Proteins are bio-macromolecules with a range of functionalities as a result of complex higher-order structures. As the end product of the “central dogma” in biology, proteins are considered the key orchestrators of life.
Developing a deep understanding of the association between protein structure and function is the key to unlocking the “code of life” and exposing the mechanisms of any given biological process.
While extracting proteins from natural sources offers valuable starting materials for characterization, protein samples can be scarce in quantity and inconsistent in quality due to sample variations that impose challenges in protein biochemistry.
This is where the concept of recombinant protein expression is particularly useful, a process that inserts a foreign gene encoding the protein-of-interest (POI) into a host cell using a vector and generates the POI by hijacking the host’s protein manufacturing machineries (Figure 1).
Discovered over 40 years ago, plasmids (typically presented as circular double-stranded DNA molecules) were identified as the primary courier of foreign genes among bacteria. Subsequently, they opened the gate to a new, advanced era of genetic engineering and manipulation to enable recombinant protein expression.
Over the course of four decades, numerous vector systems, as well as host cells, including prokaryotes such as E.coli and eukaryotes (e.g. yeast, insect cells and mammalian cells, Figure 1), have been prepared to satisfy the production of a broad spectrum of recombinant proteins close to their natural statues to advance the progress in biochemical research, vaccine and therapeutics development, industrial catalyzation and food processing,
Figure 1. Core concept of recombinant protein expression and common host cells. PTM= post-translational modification. Image Credit: Sino Biological Inc.
Challenges in recombinant protein expression
High-quality recombinant proteins are crucial starting materials for ensuring the success of research efforts and drug development campaigns. The main quality attributes of any recombinant protein include purity, oligomeric status, thermo and chemical stability, folding, post-translational modifications (PTMs), activity etc.
Due to the inherent general complexity of proteins, protein expression and purification are often complex and challenging scenarios. Specific sequence or structural features within a protein can influence the overall yield and stability of the recombinant protein product.
For example, the presence of transmembrane domains or GPI-anchor sequences often causes the mixing of target proteins with plasma membrane, which compromises the protein yield. Meanwhile, the hydrophobic nature of transmembrane domains also affects the stability of the protein.
A detergent or lipid-based stabilization reagent, therefore, is necessary throughout the purification and formulation of such proteins. Conversely, the over-expression and aggregation of the target protein might result in surprising physiological changes to the host cells, resulting in protein degradation and the production of mis-folded proteins by the host proteases.
The majority of proteins are fragile molecules vulnerable to environmental stresses throughout the expression and purification processes.
Thus, recombinant protein expression often necessitates careful planning and process optimization to establish the suitable host, culture condition and duration, as well as the best possible purification strategy to guarantee a high yield of POI with minimally sacrificed quality attributes.
Figure 2 sums up the common challenges often experienced in recombinant protein expression.
Figure 2. Key factors impacting recombinant protein expression (left) and challenges in obtaining high-quality protein-of-interest (right). TM= transmembrane domain. Image Credit: Sino Biological Inc.
Approaches to optimization
As previously mentioned, typically, a systematic optimization effort is required to formulate a practical procedure to express high-quality target proteins. However, there is no general “one-size-fits-all” approach, and the expression and purification of each protein should be customized in every case.
To demonstrate the optimization of key components in the recombinant protein expression workflow, a selection of case studies are assessed below.
Host cells define the potential folding and pattern of PTM of the recombinant protein. Depending on the attributes desired, the selection of host cells should be carefully considered.
As demonstrated in Figure 3, the attribute-of-interest for the target protein was the capacity to form unified oligomers. The protein was first expressed in insect cells due to its intracellular location, but throughout purification, higher molecular weight polymers were seen.
Figure 3. Case study — host optimization for recombinant protein expression. Image Credit: Sino Biological Inc.
However, failure to remove the polymers by way of optimizing the formulation buffer led to a switch of the expression host to E.coli. The protein expressed by the 1st E.coli strain was prone to deterioration.
A second E.coli strain with extended endogenous protease knock-out was then employed. This strain was successful in the production of the stable target protein with requisite oligomer formation.
Vectors and culture conditions
Vectors are the vehicles responsible for delivering the target genes into a host cell. They care comprised of basic components such as a multiple cloning site (MCS) to foster the gene-of-interest, a promoter to boost expression and antibiotic resistance genes to accommodate screening.
Vectors are typically optimized by commercial vendors to accomplish optimal transfection and expression efficiency. As exhibited in Figure 4, Sino Biological has set up a vector system that exceeded the performance levels of several competitors in protein expression level in HEK293.
Figure 4. Case study — optimization of vector and culture conditions to enhance protein expression. Image Credit: Sino Biological Inc.
In addition to vectors, other conditions used in the host cell culture, e.g., duration and temperature, should also be improved to capture the target protein in its intact form. Meanwhile, using additives can occasionally help increase the target protein production.
Introducing inorganics into the process, such as metal ions and co-factors, has been proven to be useful for the expression of active enzymes as a result of their stabilization effects on the protein molecules.
Particular structural features on a target protein might result in an unstable, over-expressed form of a recombinant protein.
Areas of elevated hydrophobicity, high disorders and repetitive amino acid motifs are renowned for causing protein instability, so the presence of such areas should be noted.
As long as they are not directly involved in protein function, eliminating these areas may help improve protein expression (Figure 5).
Figure 5. Case study — removal of a hydrophobic region in the protein sequence to enhance protein expression. Image Credit: Sino Biological Inc.
The rule-of-thumb that should be considered when seeking the most suitable buffer formula during purification and in protein storage is that proteins are sensitive to their surrounding chemical environment. Fluctuations in pH, ionic strength and oxidative status would have an impact on protein stability.
Throughout purification, additives are also needed at certain times to stabilize the target protein or to facilitate tag exposure. As displayed in Figure 6, the inadequate extraction of target protein (a single-pass transmembrane protein) was conducted using detergent formula 1, likely due to the poor exposure of the His-tag.
Revision of the detergent formula boosted protein extraction while in the final polishing step, another detergent (DDM) was employed as a substitute for detergent formula 2, thereby stabilizing the final protein product.
Figure 6. Case study — buffer optimization during protein purification to enhance protein recovery. Image Credit: Sino Biological Inc.
As previously mentioned, progress made in high-throughput screening necessitates an accommodating high-throughput antibody/protein expression platform to help the process of drug discovery move forward.
Conversely, the COVID-19 pandemic has shown how powerful RNA virus hyper-mutation can be, meaning appropriate tools are needed urgently to create mutant virus protein libraries to facilitate neutralizing antibody screening and assessment.
Sino Biological has created a high-throughput recombinant antibody/protein expression platform to accommodate therapeutics discovery and infectious disease research. This platform, shown in Figure 7, is predicated on HEK293.
Figure 7. General work-flow of the high-throughput antibody/protein expression platform. Image Credit: Sino Biological Inc.
Antibody/protein sequence library is synthesized via a PCR-based method and the target genes are then passed to HEK293 for expression. Purified antibodies/proteins are exposed to quality and activity evaluation, and the appropriate candidates are advanced to scale-up.
This system utilizes flasks for early culture methods, and a weekly capacity of 100~200 molecules can be produced depending on the volume of each culture.
This platform has overseen the completion of over 15 projects so far - with a maximum library size of ~600 antibodies. Virus proteins, such as influenza HA, NA and SARS-CoV-2 RBD mutates have also been generated using this platform to amplify its versatility.
Recombinant proteins are essential to the development of the current biologics landscape. Numerous factors can influence the quality and yield of recombinant proteins, including host cell, culture method, protein construct, vector, as well as purification approaches.
Recombinant expression of a protein expression is an extremely customized process as there is no “one-size-fits-all” solution. Finally, high-throughput recombinant antibody/protein expression has been accomplished by Sino Biological using HEK293 cells. This platform can be accessed for contracted research services for accelerating novel biologics discovery.
About Sino Biological Inc.
Sino Biological is an international reagent supplier and service provider. The company specializes in recombinant protein production and antibody development. All of Sino Biological's products are independently developed and produced, including recombinant proteins, antibodies and cDNA clones. Sino Biological is the researchers' one-stop technical services shop for the advanced technology platforms they need to make advancements. In addition, Sino Biological offers pharmaceutical companies and biotechnology firms pre-clinical production technology services for hundreds of monoclonal antibody drug candidates.
Sino Biological's core business
Sino Biological is committed to providing high-quality recombinant protein and antibody reagents and to being a one-stop technical services shop for life science researchers around the world. All of our products are independently developed and produced. In addition, we offer pharmaceutical companies and biotechnology firms pre-clinical production technology services for hundreds of monoclonal antibody drug candidates. Our product quality control indicators meet rigorous requirements for clinical use samples. It takes only a few weeks for us to produce 1 to 30 grams of purified monoclonal antibody from gene sequencing.
Sponsored Content Policy: News-Medical.net publishes articles and related content that may be derived from sources where we have existing commercial relationships, provided such content adds value to the core editorial ethos of News-Medical.Net which is to educate and inform site visitors interested in medical research, science, medical devices and treatments.