What are the best ways to troubleshoot protein purification issues in biotechnology research?

It is essential to ensure your recombinant protein is expressed in the subcellular compartment you anticipated and it has not adversely affected the expression system's viability. If it has, address that first. Optimization of the purification starts at construct design. Use the affinity tags and cleavage sites, which suit the systems, assays or applications you intend to utilize. Purify quickly and efficiently. Deploy well thought-out workflows, which allow for objective decision making. Using the least possible steps during purification will get you to the solution faster.

Though the biotechnology field advanced a lot over the years, still there is no one shot magical soup to devise a perfect expression experiment for a given protein/peptide of interest. I strongly believe at this juncture utilising insilico tools and accumulated large data experience will better guide us to minimize the efforts. To emphasize that the expression system is very crucial to achieve whatever the target biomolecules we are looking to purify. At the same time it is also a trade off to selectively choose your expression host down the line #what is your product/protein/peptide specified for? #also have to keep in mind the cost of goods in large scale production #quality and yields of the product

In my experience working in both academic research and industrial settings, troubleshooting protein purification starts with verifying the expression system to ensure the recombinant protein is expressed at the correct level, size, and location using techniques like SDS-PAGE, Western blot, or ELISA. It is important to optimize conditions such as the host strain, induction method, and growth parameters. In downstream processing, refining methods like affinity and ion exchange chromatography, and using systems like AKTA tangential flow filtration, can significantly improve yield and purity, as I experienced during biosimilar production at Dr. Reddy's. Additionally, addressing issues like protein aggregation, adjusting buffer conditions,

I would also include codon optimization in your workflow when developing a recombinant expression system. The same gene cannot work for all the same expression systems if there are rare codons in the given organism. For some reason, I find that codon optimization is not a commonplace practice but has become proprietary to certain businesses.

Things to consider 1. Localization of expressed protein eg. E.coli it could be Cytosolic, Inclusion body and Periplasmic space. This would determine the method used for extraction which in turn would the affect the protein quality 2. Cytosolic: you need to break the cell wall and that would release proteases which could potentially degrade your protein thus protease inhibitor addition becomes important for stability 3. Periplasmic: you dont need complete lysis thus softer methods like osmotic shock are used to avoid breaking the cell membrane and release cytosolic fractions increasing the impurities to tackle with. 4. Inclusion bodies: have the advantage of protection from proteases and lower contaminants, but the challenge is re-folding

Depending on regulatory requirements and downstream applications, this could actually be the trickiest step. It is best to make sure you have the regulatory leeway to use certain additives if they are animal-derived and if the end-product is to be used in human research. Furthermore, lysis and any type of mixing or filtering will shear the protein. This is the main cause of aggregation and low production yields. It is important to be mindful that protein sequences will be clipped at the ends with every second of mechanical shearing, even stirring. Covalent bonds are not that strong. One could re-engineer "end cap" sequences into the polypeptide, akin to telomerases for DNA. Otherwise, proteins are generally purified with 99% aggregated.

For very high purity, consider a multi-step purification scheme. Each step should be optimized to provide the best balance between yield and purity. For some applications, immobilizing the target protein directly onto a solid support can be effective. This can simplify purification and is particularly useful for proteins used in immobilized formats. Including protease inhibitors in your lysis and purification buffers can prevent degradation of the target protein. Tangential flow filtration (TFF) can be a highly efficient method for concentrating and desalting protein solutions, particularly for large volumes. For preserving protein activity, it's crucial to perform chromatography under non-denaturing conditions.

It is actually quite feasible to get highly pure protein from just one-step purification with enough gradient optimization. What is difficult to scale at this level is certainly the column size. Increasing column volumes just extends the the whole process to the point of potentially jeopardizing the quality of the protein due to extended periods of exposure to contaminants and likely proteases. Proteins, depending on the sequence of course, can be incredibly fragile and sensitive to even back pressure. I wouldn't put it past them to degrade or unfold by just looking at them the wrong way, hahah. Maybe I just work with very finicky protein targets, but that's the way it goes sometimes.

The endeavor of protein expression and purification is a considerable investment of 2-3 weeks. In order to limit time waste, it is important to check expression and yield at each step. This can be use of SDS-Page, Western Blotting against an affinity tag, or quantification protocols like DC Protein Assay before/after initial culture, separation of intended cell fraction, purification, and sample concentration. An informed production process is a replicable one.

Protein quantification can be actually quite challenging in my experience. For FDA-regulated pharma ingredients, proteins have to be quantified with an assay designed to be highly specific/selective, sensitive, rigorous, robust, etc. It seems like the only solutions for high-quality analysis of protein purity, identity, and quantity requires unconventional techniques such as CE-SDS-LIF or HPLC-SEC-LIF.

In my experience, there are very minute details that can harm your protein purification. Such as the most underrated point is the quality of the resin. Once in my work, I cared for everything from buffer conditions, temperature, pH, quantity of expressed protein, etc, but I still faced many issues during elution. After days of troubleshooting, the problem was with the quality of the resin. The resin quality was degraded due to the multiple uses and its chemistry was changed. Due to this, the protein was not binding properly and also getting lots of impurities. So, please keep checking your resin and take care of all the other factors which are mentioned by the other experts. All the best.

Great article by LinkedIn! Several factors may affect #ProteinPurification strategies & downstream process development: Protein properties: The choice of purification methods depends on the properties of the protein, such as size, charge, hydrophobicity, etc. Expression systems: Every expression system has its pros & cons in terms of feasibility, functionality, costs, etc. For eg., bacterial systems are cheaper but have no PTMs. Purification & scale-up: - Scaling up needs a keen understanding of the interplay between variables and requires stepwise adjustments & optimization. - Common issues may arise from protein instability. Focus on this issue early. Careful optimization of each step is needed for a robust purification methodology.