Maximizing Efficiency and Reliability in Recombinant Protein Selection and Characterization

When purchasing and using recombinant proteins, the selection and characterization are pivotal processes, shaping the outcome of various research endeavors and applications. This guide aims to streamline the identification of target recombinant proteins, focusing on critical factors such as molecule, species, sources (expression system), tagging, purity, concentration, functional activity, and endotoxin levels. By delving into these aspects, you can efficiently identify the most appropriate recombinant proteins with clarity and precision, ensuring optimal outcomes for their experimental pursuits.

Quickly Locate Your Target Recombinant Protein

When selecting recombinant proteins, consider certain characteristics to help you identify the most suitable option for your needs. We support several characters of Recombinant Protein as parameters when searching and selecting our products.

• Molecule: This refers to the target proteins you may study, such as interleukin or chemokines.
• Species: Species to which the molecule belongs, whether it's human (interleukin) or mouse (interleukin), among others. Studying proteins from different species offers insights into evolutionary perspectives, functional diversity, biomedical research models, drug discovery and development, ecological and environmental studies, as well as biological diversity and conservation.
• Source: The expression system, which could include bacteria (E. coli, etc.), yeast (Saccharomyces cerevisiae, etc.), or mammalian cells (HEK293, etc.). The selection of an expression system (e.g., E. coli, yeast, insect cells, mammalian cells) is dictated by the complexity of the protein, required post-translational modifications, and yield. Each system offers distinct advantages, from the high yield and simplicity of bacterial systems to the complex glycosylation patterns achieved in mammalian systems. The choice of expression system can affect the protein's folding, activity, and solubility, impacting downstream applications.
• Tag: Peptides or proteins to label target proteins for the purpose of expression, detection, tracking, and purification. Examples include His, Flag, GST, HA, as well as fluorescence proteins like GFP, YFP, and mCherry. The selection of tags (e.g., His-tag, GST, FLAG) must consider the potential impact on the protein's structure, function, and purity. Fusion tags can facilitate easier purification and detection but may need to be removed to restore native protein function, necessitating a careful balance between utility and biological relevance.

By considering these factors, you can efficiently identify the most suitable recombinant protein for your research objectives.

Purity

Higher purity is generally desirable, especially for applications where contaminating proteins could interfere with the results. Methods such as SDS-PAGE or mass spectrometry can be used to assess purity. Experiments that require higher purity of proteins typically involve detailed structural or functional analyses where any impurities could interfere with the accuracy and reliability of the results.

CSIRO, CC BY 3.0, via Wikimedia Commons

Here are some common types of experiments that necessitate high protein purity:

• Structural Biology Studies: Techniques such as X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, and cryo-electron microscopy (cryo-EM) require highly pure proteins to determine their three-dimensional structures accurately. Contaminants or impurities can interfere with the formation of well-ordered crystals or the interpretation of structural data.
• Enzyme Kinetics and Mechanistic Studies: Experiments investigating enzyme kinetics, substrate specificity, or enzyme mechanisms require pure enzymes to accurately measure catalytic rates and understand enzyme-substrate interactions. Even small amounts of contaminants can significantly affect enzyme activity and kinetic parameters.
• Protein-Protein Interaction Studies: Techniques such as surface plasmon resonance (SPR), co-immunoprecipitation, and yeast two-hybrid assays rely on pure proteins to study protein-protein interactions accurately. Contaminants could lead to false-positive or false-negative results, affecting the interpretation of interaction data.
• Biophysical Characterization: Biophysical techniques such as circular dichroism (CD) spectroscopy, fluorescence spectroscopy, and analytical ultracentrifugation require pure proteins to study their structural and thermodynamic properties accurately. Impurities can alter the spectroscopic signals or sedimentation profiles, leading to misinterpretation of data.
• Drug Discovery and Screening Assays: High-throughput screening assays for drug discovery often require pure proteins to identify potential drug candidates accurately. Impurities may interfere with the assay readouts or lead to false hits, affecting the reliability of screening results.
• In vivo Studies and Animal Models: Experiments involving in vivo administration of proteins, such as animal studies or clinical trials, require highly pure proteins to ensure safety and efficacy. Contaminants could elicit immune responses or other adverse effects in vivo, confounding experimental outcomes.

In summary, experiments requiring high protein purity typically involve detailed biochemical, biophysical, or structural analyses where the presence of contaminants could compromise the accuracy and reliability of the results.

Concentration

Accurate quantification of protein concentration is critical for experimental design, reproducibility, and interpretation of results. Methods such as UV absorption spectroscopy, Bradford assay, and BCA assay provide different levels of sensitivity, specificity, and convenience. The choice of method depends on the protein’s characteristics, the presence of interfering substances, and the required accuracy.

Functional Activity

Functional activity assays, vital for assessing the biological activity of recombinant proteins, offer insights into their roles in biology, therapeutics, or disease mechanisms.

• Enzyme Activity Assays: Measure substrate conversion rates, using methods like colorimetric or fluorometric assays, to assess enzymatic activity.
• Binding Assays: Evaluate protein-protein or protein-ligand interactions using techniques like ELISA, surface plasmon resonance, or fluorescence polarization.
• Bioassays: Assess the biological effects of proteins on living systems, such as cell proliferation or cytokine production, often through cell-based assays or animal models.
• Functional Assays in Vitro and in Vivo: Conducted in controlled environments or living organisms, respectively, to understand protein function and physiological relevance.

These assays aid in drug discovery, disease diagnosis, and understanding cellular mechanisms by providing insights into protein function and activity.

Endotoxin Level

Endotoxin, a common contaminant in protein preparations, poses significant challenges in biological applications such as cell culture and in vivo studies. It comprises various components, with lipopolysaccharide (LPS) being a prominent constituent. Assessing endotoxin levels in protein preparations is crucial, as high concentrations can trigger nonspecific immune responses and disrupt experimental outcomes.

One of the primary concerns with endotoxin contamination is its ability to induce inflammatory responses. For instance, in preparations of recombinant human heat shock protein 70 (rhHsp70), contamination with endotoxin (LSP) can lead to the release of tumor necrosis factor α (TNFα) by murine macrophages, mirroring the effects of pure LPS. This inflammatory reaction operates through receptors such as CD14 and Toll-like receptor 4, underscoring the potential for endotoxin contamination to provoke unwanted immunogenic responses in biological settings.

To mitigate the risks associated with endotoxin contamination, optimization of purification protocols is essential. Incorporating specific detergents and striving for high purity levels are effective strategies for minimizing endotoxin levels in protein preparations.

Conclusion

Navigating the intricate landscape of recombinant protein selection and characterization requires meticulous attention to detail and a comprehensive understanding of diverse factors influencing protein behavior and utility. By prioritizing parameters such as purity, concentration, functional activity, and endotoxin levels, researchers can bolster the reliability and efficacy of their experimental outcomes. This holistic approach not only enhances the quality of research findings but also accelerates progress in fields ranging from basic science to therapeutic development. As researchers continue to explore the vast potential of recombinant proteins, adherence to rigorous selection and characterization protocols remains paramount for unlocking new insights and driving innovation in biotechnology and beyond.

Still unable to decide on which recombinant protein to choose from? Contact us at inquiry@betalifesci.com.

Reference

Ghasemi A, Salari M, Pourmand M, et al. Optimization and Efficient Purification in Production of Brucella melitensis Recombinant HSP and TF Proteins With Low Endotoxin Contents. Not specified. 2013.

Gao B, Tsan MF. Endotoxin Contamination in Recombinant Human Heat Shock Protein 70 (Hsp70) Preparation Is Responsible for the Induction of Tumor Necrosis Factor α Release by Murine Macrophages. J Biol Chem. 2003;278(1):225-230.

Ecker JW, Kirchenbaum GA, Pierce SR, et al. High-Yield Expression and Purification of Recombinant Influenza Virus Proteins from Stably-Transfected Mammalian Cell Lines. Vaccines. 2020;8(2):220.

Raran-Kurussi S, Waugh DS. Expression and Purification of Recombinant Proteins in Escherichia coli with a His6 or Dual His6-MBP Tag. In: Methods in Molecular Biology. Vol 1586. Humana Press, New York, NY; 2017:255-278.

Conley AJ, Joensuu JJ, Jevnikar AM, Menassa R, Brandle JE. Optimization of elastin-like polypeptide fusions for expression and purification of recombinant proteins in plants. Biotechnol Bioeng. 2009;103(3):562-573.