Protein Expression

Recombinant protein expression in bacteria, yeast, insect, or mammalian systems

Advancements in genomics, cloning, and numerous molecular biology techniques allow researchers to express heterologous proteins in numerous biological systems. The ability to express recombinant proteins provides researchers with a wide range of powerful downstream applications for further research studies. At small scale, overexpression of proteins may facilitate studies aimed at understanding protein function; whereas, large scale protein production is essential for enzyme, antibody, and vaccine production. Determining optimal cellular growth and protein expression conditions are critical for both small- and large-scale protein expression systems. Whether it is a prokaryotic or eukaryotic expression system that is needed for post-translational modifications, the cell type will largely determine what tools and reagents are required for optimal protein expression.

Related Technical Articles

  • The extracellular (ECM) microenvironment, defined by biochemical cues and physical cues, is a deciding factor in a wide range of cellular processes including cell adhesion, proliferation, differentiation, and expression of phenotype-specific functions. For this reason, engineering the ECM microenvironment provides clear benefits in studies of cell and tissue engineering and related applications. Currently existing technology offers simple and merely adequate environments that facilitate simple cell processes such as cellular adhesion.1,2 The simple presentation of cell adhesion motifs is not optimal for controlling more integrated processes.
  • Hypoxia detection assays to measure oxygen levels in both live and fixed cells and tissues.
  • The 3xFLAG system is an improvement upon the original system by fusing 3 tandem FLAG® epitopes for a total of 22 amino acids. Detection of fusion proteins containing 3xFLAG is enhanced up to 200 times more than any other system.
  • FLAG® and 3xFLAG® expression vectors and products for bacterial and mammalian expression vectors, detection and purification of proteins.
  • Expression Vectors for Bacterial, Mammalian and Insect Cell Systems. Featured are a variety of tags, promoters and elements for secretion, transient, stable and bicistronic expression.
  • See All

Related Protocols

  • Yeasts are considered model systems for eukaryotic studies as they exhibit fast growth and have dispersed cells. Yeast cultures can be grown, maintained, and stored in liquid media or on agar plates using techniques similar to those for bacterial cultures.
  • Duolink® kits use in situ PLA®, a proximity ligation assay technology, to accurately and objectively quantify individual proteins, and their interactions and modifications in unmodified cells and tissue.
  • The video follows the simple and straightforward procedure that allows you to detect, quantify and obtain cell localization of protein interactions and their modifications in a single experiment.
  • We recommend applying the counterstaining protocol after the completion of the Amplification step in section 7.3, step 5 of the Duolink In Situ Fluorescence User Manual.
  • See All

Protein Expression Vectors

Expression vectors or plasmids are circular sequences of DNA that are commonly used by researchers as a tool to host the gene encoding their protein of interest. Plasmids containing the gene of interest are subsequently transformed or transfected into cells to overexpress the protein. Plasmids contain various useful elements that facilitate cloning, clone selection, protein expression and purification including but not limited to, a multiple cloning site (MCS), antibiotic resistance genes used for clone selection, unique tags for protein identification and purification, and strong promoter regions to drive protein expression. There are a wide variety of protein expression vectors as many of these elements are interchangeable depending on the specific application needs and the cell type used for protein expression.

Bacterial, Mammalian, and Additional Protein Expression Systems

With fast growth kinetics and plasmid transformations in E. coli in just a few minutes, bacteria are the workhorse organism for producing recombinant proteins. Bacterial protein expression relies on the 30S and 50S ribosomal subunits of the 70S bacterial ribosome. To prevent growth of plasmid-free cells, plasmids containing antibiotic-resistant genes are used as a selection method to identify and isolate bacteria that have incorporated plasmids containing the protein-encoding sequence of interest. While additional genetic sequencing is often necessary to confirm the presence of your gene sequence, numerous antibiotics that block bacterial protein synthesis are commonly used for removing plasmid-free bacteria. In addition to bacteria, insect, yeast, and mammalian cell lines are also commonly used for protein expression. However, unlike bacteria, eukaryotic cell lines contain additional molecular machinery to generate post-translational modifications (e.g. glycosylation) and are often essential for protein functionality and meaningful downstream analyses.

Recombinant Protein Expression Applications

Recombinant proteins are proteins that are encoded within the protein-expressing plasmid and, have been modified for maximal protein expression/purification or mutated to assess protein function. The ability to add, remove, or alter the protein-encoding sequence, even by a single nucleotide, provides researchers with an immensely powerful tool to investigate a large variety of fundamental research questions and elucidate the function of the protein in both healthy and disease tissues. Implications of recombinant protein expression technology extend far beyond fundamental research applications and are essential for the development life-saving therapeutics and vaccines.