Acting and reacting in life science biotechnologies
Decidedly, producing and purifying a protein is a form of art.
Indeed, each recombinant protein (rec. protein) to be produced in vitro has specific characteristics and singularities that make its production and purification most definitely challenging. In this post, I’d like to share and review a few tips and basics for optimal design of experiment when producing functional recombinant proteins and working on protein purification.
Extract from poster “Maximizing Translation of Cas9 mRNA Therapeutics by Sequence Engineering and Chemical Modification”.
For transgenesis and therapeutic perspectives, vector-free delivery of the CAS9 for gene editing is highly preferable. It avoids the risk of hitting the genome due to the vector integration. Directly using CAS9 mRNA is a top option.
Furthermore, as a new promising class of therapeutic biologics, messenger RNA are the subject of numerous studies.
This year, Matthew Porteus’s and Trilink’s teams revealed key and practical results at the ASGC with a poster you can download below.
Notably, there is a large screen of the mRNA CAS9 modifications based on the indel formation. This interesting comparison includes also CAS9 RNP and reveals the high performance of the CleanCap U-depleted 5-moU CAS9 mRNA. We can also note that for the CleanCap CAS9 mRNA HPLC purification is not required.
“Maximizing Translation of Cas9 mRNA Therapeutics by Sequence Engineering and Chemical Modification”
Download your copy of the poster here.
Shen et al (Nature Methods, 2017) explore and identify synthetic interactions among 73 cancer-associated genes. To perform their loss of function screen they combined CAS9-expressing cell lines with a sgRNA library of high titer lentiviral particles. Most of these gene interactions were subsequently validated by drug treatment. [Read more…]
Messenger RNAs – a promising class of new therapeutic biologicsAs messenger RNAs (mRNAs) are easier to deliver into cells then plasmids or viral vectors, they are useful for non-dividing cells. Inversely to the vectors, they ensure genome integrity that is recommended for cell therapeutics. mRNA are also well adapted to transient expression as required for cellular reprogramming, gene editing, and vaccines.
You might like to take a look at this poster, presented by Trilink (renowned modified nucleic acid experts) at the Keystone symposia last March (Pattern Recognition Signaling: From Innate Immunity to Inflammatory Disease). They share results on how to optimize messenger RNA for therapeutic activity.
“Innate Immune focused approaches to maximize messenger RNA therapeutic activity”
Download your copy of the poster here.
Protein biochemistry brings together a vast and varied world of methods of protein production, purification and characterization. Once you have successfully achieved the production of your protein in a selected system, you need to think about the following steps. Indeed, the quest does not stop there! The next step is purification, during which you will try to isolate your protein of interest from the surrounding contaminants while keeping it soluble and active. Your ultimate aim is to keep your protein “happy” by choosing the perfect buffer. You will be faced with a bewildering array of choices leading you to ask yourself “where do I start…?”.
Keep calm – it’s easy… [Read more…]
Gel filtration (GF), also referred as Size Exclusion Chromatography (SEC), plays a key role in the high quality purification of enzymes, polysaccharides, nucleic acids, proteins and other biological macromolecules. Gel filtration is the simplest and mildest of all chromatography techniques to separate biomolecules on the basis of difference in size. Nevertheless, the list of available columns is quite awesome and the characteristics of each of them are very different. Also, each characteristic highly influences the quality of the final purified product with important consequences in downstream applications.
I’ve put together a simple guide to help you find your GF system, together with some tips and tricks based on my experience when producing recombinant proteins and antibodies for our clients involved in the early R&D stages through to the latest phases of the bio-production flow.
If you have any questions or if you need some help, leave a message below to get in touch with our lab experts!
…or how to make your protein attractive
Biomagnetic separation is a powerful lab tool in protein biochemistry which can be used in a wide range of applications including analysis tools (for protein solubility and protein-protein interaction studies, and for testing several purification buffers…), high throughput screening (HTS) and larger protein purification scale.
Indeed, it’s an easy, quick and efficient method to purify recombinant proteins and antibodies. Note that it also can be used to remove endotoxins and abundant protein contaminants from samples. Let’s take a look together at the large number of possibilities available and how we can help you to design the most adapted protocol for your study.
Drawing blood for DNA collection
DNA is a useful source of information for research and medicine. It starts by collection, following which it is deciphered by a wide range of analysis, from simple genotyping to deep whole genome sequencing.
The most popular way to collect DNA is drawing blood. Not very pleasant for the patient, as we have all noticed, and it also requires an experienced person, a nurse. The blood must be kept cold and DNA extraction should be done not long after the sampling. Still, it brings high yields of up to 30µg od DNA.
Buccal swab-based sampling
Alternatively, saliva sampling can be used to collection DNA. Sampling is painless, but it is still not an obvious choice especially with children. Yields are highly variable, and lower than with blood. Furthermore, kits are quite expensive.
Today, there is a sampling method to easily collect DNA, whilst also providing high yields. It is based on buccal sampling. I invite you to take a look at how it works and the benefits for your studies. [Read more…]
We need to find biomarkers for prognostic, diagnostic and personalised treatment development. Notably to fight cancers that affect tissues. Since biopsies are invasive, it’s better to look for biomarkers in body fluids. Indeed, a simple blood sample becomes a kind of ‘liquid biopsy’ to reveal tissues affections. For 13 years, increasing interest has been shown for miRNA as biomarkers and it will last for sure. The 2 main reasons are that they are major regulators of cell processes and they are released from tissues into the blood. They are major biomarker candidates in serum and plasma. Thus, these circulating miRNA (cmiRNA) are the best hope for modern medicine. Still, a lot of research has to be done to determine the specific signature for each pathology, and also depending on the patient background. Obviously, cmiRNA profiling is a key step and requires sensitive and reproducible method. Sequencing, qRT-PCR, several kind of microarrays… Let’s explore together what the best approach could be. [Read more…]
Removal of contaminating bacterial chromosomal DNA in plasmid preparations (but also cosmid, BAC…) is crucial in today’s precise molecular biology experiments. In this post, a cost-effective but powerful enzyme is described for molecular biologists looking for genomic DNA-free preps.