Proteins on Michael’s Domain

Protein Drug Discovery & Development

Wed, 09 Sep 2020 00:00:00 +0000

Yesterday, I had my first Zoom lecture about protein drug discovery and development. I hope that the students did get at least something out of it. My goal is to cc-license the complete presentation but there are still a few images that I need to replace. A first attempt is rarely a great performance, and we had our fair share of technical problems. My headset failed for the first time since I bought it at the beginning of the Covid-19 pandemic. And the Zoom polling functionality disappeared before the students had any chance to use it and we did not manage to bring it up again.Here is the link to the live Google Slides: https://mjlab.fi/pddd . These will keep changing (= improving). Below you find the PDF snapshot of the presentation from the actual lecture day. The whole presentation is CC-licensed. So feel free to reuse it. All source files (mostly in Inkscape SVG format) are also available from here: https://mjlab.fi/pddd-files . I have done all the SVG files in Inkscape , but you can also open them in any browser or edit them in Adobe Illustrator. If you want to edit or extract images, the easiest is perhaps to download the presentation in Microsoft PowerPoint, LibreOffice Impress, or PDF format. I still have not figured out how to share the Google Slides presentation without making the original editable for everyone (after all, I need some control over the content of my lectures).Be aware, that at this moment, the presentation still contains eight images on Fair Use or with unknown licensing terms (even though the legal concept of fair use does not really exist outside the USA). All image sources (excluding my own images) are listed in the file README.txt with their respective licenses and source URLs. Some images are so old that I was not anymore able to locate their original URLs. Hence I am not sure about their licensing terms. I will replace these over the next few weeks when I manage to get hold of CC-licensed or public domain equivalents.

Purification and Characterization of Recombinant Proteins

Mon, 20 Mar 2017 00:00:00 +0000

We are organizing (again) a practical hands-on protein purification course from December 4th to 20th. We maximally can accommodate 16 participants, which will form groups of 2 to 4 participants. Each group needs 3 full days to go thru the practical exercises, but day 3 of the course will be overlapping with day 1 of the next group. Venue is Biomedicum Helsinki, rooms A516a1 (where the machinery is) and B318a/b (our lab). We will purify a protein (VEGF receptor 3) using a two-step protocol (affinity chromatography + gel filtration) on the the Äkta Avant FPLC device. On the third course day we’ll assay its interaction with its ligand (VEGF-C) on the ITC (isothermal calorimetry) device. We have given a similar course in 2015 ( http://www.helisci.fi/hbgs/FPLC2015/) . This new course ( https://courses.helsinki.fi/en/DPBM-135/120171139 ) will have a similar structure, but we expand it by one day to analyze protein interactions making use of the new isothermal calorimetry device of our B3P core facility. The documentation and results will be also available from here . Since we can run maximally two samples at a time (we have “only” two FPLC machines), we will have to split the participants into groups (of 2-4 students/group) and repeat the 3-day course several times depending on the number of participants.

FPLC Protein purification course

Mon, 04 Jan 2016 00:00:00 +0000

Eight postgraduate students registered for the FPLC protein purification course , which took place in December. If I learned anything, than that protein purification during a course should be done ALWAYS with a protein and a protocol, that has been used before successfully MANY times. Student-provided proteins are a great source for learning, but the time restraints of a course format did not allow us to finish the purification of these proteins during the course.Technical Problems with the new Äkta Avant 25In addition, the Äkta Avant 25 , that we newly purchased from GE Healthcare this summer, broke TWICE during the course. First the controlling computer broke (RAID failure). HP delivered the replacement drive within 24 hours and after the RAID had rebuilt itself, we could continue the course. However, during the first run after this incident, the Äkta ran into an overpressure problem. We identified a faulty flow restrictor as the cause, but we did not want to continue as we have had severe problems with air bubbles in previous runs. Therefore we performed the runs on the old Äkta Explorer 100 . GE Healthcare quickly had their service engineer check out the system, but since he did not have a spare with him, we had to wait until Monday 14.12. until the Äkta Avant was again fully functional.What we purified: soluble VEGFR-3 (VEGFR-3/Fc) and HepsinWe did purify soluble human VEGF receptor-3 (the first three domains of its extracellular domain connected to the constant Fc part of human IgG). This is a purification that we have done many times. It is equivalent to the purification of antibodies using Protein A sepharose.We had prepared in advance conditioned cell culture medium. We produce most of our proteins in insect cells (mostly Drosophila S2) and the VEGFR-3/Fc had been secreted by the S2 cells into the medium after induction of the metallothionein promoter with 1 mM Cu2+ for about 4.5 days. The preparation of the medium for purification consists only of 1) getting rid of the cells by centrifugation and 2) filtration to remove precipitates and other small particles that might clog the column. There is no need to adjust the pH.Rapid neutralization after low pH elution IS IMPORTANTWe ran the medium over a disposable 5-ml HiTrap recombinant Protein A column over night at about 1ml/min and eluted with a low pH buffer. The eluted 2-ml fractions were immediately neutralized with 400µl 1M Tris pH 8.5. Here we made a small mistake during one of the two purifications. GE Healthcare had not released the casettes for the 5-ml-collection tubes (they still have not done so even though they did promise them already for September 2015) and we used 15-ml Falcon tubes to collect 2-ml fractions, into which we had pre-aliqotted 400 µl of the neutralization solution. However, the mixing in these tubes was not efficient and the prolonged exposure to low pH resulted in a partial damage to our protein. This can be seen when comparing the size exclusion chromatograms of Group 2 versus Group 4: For Group 4 the first peak (aggregated protein) is much larger and more heterogenous compared to the same peak for Group 2.In fact, when VEGFR-3/Fc is eluted by low pH from protein A columns, it always precipitates at higher concentrations soon after elution, but dissolves again upon neutralization. This did not happen in the fractions 5.A.3 and 5.A.4 (Group 4) due to the inefficient mixing of elutate and neutralization buffer in the 15-ml-Falcon tube (the fraction size of 2 ml was probably to blame as well; 1 ml would have been better). During the run for Group 2, we removed the tubes immediately after the run had ended and thereby mixed the buffers, while for Group 4 the run finished during night time and the eluate remained largely unmixed until the morning.Alternatively, we could have eluted with a highly concentrated chaotropic salt at near-neutral pH (which we’ll do next time in case we have an automated run where the elution happens in the middle of the night). Pierce offers a (proprietary) “gentle” elution buffer of pH 6.6. I don’t know the Pierce buffer composiiton, but it is a highly concentrated solution of some chaotropic salt. 3M potassium/sodium thiocyanate or 4M magnesium chloride in buffered solutions around pH 7 are frequently used chaotropic salts for this purpose.HepsinThe student-provided proteins were challenging. First, their concentrations in the starting material was very low. While we can see clearly the protein when the VEGFR-3/Fc conditioned medium is run on a PAGE gel and stained with Coomassie, no such band is visible for the Hepsin. In addition, it appeared that a significant fraction of the protein seems not to contain the histag (anymore) and therefore is not captured with the first purification step. The fraction of Hepsin-H6 that does bind to the Ni2+ sepharose elutes already at an imidazole concentration of 20 mM, which makes washing the column challenging. The Hepsin with the longer histag (H10) survives the 20 mM imidazole wash, but it suffers also from low expression levels.Below are the chromatograms of the individual runs and the annoteded images of the Comassie-stained PAGE gels. The detailed protocol for the operation of the Äkta Avant 25 is still under preparation…

Stripping

Fri, 01 Nov 2013 00:00:00 +0000

Reprobing membranes with with a different antibody is a very common task in the lab. Various protocols exist to strip membranes and the classic method is the one that uses SDS, β-mercaptoethanol and heating. I used to do it that way, but it’s a smelly business, because β-mercaptoethanol smells like rotten eggs. Then suddenly everybody in the lab started to use the Re-Blot Plus Solution from Millipore and so did I. Until I realized by chicking the Material Safety Data Sheet that they sell cheap chemicals for a premium price. Now I make the stripping buffer myself. My 10x solution has the following composition:

Äkta Explorer FPLC core facility

Wed, 26 Jan 2011 00:00:00 +0000

Nowadays I spend much of my time at work on the purification of proteins which regulate angiogenesis and lymphangiogenesis . Therefore, I am taking care of the necessary machinery that the Molecular Cancer Biology Research Program owns: the fast protein liquid chromatography (FPLC) machine. Prof. Jorma Keski-Oja acquired about 15 years ago one of the first (and in 2010 discontinued) Äkta Explorer machines from Swedish producer Pharmacia (who merged in 1997 with Amersham to become Amersham Pharmacia Biotech, who was in turn bought by GE Healthcare in 2004). In the year 2000 the equipment moved from the Haartman Institute into the Biomedicum Helsinki . While I have maintained web pages for this piece of research infrastructure for the last five years (including online reservation and data backup), they were only available from inside the Helsinki University network.Now, I managed to have web pages about the Äkta Explorer FPLC core facility added to the web site of the Medical Faculty . At the moment, we are adding the WAVE cell culture system to our facility to be able to produce large amounts of cells/cell culture supernatant (up to 25 liters of bacterial, insect or mammalian cell culture) for protein production.

HBGS course

Mon, 10 May 2010 00:00:00 +0000

All documents related the the HBGS course Tags in protein expression, detection and purification. Most documents are available in both PDF and OpenOffice format. Feel free to repurpose the documents. They are licensed under the Creative Commons Attribution-Noncommercial-Share Alike 1.0 License .

Recombinant proteins

Fri, 25 Sep 2009 00:00:00 +0000

A dynamically updated list of proteins used to be here, but I shut down the communication to our lab’s database server due to security concerns.

Storage and Handling of Proteins

Wed, 22 Oct 2008 00:00:00 +0000