Protein production and purification

Medicilon's protein scientists have been working on protein expression and purification for many years. We can start your project even you have nothing in hand but the name of your protein. In Medicilon's laboratories, protein purification is performed in scales from micrograms and milligrams. All Protein Purification Services start with the analysis of physico-chemical and biological properties of a target protein resulting in the development of tailored procedures for its extraction, purification and characterization. Email:marketing@medicilon.com.cn web:www.medicilon.com

Protein purification

As a chromatographic procedure, IMAC has the advantages of having strong, specific binding, mild elution conditions and the ability to control selectivity by including low concentrations of imidazole in chromatography buffers. There is a broad array of common resins with slightly different binding capacities and binding strengths, but all tolerate harsh cleaning procedures (TALON Metal Affinity Resins User Manual, Clontech, 2007; the QIAexpressionist, Qiagen, 2003; and HisTrap HP, 1 ml and 5 ml (instructions), Amersham Biosciences, GE Healthcare, 2003). Most purification steps can be integrated by highperformance liquid chromatography; the most commonly used devices are the ÄKTA systems from GE Healthcare. The final purity of the protein can be optimized by controlling the ratio of recombinant protein to the column size; lower-affinity contaminants can be competed with a relative excess of the histidine-tagged recombinant protein. Accordingly, it is beneficial to determine the amount of the soluble target protein to be loaded on the column, and this can be estimated from small-scale expression trials. As a general rule, to maximize purity, one should load the column with a slight excess over the predicted binding capacity. Although not necessary, it is relatively straightforward to implement these protein purification protocols on automated chromatography systems, which have proven reliable, effective and simple to use.

Preparation of the bacterial lysate

Preparation of the bacterial lysate is a critical step. Optimal conditions maximize cell lysis and the fraction of the recombinant protein that is extracted while minimizing protein oxidation, unwanted proteolysis and sample contamination with genomic DNA. Mechanical lysis by high-pressure homogenization or sonication, or lysis by freeze-thaw procedures with lysozyme are equivalent in most cases. The lysis buffer should contain a strong buffer (50–100 mM phosphate or HEPES) to overcome the contribution of the bacterial lysate, high ionic strength (equivalent to 300–500 mM NaCl) to enhance protein solubility and stability, protease inhibitors and a reducing agent such as Tris(2-carboxyethyl) phosphine hydrochloride (TCEP) to prevent oxidation of the protein. Loading large amounts of bacterial lysate (>1 l culture volume) on small (<1 ml) affinity columns may require prior removal of any particulate or viscous material. This can be accomplished by using enzymes that degrade nucleic acid and cell-wall material, such as DNase or Benzonase (Merck/EMD) and lysozyme, respectively. Some of the enzymes used in lysis are less active in the presence of reducing agents or high salt concentration; optimal lysis may require sequential addition of the components. Clarified lysates can also be filtered before loading on the affinity columns. IMAC purification is performed in phosphate buffer, pH 8.0 and an ionic strength equivalent to 300–500 mM NaCl. HEPES buffer (and, to a lesser extent, Tris buffer) at pH 7.5–8.0 can also be used. It has been consistently observed that conditions of high ionic strength (for example, 500 mM NaCl) maintain solubility and stability of the widest variety of proteins. Indeed, a substantial fraction of proteins precipitate if the salt concentration is reduced to physiological levels, particularly as the protein becomes more pure and concentrated. The choice of NaCl as the salt is mainly historical and, although not systematically explored, there is no reason to believe that sodium and chloride are optimal. Indeed, sodium and chloride levels in the cell are very low and are probably never the physiologically relevant counter-ions for intracellular proteins. A modest amount of imidazole (see resin manufacturer’s recommendations) should be included in the cell extraction buffer to reduce binding of less histidine-rich proteins to the IMAC column. For intracellular proteins, care should be taken to maintain a reducing environment. TCEP, unlike dithiothreitol (DDT), is compatible with all known IMAC matrices. Finally, inclusion of glycerol (10%) during protein purification enhances the solubility and stability of many proteins.

Chromatography

After the lysate is loaded on the IMAC column, it should be washed with buffer including an intermediate concentration of imidazole (see manufacturer’s instructions), which will elute weakly bound contaminants without sacrificing large amounts of the recombinant protein. It is sometimes necessary to optimize the wash step with respect to the concentration of imidazole as well as the volume of the wash. Finally, the recombinant protein should be with a step gradient (for example, 300 mM imidazole). If EDTA and DTT are added after IMAC; add the EDTA first to sequester any nickel that has leached off and that could react with the DTT. The choice of gel filtration as the next step may be surprising, considering its lower resolving power compared with ion exchange or other adsorption chromatography methods, but this step is often sufficient after IMAC if the protein was abundant in the lysate.Moreover, gel filtration is more generic, can be performed in any buffer condition, and can be used to resolve the oligomerization state of the target protein. In some cases, if the protein is judged insufficiently pure for the intended purpose, one can remove the tag with a histidine-tagged TEV protease and perform IMAC again as an additional ‘generic’ purification step, collecting the recombinant protein in the flowthrough. This step very efficiently removes histidine-rich proteins derived from the expression host, which may have copurified in the primary IMAC procedure, as well as the cleaved tag and the histidinetagged protease.

Protein characterization

Characterizing the purified protein in some detail reduces the risk of wasting resources on protein material of inadequate quality. It also provides a means to ensure that different batches of the same protein have similar properties. Below, we outline a simple, generic protein characterization protocol that allows the experimentalist to judge whether the correct protein has been purified, whether additional molecular species are present and to estimate the approximate protein concentration. Other characterization methods that are very informative but not as widely applied, such as mass spectrometry, static or dynamic light scattering, and measuring protein thermal stability, are described in Supplementary Methods.

Inspection of gel filtration chromatogram

If size exclusion chromatography was used as the last purification step, a close look at the chromatogram is essential. Symmetric elution profiles are characteristic of homogeneous proteins, whereas asymmetric profiles reflect inhomogeneous, or partially aggregated, samples (Fig. 2), or whether the column itself is in poor condition. The elution profiles will also reveal the primary oligomerization state. The presence of additional oligomerization states may be of biological significance, or may be a sign of nonspecific aggregation. If the protein elutes in the void volume of the chromatogram, the protein is most likely forming large, nonspecific aggregates, which may be an indication of improper folding and compromised activity. It is also of value to analyze individual peaks by SDS-PAGE or mass spectrometry to analyze the protein in each peak.

SDS-PAGE analysis

After protein purification, samples should be resolved by denaturing SDS-PAGE. If stained with a dye such as Coomassie brilliant blue, the intensity of the bands will usually be proportional to the amount of protein53. This allows the purity of the sample to be estimated and whether the purified protein is of the expected size.

UV absorption spectroscopy

To quantify the amount and concentration of purified protein, the simplest and most common method is the Bradford assay53, which measures the binding of Coomassie brilliant blue to the protein. As some proteins bind the dye anomalously, it is also useful to measure the UV absorption at A280 and calculate the concentration of the protein by using the predicted molar extinction coefficient at A280. By taking a UV absorption spectrum, it is also possible to uncover contamination with DNA or RNA, or reveal common copurifying cofactors (for example, NAD, FAD, heme).

Storing purified protein

Aliquots of the protein to be stored should be placed in thin-walled PCR plastic tubes, frozen in liquid nitrogen and stored at −80 °C. Small aliquots should be frozen to avoid damaging freeze-thaw cycles, and aliquots should be thawed on ice. Concentrated proteins (for example, >1 mg/ml) tend to be more stable to freeze-thaw cycles. Proteins are usually concentrated using centrifuge-driven filter devices with adequate molecular weight size cutoffs. Care should be taken during centrifugation to avoid local over-concentration and irreversible precipitation or aggregation of the protein on the filtration membrane. It is advisable to explore the stability of the protein to concentration and freeze-thaw cycles before processing the entire batch. The frozen and thawed sample should be compared with protein that was not frozen for biochemical activity, visible precipitation, changes in physical properties or crystallization characteristics. In our collective experience, relatively few proteins are irreversibly inactivated by one freeze-thaw cycle. In those rare instances, the protein can be stored at 4°C for short periods of time, at −20 °C in high concentrations of glycerol, or as an ammonium sulfate suspension.