High-performance Hydrophobic Interaction Chromatography Research Articles

Refolding with Simultaneous Purification of Recombinant Core Streptavidin Using Single-step High-performance Hydrophobic Interaction Chromatography

Streptavidin has applied to many areas including detection, purification, labeling, crosslinking and immobilization resulting in a high demand on its production. In this study, we report a method for preparation of recombinant core streptavidin (cSAV) protein using highperformance hydrophobic interaction chromatography (HPHIC). Firstly the cSAV was successfully cloned and expressed in Escherichia coli as inclusion bodies. A bifunctional stationary phase mainly working as HIC mode accompanied by weak anion exchange chromatography (WAX) was prepared using β-phenylethylamine (PEA) as a ligand. The denatured cSAV was then refolded and simultaneously purified by PEA hydrophobic interaction chromatography (PEA-HIC). The mass recovery and purity of cSAV by single-step were 30.2% and 98%, respectively. The bioactivity was determined to be 13.2 U/mg by biotin binding capacity assay. This method provides a new possibility for fast separation with simultaneous renaturation of cSAV.

Preparation of a silica-based high-performance hydrophobic interaction chromatography stationary phase for protein separation and renaturation.

In this work, based on the structural characteristics of bio-membrane molecules, a novel type of high-performance hydrophobic interaction chromatography stationary phase was prepared using cholesterol as a ligand. Investigating the separation performance of this stationary phase, the effect of pH and salt concentration of the mobile phase on the retention time, the absorption capacity, and the hydrophobic ability revealed that this stationary phase had a high loading capacity and moderate hydrophobic interactions compared with four different hydrophobic interaction chromatography stationary phase ligands. Five types of standard proteins could be baseline separated with a great selection for protein separation. When 3.0 M urea was added to the mobile phase, it could be refolded with simultaneous purification of denatured lysozyme by one-step chromatography. The mass recovery of lysozyme reached 89.5%, and the active recovery was 96.8%. Compared with traditional hydrophobic interaction chromatography, this new stationary phase has a good hydrophobic ability and a significant refolding efficiency.

Expression and purification of recombinant human serum albumin from selectively terminable transgenic rice

Human serum albumin (HSA) is widely utilized for medical purposes and biochemical research. Transgenic rice has proved to be an attractive bioreactor for mass production of recombinant HSA (rHSA). However, transgene spread is a major environmental and food safety concern for transgenic rice expressing proteins of medical value. This study aimed to develop a selectively terminable transgenic rice line expressing HSA in rice seeds, and a simple process for recovery and purification of rHSA for economical manufacture. An HSA expression cassette was inserted into a T-DNA vector encoding an RNA interference (RNAi) cassette suppressing the CYP81A6 gene. This gene detoxifies the herbicide bentazon and is linked to the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) cassette which confers glyphosate tolerance. ANX Sepharose Fast Flow (ANX FF) anion exchange chromatography coupled with Butyl Sepharose High Performance (Butyl HP) hydrophobic interaction chromatography was used to purify rHSA. A transgenic rice line, HSA-84, was obtained with stable expression of rHSA of up to 0.72% of the total dry weight of the dehusked rice seeds. This line also demonstrated high sensitivity to bentazon, and thus could be killed selectively by a spray of bentazon. A two-step chromatography purification scheme was established to purify the rHSA from rice seeds to a purity of 99% with a recovery of 62.4%. Results from mass spectrometry and N-terminus sequencing suggested that the purified rHSA was identical to natural plasma-derived HSA. This study provides an alternative strategy for large-scale production of HSA with a built-in transgene safety control mechanism.

Responsive membranes for hydrophobic interaction chromatography

Poly N-vinylcaprolactam (PVCL) chains were grown from the surface of regenerated cellulose membranes using atom transfer radical polymerization (ATRP). Modified membranes were characterized using scanning electron microscopy, infrared spectroscopy, X-ray photoelectron spectroscopy and by measuring surface contact angles. The grafting degree of PVCL increases with increasing ATRP initiator immobilization time. PVCL is a thermo-responsive polymer with a lower critical solution temperature (LCST) that depends on the concentration of salt ions present in solution. The LCST decreases below room temperature in buffer containing 1.8M (NH3)2SO4 where the polymer adopts a more hydrophobic/collapsed conformation. At low ionic strength, the LCST remains above room temperature. Contact angles for PVCL in 20mM phosphate buffer with and without 1.8M (NH3)2SO4 were determined. Contact angles in the buffer with high ionic strength were higher than those in low ionic strength.Adsorption and desorption of bovine serum albumin (BSA) and immunoglobulin G (IgG) have been investigated. Loading was conducted in high ionic strength buffer. Elution was conducted in low ionic strength buffer. By using a responsive ligand that changes its conformation during loading and elution, high protein recoveries were obtained. Ligands that respond to solution ionic strength show promise for high performance hydrophobic interaction chromatography.

Refolding and purification of recombinant human granulocyte colony-stimulating factor using hydrophobic interaction chromatography at a large scale

High level expression of recombinant human granulocyte colony-stimulating factor (rhG-CSF) in Escherichia coli (E. coli) usually forms insoluble and inactive aggregates, i.e. inclusion bodies. In the present work, high performance hydrophobic interaction chromatography (HPHIC) was applied to the refolding of rhG-CSF, which was solubilized by 8.0molL−1 urea from the inclusion bodies. First a laboratorial scale column (10mm×20mm I.D.) was employed to study the refolding process. Several factors, including concentration of ammonium sulfate, pH of the mobile phase and flow rate, were investigated in details. The results indicated that the rhG-CSF produced by E. coli could be successfully refolded with simultaneous purification by using HPHIC. The refolding process was further scaled up by using a large column (50mm×200mm I.D.). 200mL of rhG-CSF solution solubilized by 8.0molL−1 urea, with a total amount of protein around 1.6g, could be loaded onto the large column at one time. Under these conditions, the obtained rhG-CSF had a specific activity of 2.3×108IUmg−1 and a purity of 95.4%, the mass recovery during the purification was 36.9%. This work might have great impact on practical production of rhG-CSF, and it also shed a light on protein refolding using liquid chromatography at large scales.

Fast preparation of recombinant human stem cell factor from inclusion bodies using different hydrophobic interaction chromatographic columns

A method was developed to increase the recovery of recombinant human stem cell factor (rhSCF) from inclusion bodies using high performance hydrophobic interaction chromatography (HPHIC). The target protein was first solubilized in 8.0 mol/L urea solution, and was purified and refolded simultaneously by HPHIC with different chromatographic cakes. Experimental conditions, such as the ligand structures of stationary phase and the composition of mobile phase, were optimized. Under the optimal conditions, high mass recoveries and specific activities of rhSCF were acquired, the purities of rhSCF were above 95.5%, and the mass recoveries of rhSCF were above 49.6%. The final product was also verified as monomer by size exclusion chromatography and matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS). These results provided further evidence that HPHIC is an effective tool in the refolding and purification of recombinant proteins.

Renaturation with simultaneous purification of the recombinant human Flt3 ligand from inclusion bodies by high performance hydrophobic interaction chromatography

Flt3 ligand (FL) is a class of cytokines with the functions of promoting early hematopoiesis. It has important clinical value in promoting growth and development of hematopoietic cells and hematopoietic mobilization. In order to obtain large quantities of recombinant human FL (rhFL) by genetic engineering methods for clinic and research, in this work, rhFL was expressed in E. coli as inclusion bodies. The inclusion bodies were recovered, cleaned and solubilized in 8 mol/L urea, the solubilized rhFL was renatured by high performance hydrophobic interaction chromatography (HPHIC) with simultaneous purification, the retention feature and renaturation regularity were studied. The results showed that when the denatured protein concentration was 8.51 g/L, and the end group of stationary phase was PEG800, under the conditions of mobile phase of pH 7.0 and with the addition of 4 mol/L urea, 1.8 mmol/L glutathione (GSH) and 0.3 mmol/L oxidative glutathione (GSSG), a mass recovery of 36.9% and a purity of 94.5% were obtained after refolding with simultaneous purification. The obtained rhFL was successfully renatured with simultaneous purification in only one step of HPHIC, and it provided a foundation for the manufacturing of high quality rhFL.

Effect of Mobile Phase on the Mass Recovery of rhIFN-γ in Protein Folding Liquid Chromatography

Protein refolding is a key process for the production of recombinant proteins from E. coli. One of the effective techniques for protein refolding is through liquid chromatography named as protein folding liquid chromatography (PFLC). Mobile phase composition is one of the most important factors affecting the efficiency of protein renaturation in PFLC. Optimization of mobile phase composition in PFLC for protein renaturation with simultaneous purification is thus much more important than that in usual liquid chromatography. This study presents an approach with regard to the increase of protein production through optimizing the mobile phase composition of PFLC. Recombinant human interferon-gamma (rhIFN-gamma) was purified with simultaneous renaturation by high performance hydrophobic interaction chromatography (HPH-IC) using a stationary phase with an end group of PEG-200. Effects of mobile phase composition, gradient elution mode, and flow rate on the recoveries of mass and bioactivity of rhIFN-gamma were investigated. It was found that these factors were very important for refolding with simultaneous purification of rhIFN-gamma by PFLC. This study highlights the importance of optimizing the mobile phase composition in PFLC for increasing the recovery of protein. Taking 3.0 mol/L (NH4 )2 SO4 + 0. 05 mol/L KH2 PO4 (pH 7. 0) and 0. 05 mol/L KH2 PO4 (pH 7. 0) as mobile phases A and B, respectively, the highest mass recovery was obtained under a nonlinear gradient elution for 35 min.

Effect of ligand structure of stationary phase of high performance hydrophobic interaction chromatography on renaturation efficiency of GuHCl-denatured α-chymotrypsin

The renaturation of the denatured α -chymotrypsin (α-Chy) with 1.7 mol · L−1 guanidine hydrochloride (GuHCl) by three kinds of stationary phase of high performance hydrophobic interaction chromatography (STHIC) with a comparable hydrophobicity but different ligand structures was investigated. The obtained result indicates that the ligand structures of the three STHIC contribute to the renaturation efficiency of α-Chy in the order of the end ligands PEG-600> phenyl group > tetrahydrofurfuryl alcohol (THFA).

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.

High-performance hydrophobic interaction chromatography.

High-performance hydrophobic interaction chromatography.

Assessment of purity and quantification of plasmid DNA in process solutions using high-performance hydrophobic interaction chromatography

A hydrophobic interaction HPLC method was developed for the quantification of plasmid DNA and assessment of its purity in crude Escherichia coli lysates and other process streams. A Phenyl Sepharose Source (Amersham Biosciences) column was used to separate the double-stranded plasmid DNA molecules from the more hydrophobic impurities present in the process streams. The method is rapid (each analysis takes 7 min), reproducible, easy to perform and does not require previous digestion of RNA in samples with RNase or other pre-treatment. Furthermore, it is capable of handling heavily contaminated samples, with less than 5% of plasmid DNA thus constituting a good alternative to other less robust analytical techniques currently in use.

Studies on Renaturation with Simultaneous Purification of Recombinant Human Proinsulin from E. coli with High Performance Hydrophobic Interaction Chromatography

The renaturation with simultaneous purification of recombinant human proinsulin (rh‐proinsulin) expressed in E. coli by high performance hydrophobic interaction chromatography (HPHIC) was investigated. The result indicates that the reduced/denatured rh‐proinsulin, extracted with 8.0 mol L−1 urea solution in the presence of β‐mercaptoethanol can be renatured and purified, simultaneously, in 45 min with HPHIC, resulting in the purity and mass recovery being more than 90% and 94%, respectively. The disulfide bonds of rh‐proinsulin can correctly form on the HPHIC column without the presence of reduced and oxidized glutathione (GSH, GSSG). The renaturation efficiency of rh‐proinsulin with HPHIC was tested by enzyme cleavage in order to obtain insulin. The result was also confirmed with RPLC, SDS–PAGE, and MALDI‐TOF, respectively. The renatured and purified rh‐proinsulin can directly be enzyme‐cleaved in the collected fraction containing the rh‐proinsulin. Thus, the technology for the renatured and purified rh‐proinsulin is very simple and fast.

Determination of free energy of protein folding on liquid-solid interface

Based on the fact that the stoichiometric displacement model for retention of solute and the total adsorption free energy of solute on a solid surface can be divided into two components, net adsorption and net desorbed energies, a new principle and an equation for calculating the free energy of protein folding, ΔΔGF, on the solid surface are proposed. With high-performance hydrophobic interaction chromatography (HPHIC), an experimental method for determining the ΔΔGF is established. Lysozyme and α-amylase have been selected as examples to test the new method, and their ΔΔGF on the HPHIC stationary phase surface are found to be much higher than that reported from a solution. In addition, the ΔΔGF of the two proteins are found to increase with the concentration of denaturing agent employed. The average standard deviations, ±4.7% for lysozyme and ±3.0% for α-amylase, indicate that the new method has a satisfactory reproducibility and reliability.

“BPSA,” a specific molecular form of free prostate-specific antigen, is found predominantly in the transition zone of patients with nodular benign prostatic hyperplasia

Objectives. The biologic mechanism for the increased proportion of noncomplexed (“free”) prostate-specific antigen (PSA) found in the serum of patients with benign prostate disease is unknown. We recently reported that most of the PSA found in benign, hyperplastic, and cancerous prostatic tissue is in the free form. To determine whether specific molecular forms of free PSA are associated differentially with normal, hyperplastic, or cancerous prostatic tissue, we have further characterized the free PSA in each type of prostatic tissue. Methods. PSA was purified by immunoaffinity chromatography from matched prostatic tissue samples of peripheral zone cancer (PZ-C), PZ noncancer (PZ-N), and transition zone (TZ) tissue from 10 large-volume (greater than 50 g) and 8 small-volume (less than 25 g) radical prostatectomy specimens. Eight TZ specimens obtained during transurethral resection of the prostate for benign prostatic hyperplasia (BPH) were also analyzed. The different molecular forms of PSA were further resolved by high-performance hydrophobic interaction chromatography. Clipped forms of PSA were identified by N-terminal amino acid sequencing. Results. More than 99% of the PSA in prostatic tissues was in the free, noncomplexed form. Specimens from the prostate TZ were found to contain elevated levels of an altered form of PSA, which we designated BPSA. Purified BPSA contained a distinctive cleavage at lysine 182. The median percent BPSA (%BPSA) was 11.4 in the TZ of specimens with nodular BPH compared with a %BPSA of 4.1 in the TZ of specimens without nodular BPH ( P <0.0014). The median %BPSA levels of the PZ-N and PZ-C tissues ranged from 3.2 to 4.9 and were not significantly different from one another or from the %BPSA level of TZ tissues without nodular BPH. Conclusions. We have identified a specific molecular form of clipped free PSA, called BPSA, that is increased within the prostatic TZ of patients exhibiting nodular BPH. Higher levels of percent free PSA in serum have been found to correlate strongly with prostate volume, which in turn is closely associated with the progressive enlargement of nodular BPH tissue within the TZ of the prostate. Thus, it is possible that a proportion of the serum percent free PSA found in patients with BPH may be composed of BPSA released into the serum.

Hydrophobic interaction chromatography and capillary zone electrophoresis to explore the correlation between the isoenzymes of salivary α-amylase and dental caries

High-performance hydrophobic interaction chromatography (HIC) and capillary zone electrophoresis (CZE) were utilized in an effort to find the correlation between the composition of some isoenzymes of human salivary α-amylase (HSA) and dental caries. The mixture of more than three isoenzymes of HSA, fractionated from human parotid saliva with HIC, was further separated by CZE at the optimum pH 6.50. The composition and relative quantity of these isoenzymes were compared between two groups of individuals with different caries-susceptibility. It is found that the present frequency of peak II on CZE in the caries-free group was higher than that in the caries-active group and the relative quantities of peak III and peak IV showed remarkable differences ( p<0.05) between the two groups. These results may indicate that the composition of HSA isoenzymes is related to the occurrence of dental caries. However, more work should be done to further affirm this correlation between the isoenzymes of salivary α-amylase and dental caries.

SEPARATION OF AMINO ACIDS AND PEPTIDES BY HIGH PERFORMANCE HYDROPHOBIC INTERACTION CHROMATOGRAPHY

Chromatographic behaviors of amino acids and peptide derivatives were examined by high performance hydrophobic interaction chromatography(HIC) on two different packings,TSKgels Phenyl–5PW and Ether–5PW. Most amino acids hardly retained and eluted at an elution volume being the same as the column volume even with an initial buffer for hydrophobic interaction chromatography; aromatic amino acids showed weak retention. On the other hand, peptides retained in the column and their elution was roughly in the order of the molecular mass, although some of them showed extraordinary elution behaviors. Kunitz bovine pancreas trypsin inhibitor showed weak retention in spite of having larger molecular mass, which might be due to the steric conformation. Some peptides showed considerably stronger retention even under ordinary conditions, and the addition of organic modifier (20 % acetonitrile) in eluent was required for their elution. HIC using less hydrophobic packings examined was found to be adequate to separate peptides quantitatively, and this might be useful in removing peptides from contaminants of amino acids and proteins.

The Influence of Urea on the Retention of Protein in Hydrophobic Interaction Chromatography

Abstract The retention behaviour of eight proteins in high performance hydrophobic interaction chromatography (HPHIC) in a mobile phase consisting of ammonium sulphate-potassium dihydrogen phosphate containing urea was studied. By using the stoichiometric displacemant model for retention (SDM-R) of solute in liquid chromatography, and by measuring surface tention of the mobile phase in the presence of urea, also and UV-spectra of proteins, it was found that in the presence of urea in the mobile phase of HIC, even though the urea somewhat strong solvent in terms of result, the change of the conformation of protein molecular in hydrophobic interaction systems due to the presence of urea is considered as the major contribution to the retention of proteins.

Salt Concentration Effects in High-Performance Hydrophobic-Interaction Chromatography in Comparison with NMR of Proteins in Solution

Abstract The effects of salt concentration on the chromatographic behavior of cytochrome C, ribonuclease A, and α-chymotrypsinogen A in hydrophobic interaction chromatography (HIC) has been examined by isocratic elutions on a Bio-Gel TSK Phenyl 5 PW column. In some cases, conformational variations were manifest chromatographically by reproducible changes in peak shape and appearance of multiple peaks as a function of sodium sulfate concentration in the mobile phase. A parallel study by proton nuclear magnetic resonance (NMR) spectroscopy on the salt concentration dependence of the spectral property of these proteins is in agreement with the possible contribution of the mobile phase composition to the observed chromatographic behavior.

The purification of early‐pregnancy factor to homogeneity from human platelets and identification as chaperonin 10

Early-pregnancy factor (EPF), first discovered in the early stages of gestation, is associated with and necessary for cell proliferation in a wide variety of biological situations. Like many other growth factors, EPF is present in platelets, and, by titration studies with a neutralising anti-EPF monoclonal antibody, platelets were identified as an extremely rich source of this growth factor. EPF has been purified from clinically outdated human platelets by heat extraction, ion-exchange and affinity chromatographies on SP-Sephadex and heparin-Sepharose respectively, high-performance hydrophobic interaction chromatography and three reverse-phase HPLC steps, with an average yield of 15 micrograms/100 platelet units (equivalent to approximately 50 1 blood). Using SDS/PAGE, EPF migrated as a single band with approximate M(r) 8500, coincident with biological activity. Mass spectrometry provided an accurate and precise determination of the molecular mass as M(r) 10843.5 +/- 2, along with definitive evidence of the homogeneity of the preparation. Attempts at Edman degradation indicated that the molecule was blocked at the N-terminus and sequencing of proteolytic fragments was undertaken. The amino acid sequence of approximately 70% of the molecule was determined which, with a single exception, is identical with rat chaperonin 10. This structural relationship was shown to extend to functional identity by studies using chaperonin 10 and its functional associate chaperonin 60. Investigations with the latter confirmed that chaperonin 10 is the moiety in pregnancy serum which initiates response in the EPF bioassay. Our studies identify EPF as a member of the highly conserved heat-shock family of molecules and demonstrate a molecular chaperone performing an extracellular role.