Xindu Geng

Northwest University, Ch’ang-an, Shaanxi, China

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Publications (56)89.2 Total impact

  • Xindu Geng · Xiaodan Jia · Peng Liu · Fei Wang · Xiaoming Yang
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    ABSTRACT: 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.
    No preview · Article · Sep 2015 · The Analyst
  • Ruijuan Niu · Yi Min · Xindu Geng
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    ABSTRACT: 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 (10 mm) 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. Copyright © 2014 John Wiley & Sons, Ltd.
    No preview · Article · Aug 2014 · Biomedical Chromatography
  • Lili Wang · Xindu Geng
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    ABSTRACT: 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.
    No preview · Article · Nov 2013 · Amino Acids
  • Chaozhan Wang · Xindu Geng
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    ABSTRACT: 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 8.0 mol L−1 urea from the inclusion bodies. First a laboratorial scale column (10 mm × 20 mm 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 (50 mm × 200 mm I.D.). 200 mL of rhG-CSF solution solubilized by 8.0 mol L−1 urea, with a total amount of protein around 1.6 g, could be loaded onto the large column at one time. Under these conditions, the obtained rhG-CSF had a specific activity of 2.3 × 108 IU mg−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.
    No preview · Article · Dec 2012 · PROCESS BIOCHEMISTRY
  • Fei Wang · Yi Min · Xindu Geng
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    ABSTRACT: 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.
    No preview · Article · Nov 2012 · Journal of Separation Science
  • Xiaodan Jia · Congyu Ke · Xuan Sun · Xindu Geng
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    ABSTRACT: Mixed-mode chromatography (MMC) is a type of chromatographic method in which multiple-mode take place between the stationary phase and solute in the feed. MMC has been found to have many advantages and thus became a research hot point. However, before going wide applications, the synthesis of new MMC column and/or the selection of some commercial columns having the character of MMC need to be established. Based on the fact that some commercial ion-exchange columns perform the separation behavior of hydrophobic interaction chromatography (HIC), one of four commercial weak-cation exchange (WCX) column, a two-dimensional mode (WCX, HIC) column called as 2D column, and standard proteins were selected to investigate and compare their retention behaviors. The two type columns have some common characters: proteins can be separated by either WCX, or HIC mode, but have some quite different column efficiencies, and the profiles of "U-shape" elution curve. The width of the "U-shape" curve was experimentally found to relate to the chromatographically dynamic factor, i.e., the broader the "U-shape" curve, the narrower the chromatographic peak obtains, while the magnitude of the critical point of the "U-shape" curve relates to the protein retention, a larger value of a critical point corresponds to a stronger retention. An intensive comparison between the two types of column efficiency was carried out by the peak capacity of proteins. The experimental results showed that the 2D column has much more number of peak capacity than the commercial WCX column, either for one-dimensional, or two-dimensional separation. However, for the separation of a group proteins, the elution order is dominated by the comprehensive consideration of the peak capacity, the dynamic, and thermodynamic factors. Finally the nomenclature of the various stationary phases of MMC and/or 2D column employed in two-dimensional liquid chromatography were suggested. Although, in terms of column efficiency, none of the four commercial WCX column could be compared to the 2D column, at least, two of them as "quasi-2D column" may be employed for protein separation with both of WCX and HIC modes by means of either off-line in all cases, or on-line in some cases. However, none of them has been found to approach to the criteria of the 2D column. The nomenclature and expressions of MMC and 2D columns were suggested and the difference between the MMC and 2D columns were also explained. © 2012 Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences.
    No preview · Article · Mar 2012 · Acta Chimica Sinica -Chinese Edition-
  • Yun Yang · Xindu Geng
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    ABSTRACT: 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.
    No preview · Article · Dec 2011 · Journal of Chromatography A
  • Xiaoming Yang · Xindu Geng
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    ABSTRACT: 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.
    No preview · Article · Mar 2011 · Se pu = Chinese journal of chromatography / Zhongguo hua xue hui
  • Lili Wang · Chaozhan Wang · Xindu Geng
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    ABSTRACT: 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.
    No preview · Article · Jan 2011 · Se pu = Chinese journal of chromatography / Zhongguo hua xue hui
  • Quan Bai · Xindu Geng
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    ABSTRACT: 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.
    No preview · Article · Jan 2011 · Methods in molecular biology (Clifton, N.J.)
  • Fei Wang · XinDu Geng
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    ABSTRACT: A new approach for separation of intact proteins with high resolution, high speed and high sample loading (“three highs”) by non-porous packings in high performance liquid chromatography is presented. A chromatographic cake having larger diameter than its thickness is firstly employed to completely separate 1.0 and 40 μg of the mixture of 7 standard proteins within 1 min under conventional chromatographic conditions. Also, 0.5 mg of the mixture was almost completely separated within 1 min, accomplishing the “three high” purpose. This research expands the application utilized for separation of intact proteins with non-porous packings. A smaller geometric size of chromatographic cake packed with much smaller particles of non-porous packings may improve results. Keywordsliquid chromatography-protein separation-non-porous separation media-chromatography cakes-proteomics
    No preview · Article · Jun 2010 · Chinese Science Bulletin
  • Jia Jia · Lili Wang · Dong Gao · Xindu Geng
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    ABSTRACT: 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.
    No preview · Article · Jun 2010 · Se pu = Chinese journal of chromatography / Zhongguo hua xue hui
  • Yi Min · Gang Chen · Xindu Geng
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    ABSTRACT: Based on the fact that the resolution of intact protein separation is almost independent of column length, the effect on the resolution for intact protein separation causing from dynamic factors in hydrophobic interaction chromatography (HIC) was investigated. A concept of "conditional plate height" (H) for protein separation is firstly suggested for characterizing this effect for protein separation under linear gradient elution. Standard proteins were separated with conventional chromatographic column and chromatographic cake, and the plot of the H vs the linear velocity of mobile phase (u) was made, respectively. It was found that the obtained plot is similar to the conventional van Deemter Plot but has some differences. The optimized u corresponding to the minimum H was determined to be approximate 0.2 mm/s for the chromatographic cake and 1-3 mm/s for the conventional column. Furthermore, in comparison with the latter, optimized u value for the former has much broader range. Based on this fact, the resolutions and speeds for standard protein separation between the chromatographic cake packed with silica-base HIC material and the conventional column packed with soft HIC media were compared. The chromatographic cake (10 mm x 20 mm i.d.) was found to perform a complete separation of seven standard proteins in 10 min, while with the latter (55 mm x 12 mm i.d.) only five standard proteins can be completely separated in 140 min, even though the sample load for the former having bed volume of 3.14 mL, five times of that of the latter. The HIC chromatographic cake was also employed for the renaturation with simultaneous purification of the recombinant human granulocyte colony stimulating factor. The obtained purity was > or = 97%, mass recovery was 39%, specific bioactivity was 1 x 10(8) IU/mg with only one step HIC in 50 min. It would be expected that when a kind of packings having very small particle size is packed into a chromatographic cake with diameter to be greater than its thickness and is employed to separate, and/or renature proteins, a result of high speed and high resolution with simultaneous renaturation under high protein loading ("three H" target) could be obtained.
    No preview · Article · Sep 2009 · Se pu = Chinese journal of chromatography / Zhongguo hua xue hui
  • Peng Liu · Haiya Yang · Xindu Geng
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    ABSTRACT: Using four commercial weak anion-exchange chromatography (WAX) columns and 11 kinds of different proteins, we experimentally examined the involvement of hydrophobic interaction chromatography (HIC) mechanism in protein retention on the WAX columns. The HIC mechanism was found to operate in all four WAX columns, and each of these columns had a better resolution in the HIC mode than in the corresponding WAX mode. Detailed analysis of the molecular interactions in a chromatographic system indicated that it is impossible to completely eliminate hydrophobic interactions from a WAX column. Based on these results, it may be possible to employ a single WAX column for protein separation by exploiting mixed modes (WAX and HIC) of retention. The stoichiometric displacement theory and two linear plots were used to show that mechanism of the mixed modes of retention in the system was a combination of two kinds of interactions, i.e., nonselective interactions in the HIC mode and selective interactions in the IEC mode. The obtained U-shaped elution curve of proteins could be distinguished into four different ranges of salt concentration, which also represent four retention regions.
    No preview · Article · Aug 2009 · Journal of Chromatography A
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    Congyu Ke · Jianjun Li · Zhenling Liu · Xindu Geng
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    ABSTRACT: 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.
    Preview · Article · Mar 2009 · International Journal of Molecular Sciences
  • Chaozhan Wang · Lili Wang · Xindu Geng
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    ABSTRACT: Immobilized metal ion affinity chromatography (IMAC) is a new technique for protein refolding, but it is limited to the refolding of fusion proteins with histidine affinity tags. In the present work, a non-fusion recombinant protein, recombinant human granulocyte colony-stimulating factor (rhG-CSF) expressed in Escheriachia coli (E. coli) in the form of inclusion bodies was successfully refolded with simultaneous purification by IMAC. rhG-CSF inclusion bodies solubilized in 8.0 mol/L urea was injected into a Cu(II)-iminodiacetic acid (IDA)-IMAC column, the soluble and active form of rhG-CSF in aqueous solution was obtained after desorbed from the column by linear increase of imidazole concentration. Several factors in the refolding process, including urea concentration and pH of mobile phases, type of buffer, glycerol concentration and loading sample volume, were investigated, respectively. When 200 μL of denatured/reduced rhG-CSF solution at a total protein concentration of 2.8 mg/mL was loaded on the IMAC column, rhG-CSF with a specific activity of 2.3 × 108 IU/mg and a mass recovery of 39% was obtained after IMAC refolding, and rhG-CSF was also purified during this chromatographic process, its purity was determined to be 97%.
    No preview · Article · Feb 2009 · Biochemical Engineering Journal
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    ABSTRACT: This paper reports the on-line separation of native (N) proteins by two-dimensional liquid chromatography (2D-LC) using a single column with one phase (called 2D column). The 2D column exhibits excellent resolution, selectivity, and retention of proteins in the N state and functions in two retention modes--hydrophobic interaction chromatography (HIC) and weak-cation exchange chromatography (WCX). We describe a new approach to on-line buffer exchange and collection of fractions from the first retention mode and their quantitative re-injection into the same column, followed by re-separation in the second retention mode. Thus, liquid chromatography in a closed system and in an on-line manner could be successfully carried out. This method was termed on-line protein separation by 2D-LC using only a single column (on-line 2D-LC-1C). The applicability of this method was experimentally demonstrated using standard proteins and a human serum sample. The total hypothetical maximum possible peak capacity n(c,total) and total sample peak capacity n(c,total)(*) of the 2D column were 329 and 199, respectively. By comparison against several popular commercially available columns, it was found that the 2D column had not only comparable resolution and better selectivity but also some unique characteristics. This 2D-LC-1C method could be applied to the fast purification of intact proteins in the N state, such protein drugs from natural products, and recombinant proteins and also for the fast pre-fractionation of intact proteins in the "top-down" MS strategy in proteomics.
    No preview · Article · Feb 2009 · Journal of Chromatography A
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    Xindu Geng · Lili Wang
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    ABSTRACT: 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.
    Preview · Article · May 2008 · Journal of Chromatography B
  • Dan Wu · Dong Gao · Quan Bai · Xindu Geng
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    ABSTRACT: The renaturation with simultaneous purification of recombinant human interferon-gamma (rhIFN-gamma) expressed as inclusion bodies in Escherichia coli (E. coli) was accomplished by the stationary phase of hydrophobic interaction chromatography (STHIC) with the end group of poly(ethylene glycol) (PEG)(PEG200) packed in a chromatographic column and a chromatographic pie by nonlinear gradient, separately. In order to provide more selections for the chromatographic separation of rhIFN-gamma from different sources, the chromatographic behavior of rhIFN-gamma in reversed-phase liquid chromatography, ion-exchange chromatography and immobilized-nickel affinity chromatography were also studied. The fraction of the renatured and purified rhIFN-gamma from HIC was desalted by the size exclusion chromatography, subsequently freeze-dried to powder. With matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS), monomeric and dimeric rhIFN-gamma were found in the powder due to the freeze-dried process and their relative molecular masses were 17 184.0 and 34 204.4, respectively. With the bioactivity assay by cytopathic effect inhibition (CPEI), the specific bioactivity of rhIFN-gamma was 9.5 x 10(8) IU/mg, which was higher than that of the required criteria in the phar macopoeia of China, because the presence of dimeric rhIFN-gamma which has much higher specific bioactivity than its monomer in the powder. The obtained mass recovery, purity, specific bioactivity of the purified monomeric rhIFN-y were 93.7%, > 95%, and 4.3 x 10(7) IU/mg, respectively. The results showed that the renaturation with simultaneous purification of rhIFN-gamma by PEG200-STHIC is a kind of efficient method.
    No preview · Article · Mar 2008 · Se pu = Chinese journal of chromatography / Zhongguo hua xue hui
  • Chaozhan Wang · Jiahua Liu · Lili Wang · Xindu Geng
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    ABSTRACT: 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.
    No preview · Article · Mar 2008 · Applied Biochemistry and Biotechnology