Researchers that have overexpressed and purified recombinant proteins from E. coli knows are familiar with inclusion bodies. Inclusion bodies are insoluble aggregates of overexpressed recombinant protein in the bacterial cells. Some protein are prone to aggregation and others are not. The proteins that don’t aggregate usually are correctly folded and retains biological activity. On the other hand, inclusion bodies are aggregates of protein and are present in form of aggregates of high density within bacterial cytoplasm. To retrieve active recombinant protein from inclusion bodies is often a long and cumbersome process which requires following essential steps:
- Purification of inclusion bodies to homogeneity
- Solubilization of inclusion bodies
- Refolding of the solubilized proteins
- Purification of the refolded protein
Although, these steps substantially lengthen the protein purification process and adds to overall cost of protein purification, they are unavoidable for purification of some proteins. Refolding and processing of inclusion bodies will be the topic of discussion in another blog article. First, let’s understand some key concepts about inclusion bodies and ways to reduce or eliminate inclusion bodies formation at the protein induction step itself.
Why do Inclusion bodies form?
As mentioned earlier certain proteins have an intrinsic tendency to form insoluble aggregates, sometimes even single amino acid mutation can turn a soluble protein into inclusion bodies. Presence of long hydrophobic stretches in certain proteins has been shown to be associated with formation of inclusion bodies. Furthermore, it has been experimentally validated that certain conditions that favour high rate of protein synthesis such as high temperature and higher inducer concentration are responsible for formation of inclusion bodies inside E. coli cells.
Therefore, the first thing to try to avoid formation of inclusion bodies is to lower the temperature of bacterial cultures usually between 16°C-20°C and use IPTG at lower concentrations. Usually screening at pilot level with different IPTG concentrations/temperatures helps to identify the optimal conditions. More recently a cold shock method has been suggested to lower the formation of inclusion bodies. This involves incubating the culture for 1hr at 4°C or on ice just before IPTG or inducer is added. The cold shock treatment compels the bacterial metabolism to go down and elicits formation of cold shock proteins, the overall effect is slowing down of protein synthesis which leads to expression of protein in soluble form and prevents formation of inclusion bodies. If all above attempts fail to solubilise protein of interest, then the protein can be expressed along with presence of soluble tags such as GST tag and MBP tag to prevent formation of inclusion bodies by the protein.
Unlike eukaryotic cells, E. coli cells don’t have intracellular protein sorting compartments and very stringent protein quality control mechanisms and expression of recombinant proteins under strong promoters, high temperatures and high inducer concentrations can lead to exhaustion of E. coli’s protein quality control mechanisms which in turn prompts formation of inclusion bodies.
Inclusion bodies are not always made up of insoluble aggregates of protein. It has been suggested that even inclusion bodies can have proteins in their native form retaining their secondary structures, many inclusion bodies have been found to have biologically active proteins. Such inclusion bodies have bene called non classical inclusion bodies. Furthermore, NMR studies involving H and D exchange have revealed that inclusion bodies are dynamic entities and may have an equilibrium between unfolded and natively folded states of protein. Moreover, it has been found that by using certain strategies, formation of non-classical inclusion bodies can be induced, for example inducing at lower optical densities, inducing with low IPTG concentrations, inducing for a shorter period. Contrary to classical inclusion bodies which can only be solubilised by harsh conditions such as high concentrations of Urea or Guanidinium Hydrochloride, non-classical inclusion bodies can be solubilised using milder conditions leading to retrieval of natively folded proteins that retains the biological activity.
Are inclusion bodies always bad?
Formation of inclusion bodies is not always a bad thing. Inclusion bodies contain a lot of overexpressed protein of interest (in high purity), on an average the inclusion bodies usually have more protein than soluble proteins per litre of E. coli culture. In fact, many processes deliberately induce formation of inclusion bodies by their recombinant protein of interest to obtain higher yields. Certain proteins are extremely sensitive to intracellular proteases and proteins expressed as inclusion bodies are protected from protease action. Similarly, certain toxic proteins are best expressed as inclusion bodies because of biological inertness. Inclusion bodies are known to adopt a cross B sheet structure which is very similar to amyloids formed in neurodegenerative diseases. They serve a good research template to investigate the chemical and physical aspects of amyloid formation.
How to solubilise inclusion bodies?
As mentioned earlier, inclusion bodies are resilient to solubilisation and usually 8M urea or 6 molar Guanidinium hydrochloride is used for the same. Furthermore, 1-10mM of DTT is required if protein contains a lot of disulphide bonds. Prior to solubilisation, inclusion bodies are usually treated with tween-20 or Triton X-100 to clean the inclusion bodies. Usually different formulations are required to solubilise inclusion bodies of different proteins and no single formulation works for all.
G biosciences has a proprietary formulation to meet all needs concerning IB solubilisation with minimal effort. The IBS‐HPTM Buffer kit gently solubilizes both hydrophilic and hydrophobic proteins from the inclusion bodies.
The supplied IBS‐HPTM Buffer contains urea, thiourea and SDS. IBS‐HPTM Buffer is compatible for protein estimation with our NITM protein assay (Cat. # 786‐005) and running SDS‐PAGE analysis using PAGE‐PerfectTM kit (Cat. # 786‐123).