Brief Discussion Of Several Unique Methods Of Hollow Fiber in Inclusion Bod Purification

Inclusion Bodies (IB) are high-density, insoluble protein particles wrapped in membranes formed when foreign genes are expressed in prokaryotic cells, especially in Escherichia coli. When observed under a microscope, IB is a high-refraction region, which is obviously different from other components in the cytoplasm.

Inclusion body formation is relatively complex, related to the rate of protein production in the cytoplasm, the concentration of newly generated peptides is high, and there is not enough time to fold, so as to form amorphous protein aggregates. The inclusion bodies are basically composed of proteins, more than 50% of which are cloned products. The primary structure of these products is completely correct, but the three-dimensional configuration is wrong, so there is no biological activity. The inclusion body size is 0.5-1μm, insoluble in water, only soluble in denaturants such as urea, guanidine hydrochloride, etc.

In E. coli, inclusion bodies can occur in two locations of the cell: cytoplasm and peripheral cytoplasm. The location and characteristics of inclusion bodies in the cell depend on how proteins are expressed.

The inclusions in the cytoplasm of E. coli generally range in diameter from 0.2 to 1.5μm, and different proteins have different diameters, such as the size of interferon is 0.811μm, and the size of prorennet is 1.281μm. In some cases, the diameter of some inclusion bodies is larger than the diameter of E. coli, which giving E. coli a protrusion. In general, a cell has only one inclusion body.

 

Inclusion body purification

 

The inclusion body purification process is generally:

Cell crushing:

The general technology of cell crushing is: high-speed tissue crushing, glass homogenizer homogenization, ultrasonic treatment, repeated freeze-thaw method, chemical treatment (generally using lysozyme treatment).

 

Inclusion body washing:

Inclusion bodies are inactive solid particles formed by intracellular agglutination of proteins expressed by bacteria, which usually exist in amorphous, insoluble form. In the inclusion body, the true target protein only accounts for about 50%, and the rest contains lipids, lipopolysaccharides, nucleic acids and heteroproteins, which are bound to the inclusion body and affect the renaturation of the inclusion body protein. Therefore, washing before denaturation is a very necessary step.

In addition, the washing of inclusion bodies is helpful to increase the refolding yield of recombinant proteins. By removing impurities, obstacles in the renaturation process can be reduced, so that the protein can be more efficiently folded and assembled correctly, thereby improving the activity and function of the protein‌.

Washing commonly used less than 1% of the neutral detergent, such as Tween, Triton, Urea and NP40 plus EDTA and reducing agents 2-mercaptotreitol (DTT), β-mercaptoethanol repeated many times, because the detergent washing capacity is enhanced with the increase of the ionic strength of the solution, in the washing of inclusion bodies can be added NaCl to increase the ionic strength.

Inclusion bodies can be used to remove other components of the cell breakdown solution by centrifugation or filtration, both of which take advantage of different physical properties of inclusion bodies.

 

Centrifugation:

Inclusion body proteins are much denser than the same volume of cell fragments, so the inclusion body can be separated from the rest of the cell by means of centrifugation. Continuous centrifugation is the most frequently used operation for obtaining inclusion bodies in industrial production. Since the density of the cell fragments is smaller than that of the inclusion body, the sedimentation rate is smaller than that of the inclusion body. Continuous suspension and centrifugation can centrifuge most of the inclusion body, while the cell fragments are gradually removed.
Filtration (tangential flow filtration) : Due to the different molecular sizes of inclusion bodies and soluble proteins, the method of filtration can be used, which can reduce operating costs and make it easy to scale up. Tangential flow filtration (TFF) is driven by the transmembrane pressure difference. Substances and impurities smaller than the membrane pore size pass through the membrane, while impurities such as cells with larger particles are trapped. The membrane aperture generally used for filtration washing is 0.1μm. Hollow fiber tangential flow microfiltration can directly deal with high solid content of liquid material, fewer steps, simple operation, the membrane can be used repeatedly through cleaning, reducing equipment investment and operating costs, in line with the requirements of modular automated production.

 

The following is an application case for inclusion body washing using Guidling hollow fiber columns.

We used 94cm2 0.1-0.45μm hollow fiber to concentrate 250mL liquid (target protein molecular weight 17kd). In the whole washing process, the recovery rate of target protein and membrane flux depletion were investigated.

0-71min is the concentration process, 71-224min is the washing and filtration process. During the whole microfiltration process, TMP gradually increased, the liquid inlet speed remained unchanged, and the average material flux was 12LMH.

The results showed that the target protein was completely trapped, and the recovery rate of target protein was more than 90%. Guidling hollow fiber columns are stable and suitable for this application.

 

Inclusion body dissolution:

Inclusion bodies are generally dissolved under the condition of denaturating agent urea or guanidine hydrochloride, and the dissolved inclusion body proteins are completely denaturated, that is, except the primary structure and covalent bonds are retained, all hydrogen bonds and hydrophobic bonds are destroyed, and hydrophobic side chains are completely exposed.

Urea and guanidine hydrochloride are medium strength denaturants, which are easily removed by dialysis and ultrafiltration. The general concentration of urea 8-10M, guanidine hydrochloride 6-8M. Urea dissolution has the advantages of non-ionization, neutral, low cost, removal of protein after renaturation will not cause a large amount of protein precipitation, and dissolved inclusion bodies can be purified by a variety of chromatographic methods, so it has been widely used.

 

Inclusion body protein refolding:

The dissolved recombinant protein must be folded correctly to form a functional protein. Techniques for renaturation include dilution of protein solution to near neutral, removal of denaturant, renaturation on column, and gel filtration chromatography. Among them, diluting protein solution to nearly neutral and removing denaturant is a common classical method of renaturation, especially dilution renaturation method has the highest utilization rate.

Denaturant removal:

Dialysis: The advantage is that the volume does not increase, and the removal rate of denaturant is controlled by gradually reducing the concentration of external permeable liquid, but it takes a long time and is easy to form inactive protein aggregates, which is not suitable for large-scale operation and cannot be applied to the production scale.

Ultrafiltration (TFF) : easy to control the dialysis speed, whether in research and development, pilot test or production, can remove denaturants (liquid change + concentration) through ultrafiltration (TFF).

 

Inclusion body protein purification:

The methods of protein purification after renaturation are similar to those of soluble protein purification, namely ion exchange chromatography, gel filtration chromatography, affinity chromatography, ammonium sulfate salt-out precipitation, etc.

Inclusion bodies have no biological activity, there is no need to worry about the loss of protein activity, and large amounts of expression will not lead to cell death, and maximize resistance to protease attack. Jiuling hollow fiber can be flexibly applied in the downstream purification process of inclusion bodies. In the inclusion body washing step, excellent performance, compared with centrifugal washing, the use of nine age technology microfiltration hollow fiber for tangential flow filtration washing can greatly shorten the process time, and the general recovery rate can reach more than 90%. Jiuling hollow fiber can also be used in the protein renaturation step of inclusion bodies. The turbidity of the feed liquid filtered by microfiltration hollow fiber will be significantly reduced, and the protein recovery rate is high; The use of ultrafiltration hollow fiber can not only remove denaturants, but also shorten the chromatographic sample loading time, so as to save time, reduce the volume of filler and reduce the production cost. The recovery rate of microfiltration/ultrafiltration hollow fiber varies with different materials, and the general recovery rate can reach 90-95%.

Guidling Technology welcomes you to request test kits for related reviews.

 

About Guidling

Guidling Technology is a national high-tech enterprise focusing on biopharmaceuticals, cell culture, purification and concentration of biomedicine, diagnosis and industrial fluids. We have successfully developed centrifugal filter devices, ultrafiltration & microfiltration cassettes, virus filter, TFF system, depth filter, hollow fiber, etc. Which fully meet the application scenarios of biopharmaceuticals, cell culture, and so on. Our membranes and membrane filters are widely used in concentration, extraction and separation of pre-filtration, microfiltration, ultrafiltration and nanofiltration. Our many product lines, from small, single-use laboratory filtration to production filtration systems, sterility testing, fermentation, cell culture and more, meet the needs of testing and production. Guidling Technology is looking forward to cooperating with you!