lab, read an article for my pedagogy studies, have a meeting, pick up the kids, make dinner, put them to bed, make/drink/make/drink coffee and try to get 4000-6000 words down this evening.

Wish me luck!