Two variables dominating the retention of intact proteins under gradient elution with simultaneous ultrafast high-resolution separation by hydrophobic interaction chromatography.

The retention of intact proteins under gradient elution in hydrophobic interaction chromatography (HIC) was found to be governed by two variables, the steady region (SR) and the migration region (MR). In the SR, the proteins are immobilized by the strong interactions with the stationary phase such that the retention time is independent of the column length. In the MR, the proteins also interact with the stationary phase, but they move normally, thus the retention time depends on their partition coefficients and the column length. The SR can be used as an operation space (OP) for high-throughput protein analysis by 1D-LC using short columns at high flow rates to maintain a high resolution. The OP can also be employed for all assisted operations in online 2D-LC. Based on the steady region/migration region optimization strategy developed in this study, five successive complete separations of seven intact proteins were performed in a HIC cake in less than 5 min, and a crude extract of ribonuclease A from bovine pancreas was purified using online 2D-LC to 95.8% purity with 93.2% mass recovery in 45 min. This approach can be used to expedite the purification of drug-target proteins and should therefore be of interest to the pharmaceutical industry.

Protein renaturation with simultaneous purification by protein folding liquid chromatography: recent developments.

Protein folding liquid chromatography (PFLC)is a powerful tool for protein refolding with simultaneous purification. We review its recent progress in liquid chromatography and molecular biology, primarily involving the validation of PFLC refolding of proteins containing multiple disulphide bonds, the application of mixed-mode chromatography, PFLC in molecular biology. Representative examples are described.

Fast separation of native proteins using sub-2 µm nonporous silica particles in a chromatographic cake.

A novel method for the fast separation of native proteins was investigated using sub-2 µm nonporous silica packing inside a chromatographic cake having a diameter much larger than its thickness. Various silica-based particles ranging from 630 nm to 1.2 µm were synthesized and chemically modified with polyethylene glycol 600. The packing material was laterally packed into a series of chromatographic cakes containing the same diameter (10mm) and different thicknesses, ranging from 2 to 10 mm, and tested by hydrophobic interaction chromatography. The results showed that the sub-2 µm NPS particles in a small chromatographic cake were found to have a high efficiency at a flow rate of 10 mL/min and a backpressure of <20 MPa. The effect of the thickness of the chromatographic cake on the resolution of the proteins was also investigated and it was found that too short a column length could dramatically decrease the protein resolution; the minimum column length was also qualitatively evaluated. The presented method is expected to be useful for routine analysis of native and/or intact proteins in hospitals and as a tool for the fast screening protein drugs and optimization of experimental laboratory conditions.

Fast separations of intact proteins by liquid chromatography.

Scientists working in many fields require fast separations of intact proteins using liquid chromatography. The fast separations here concern not only the separation step alone but also the complete chromatographic process, including column regeneration, system equilibration, and buffer exchange, in one- and two-dimensional liquid chromatography in addition to fast purification technologies predominantly on the analytical scale with some unique examples on the preparative and industrial scales. This comprehensive review discusses recent developments in methodologies, packing materials, column techniques, and purification technologies in the field of rapid liquid chromatography of intact proteins. Some typical examples are summarized in the tables.

A novel thermodynamic state recursion method for description of nonideal nonlinear chromatographic process of frontal analysis.

A novel thermodynamic state recursion (TSR) method, which is based on nonequilibrium thermodynamic path described by the Lagrangian-Eulerian representation, is presented to simulate the whole chromatographic process of frontal analysis using the spatial distribution of solute bands in time series like as a series of images. TSR differs from the current numerical methods using the partial differential equations in Eulerian representation. The novel method is used to simulate the nonideal, nonlinear hydrophobic interaction chromatography (HIC) processes of lysozyme and myoglobin under the discrete complex boundary conditions. The results show that the simulated breakthrough curves agree well with the experimental ones. The apparent diffusion coefficient and the Langmuir isotherm parameters of the two proteins in HIC are obtained by the state recursion inverse method. Due to its the time domain and Markov characteristics, TSR is applicable to the design and online control of the nonlinear multicolumn chromatographic systems.

A new approach for characterizing the intermediate feature of α-chymotrypsin refolding by hydrophobic interaction chromatography.

A new approach for characterizing the intermediate of urea-denatured alpha-chymotrypsin (alpha-Chy) by hydrophobic interaction chromatography (HIC) is presented. The contact surface region (Z, S), affinity (logI), and the character of interaction force (j) of the alpha-Chy to the stationary phase of HIC (STHIC) between the intermediate (M) and native (N) states were found to be quite different as urea concentration (C(urea)) changes. With the changes in C(urea), a linear relationship between logI and Z was found to exist only for its N state, not for M state, indicating the interaction force between alpha-Chy in N state to the STHIC to be non-selective, but selective one for its M state. Also, the measured magnitude of both logI and Z in M state is only a fifth of that in N state. All three parameters were employed to distinguish protein in the N state from that in the M state. It would be expected that this result could be employed to distinguish any kind of non-functional protein having correct three-, or four-dimensional molecular structure from their stable M state of any kinds of proteins, and/or other proteins in proteome investigation, separation process of protein, and intensively understanding the intrinsic rule of protein folding in molecular biology.

Expression, refolding, and purification of a truncated human Delta-like1, a ligand of Notch receptors.

The Notch signaling pathway plays a pivotal role in proliferation, apoptosis, and cell fate specification in both embryonic and postnatal development, and is a potential therapeutic target for human diseases such as cancer. To express in Escherichia coli and purify soluble fragment of human Delta-like1 (hDll1), we cloned two extracellular fragments of hDll1 [hDll1 (127-225) and hDll1 (26-225)]. The hDll1 (127-225) fragment was successfully expressed in E. coli as a GST fusion protein (GST-hDll1). The GST-hDll1 protein, which was expressed as inclusion bodies after induction by IPTG, was refolded and purified simultaneously using affinity chromatography and size exclusion chromatography. The purified GST-hDll1 was of more than 95% purity, and had a molecular weight of 39kDa. Reporter assay showed that GST-hDll1 could activate a reporter gene that is dependent on Notch activation. Therefore, using the E. coli expression system and different chromatography systems, we successfully expressed, refolded, and purified a biologically active GST-hDll1, which might be potentially useful for therapy and studying the Notch pathway.

High recovery refolding of rhG-CSF from Escherichia coli, using urea gradient size exclusion chromatography.

Protein folding liquid chromatography (PFLC) is a powerful tool for simultaneous refolding and purification of recombinant proteins in inclusion bodies. Urea gradient size exclusion chromatography (SEC) is a recently developed protein refolding method based on the SEC refolding principle. In the presented work, recombinant human granulocyte colony-stimulating factor (rhG-CSF) expressed in Escheriachia coli (E. coli) in the form of inclusion bodies was refolded with high yields by this method. Denatured/reduced rhG-CSF in 8.0 mol.L(-1) urea was directly injected into a Superdex 75 column, and with the running of the linear urea concentration program, urea concentration in the mobile phase and around the denatured rhG-CSF molecules was decreased linearly, and the denatured rhG-CSF was gradually refolded into its native state. Aggregates were greatly suppressed and rhG-CSF was also partially purified during the refolding process. Effects of the length and the final urea concentration of the urea gradient on the refolding yield of rhG-CSF by using urea gradient SEC were investigated respectively. Compared with dilution refolding and normal SEC with a fixed urea concentration in the mobile phase, urea gradient SEC was more efficient for rhG-CSF refolding--in terms of specific bioactivity and mass recovery, the denatured rhG-CSF could be refolded at a larger loading volume, and the aggregates could be suppressed more efficiently. When 500 microL of solubilized and denatured rhG-CSF in 8.0 mol.L(-1) urea solution with a total protein concentration of 2.3 mg.mL(-1) was loaded onto the SEC column, rhG-CSF with a specific bioactivity of 1.0 x 10(8) IU.mg(-1) was obtained, and the mass recovery was 46.1%.

Liquid chromatography of recombinant proteins and protein drugs.

Many recombinant proteins (rPRTs) have a high bioactivity and some of them may eventually be classified as drugs beneficial to human health, recombinant human protein drugs (rPDs). rPDs are a high-technology product with all the associated economic benefits, therefore the liquid chromatography (LC) of rPRT is different from that of proteins isolated in laboratory scale for purely research purposes. The design of a purification scheme for an rPRT depends on the intended function of the purified rPRT, as a pure sample for research in small scale, or as a product for industrial production. This review paper mainly deals with the latter instance, producing rPD at a large scale. Pharmaceutical economics is considered not only for each step of purification, but also the whole production process. This strategy restricts the content of this review paper to the factors affecting the optimization source, the character of rPRT in up-stream technology and the purification of the rPRT in down-stream production. In the latter instance, the purification step is required to be as efficient as possible and LC is the core of the refined purification method, which is either a single LC method or combination of LC methods, sometimes, it may be a combination of LC and other non-LC separation methods comprising an optimized purification technology. Here some typical examples of rPRT purification at the large scale, recent developments, such as protein folding liquid chromatography, short column chromatography, and new packing material and column techniques are introduced.

Solubilization and refolding with simultaneous purification of recombinant human stem cell factor.

Recombinant human stem cell factor (rhSCF) was solubilized and renatured from inclusion bodies expressed in Escherichia coli. The effect of both pH and urea on the solubilization of rhSCF inclusion bodies was investigated; the results indicate that the solubilization of rhSCF inclusion bodies was significantly influenced by the pH of the solution employed, and low concentration of urea can drastically improve the solubilization of rhSCF when solubilized by high pH solution. The solubilized rhSCF can be easily refolded with simultaneous purification by ion exchange chromatography (IEC), with a specific activity of 7.8 x 10(5) IU x mg(-1), a purity of 96.3%, and a mass recovery of 43.0%. The presented experimental results show that rhSCF solubilized by high pH solution containing low concentration of urea is easier to be renatured than that solubilized by high concentration of urea, and the IEC refolding method was more efficient than dilution refolding and dialysis refolding for rhSCF. It may have a great potential for large-scale production of rhSCF.

[DSC and FTIR study of adsorbed lysozyme on hydrophobic surface].

During a process of hen egg white lysozyme adsorption and folding on a moderately hydrophobic surface (PEG-600), the effects of salt((NH4)2SO4) concentrations, surface coverage and denaturant (guanidine hydrochloride, GuHCl) concentrations on thermal stability and the changes in the molecular conformation of adsorbed native and denatured lysozyme without aqueous solution were studied with a combination of differential scanning calorimetry (DSC) with FTIR spectroscopy. The results showed that temperature due to endothermic peaks was reduced and the disturbance increased at higher temperature with the increase in salt concentration and surface coverage of adsorbed protein. beta-Sheet and beta-Turn stucture increased while alpha-Helix structure decreased after the adsorption. The peaks corresponding to both C-C stretching frequency in 1400-1425 cm(-1) and amide I band frequency in 1650-1670 cm(-1) of adsorbed denatured lysozyme can be detected in FTIR spectra while that due to amide I band frequency of adsorbed native lysozyme almost can't be observed. Adsorption resulted in structural loss of adsorbed native lysozyme, whose performance was less stable.

An approach for increasing the mass recovery of proteins derived from inclusion bodies in biotechnology.

A method for increasing the mass recovery of therapeutic proteins produced by E. coli using liquid chromatography was investigated. Recombinant human interferon-gamma (rhIFN-gamma) produced by E. coli was selected as a model therapeutic protein, and hydrophobic interaction chromatography (HIC) was performed as a model for liquid chromatography. Using seven types of stationary phase hydrophobic interaction chromatography (STHIC) with different end groups, the effect of the stationary phase on the mass recovery during protein folding by liquid chromatography (LC) and the causes of mass loss of rhIFN-gamma during its folding with simultaneous purification were investigated. Also strategies for increasing mass recovery are proposed. The results demonstrate that the mass recovery of rhIFN-gamma increases with the decreasing hydrophobicity for six STHIC with end groups of PEG-200, PEG-400, PEG-600, PEG-1000, furfural, and phenyl, except for PEG-1000. However, for oxethyl and PEG-600, even though the same diol end group is bonded to PEG-600, so long as the PEG-600 is modified by acetyl chloride, it can effectively enhance the mass and bioactivity recovery of rhIFN-gamma compared to the PEG-600 column. The effect of sample size including both mass and volume on the mass recovery of the rhIFN-gamma was also investigated. Last, redissolving the target protein that has irreversibly adsorbed to the stationary phase and re-injecting it onto the column is an approach for increasing mass recovery.

Renaturation and purification of rhGM-CSF with ion-exchange chromatography.

The renaturation and purification of recombinant human granulocyte macrophage colony stimulation factor (rhGM-CSF) expressed in Escherichia coli with strong anion-exchange chromatography (SAX) were studied. The effects of pH values, ratios of concentrations of GSH/GSSG, and urea concentrations in the mobile phase on the renaturation and purification of rhGM-CSF with SAX were investigated, respectively. The results show that the above three factors have remarkable influences on the efficiency of renaturation and mass recovery of rhGM-CSF. The addition of GSH/GSSG in the mobile phase can improve the formation of correct disulfide bonds in rhGM-CSF so that its renaturation yield increases. In addition, to enhance the mass recovery of rhGM-CSF with SAX, the low concentration of urea was added in the mobile phase to prevent denatured protein aggregation. Under the optimal conditions, rhGM-CSF was renatured with simultaneous purification on SAX column within 30 min only by one step. After that its specific bioactivity, mass recovery, and purity reached 1.66 x 10(7) IU x mg, 58.8%, and 96.2%, respectively.

Protein folding liquid chromatography and its recent developments.

The ultimate goal of proteomics is to identify biologically active proteins and to produce them using biotechnology tools such as bacterial hosts. However, proteins produced by Escherichia coli must be refolded to their native state. Protein folding liquid chromatography (PFLC) is a new method developed in recent years, and it is widely used in molecular biology and biotechnology. In this paper, the new method, PFLC is introduced and its recent development is reviewed. In addition the paper includes definitions, advantages, principles, applications for both laboratory and large scales, apparatus, and effecting factors of PFLC. In addition, the role of this method in the future is examined.

Refolding and purification of interferon-gamma in industry by hydrophobic interaction chromatography.

A new technology for renaturation with simultaneous purification of the recombinant human interferon-gamma (rhIFN-gamma) in downstream of biotechnology is presented. The strategies to develop the new technology in industry scale were suggested. Based on chemical equilibrium and molecular interactions, the principle of rhIFN-gamma refolding by HPHIC was described. The kind of stationary and mobile phases were evaluated and found the former to contribute to the rhIFN-gamma refolding more than the latter. The extract containing the rhIFN-gamma in gram scale in 7.0 mol L(-1) guanidine hydrochloride solution of 700 mL was directly pumped into a unit of simultaneous renaturation and purification of proteins (USRPP, 10 x 300 mm i.d.) packed by small particle packings of hydrophobic interaction chromatography and a satisfactory recovery of bioactivity and mass of the rhIFN-gamma was obtained. With flow rate 100 mL min(-1) and a gradient elution by only one step in 4h, the purity and specific bioactivity approach to 95% and 8.7 x 10(7) IU(-1) mg, respectively. To evaluate the goodness of the presented new technology in this study, a usual method with the renaturation by dilution method firstly and then purification with a series of LC in literature was employed to compare with each other. The obtained result in terms of purity, recoveries of mass and bioactivity, cost time as well as expenses, the former is much better than the latter. Comparing the total bioactivity of rhIFN-gamma in the extract before to that after the renaturation by the USRPP, the total bioactivity of rhIFN-gamma increased 62-fold.

Preparation of hydrophobic interaction chromatographic packings based on monodisperse poly(glycidylmethacrylate-co-ethylenedimethacrylate) beads and their application.

The monodisperse, poly(glycidylmethacrylate-co-ethylenedimethacrylate) beads with macroporous in the range of 8.0-12.0 microm were prepared by a single-step swelling and polymerization method. The seed particles prepared by dispersion polymerization exhibited good absorption of the monomer phase. The pore size distribution of the beads was evaluated by gel permeation chromatography and mercury instrusion method. Based on this media, a hydrophobic interaction chromatographic (HIC) stationary phase for HPLC was synthesized by a new chemically modified method. The prepared resin has advantages for biopolymer separation, high column efficiency, low column backpressure, high protein mass recovery and good resolution for proteins. The dynamic protein loading capacity of the synthesized HIC packings was 40.0 mg/ml. Six proteins were fast separated in less than 8.0 min using the synthesized HIC stationary phase. The HIC resin was firstly used for the purification and simultaneous renaturation of recombinant human interferon-gamma (rhIFN-gamma) in the extract solution containing 7.0 mol/l guanidine hydrochloride with only one step. The purity and specific bioactivity of the purified of rhIFN-gamma was found more than 95% and 1.3 x 10(8) IU/mg, respectively.

Purification of recombinant bovine normal prion protein PrP(104-242) by HPHIC.

Purification of the prion protein (PrP) is a major concern for biological or biophysical analysis as are the structural specificities of this protein in relation to infectivity. A simple and efficient method for purification of recombinant bovine normal prion protein containing residues 104-242, PrP(104-242) expressed in Escherichia coli by high performance hydrophobic interaction chromatography (HPHIC) was presented in this work. The solution containing denatured and reduced protein in 8.0 mol/L urea extracted from the inclusion body was directly injected into the HPHIC column, aggregates were prevented by the interaction between the denatured PrP(104-242) molecules and the stationary phase during the chromatographic process, the soluble form of PrP(104-242) in aqueous solution was obtained after desorbed from the column. Several factors, including pH value, types of stationary phase and salt, and gradient mode, influencing the purification results were investigated. Optimal conditions were obtained for the purification of PrP(104-242) by HPHIC. This procedure yield PrP(104-242) of a purity of 96% with a recovery of 87%, respectively, for a single step purification of 40 min.

Protein folding liquid chromatography.

A method for carrying out protein folding with simultaneous separation by protein folding liquid chromatography (PFLC) is described herein. Furthermore, a two-dimensional chromatographic column, termed a 2D column, which can be independently employed for accomplishing PFLC in either weak cation exchange mode or hydrophobic interaction chromatography mode is reported. The content of this chapter describes the most commonly employed methods and operations of PFLC, such as the use of urea or guanidine hydrochloride as a denaturant with the protein in either the reduced or oxidized state and solving problems caused by the formation of the precipitates during protein folding. The PFLC can be performed using conventional chromatographic columns and a new chromatographic cake. A protocol for fast renaturation with simultaneous purification of inclusion body protein of the recombinant human interferon-gamma to obtain purity ≥95% and high specific bioactivity in a single step and in 1 h is introduced.

[Recent development for purification of active proteins from bovine pancreas with liquid chromatography].

Many active proteins exist in bovine pancreas and some of them have become protein drugs for human heath. These protein drugs sourcing from bovine pancreas are also high-tech product having high economic benefit. In the modern biological technology, the preparation of most active protein products relies on various liquid chromatographic techniques. The recent development of extraction of the active proteins from bovine pancreas and their separations and purifications, mainly with chromatographic methods are reviewed in this paper. It would be expected to be helpful for the preparation and application of the active proteins from natural products.

Mixed-mode chromatography and its applications to biopolymers.

Mixed-mode chromatography is a type of chromatography in which a chromatographic stationary phase interacts with solutes through more than one interaction mode. This technique has been growing rapidly because of its advantages over conventional chromatography, such as its high resolution, high selectivity, high sample loading, high speed, and the ability to replace two conventionally corresponding columns in certain circumstances. In this work, some aspects of the development of mixed-mode chromatography are reviewed, such as stationary phase preparation, combinations of various separation modes, separation mechanisms, typical applications to biopolymers and peptides, and future prospects.