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1.
Ting Zhang ;Xiaojing Xu ;Liang Shen ;Yanye Feng ;Zhong Yang ;Yaling Shen ;Jufang Wang ;Weirong Jin ;Xiaoning Wang 《Acta biochimica et biophysica Sinica》2009,(12):1044-1052
Overexpression of foreign proteins in Escherichia coli often leads to the formation of inclusion bodies (IBs), which becomes the major bottleneck in the preparation of recombinant proteins and their applications. In the present study, 36 proteins from IBs were refolded using a simple refolding method. Refolding yields of these proteins were defined as the percentage of soluble pro- teins following dilution refoiding in the amount of denatured proteins in the samples before diluting into refolding buffer. Furthermore, a mathematical model was deduced to evaluate the role of biochemical proper- ties in the protein refolding. Our results indicated that under the experimental conditions, isoelectric point of proteins might be mostly contributing to the high effi- cacy of protein refolding since the increment of one unit resulted in a decrease of 14.83% in the refolding yield. Other important mediators were components of protein secondary structure and the molecular weight (R2= 0.98, P = 0.000, F-test). Six proteins with low efficiency in the protein refolding possessed relatively low isoelectric points. Furthermore, refolding yields of six additional proteins from IBs were predicted and further validated by refolding the proteins under the same conditions. Therefore, the model of protein refold- ing developed here could be used to predict the refold- ing yields of proteins from IBs through a simple method. Our study will be suggestive to optimize the methods for protein refoiding from IBs according to their intrinsic properties. 相似文献
2.
Refolding of protein inclusion bodies directly from E. coli homogenate using expanded bed adsorption chromatography 总被引:3,自引:0,他引:3
To avoid the intrinsic problem of aggregation associated with the traditional solution-phase refolding process, we proposed
a solid-phase refolding method integrated with the expanded bed adsorption chromatography. The model protein was a fusion
protein of recombinant human growth hormone and a glutathione S-transferase fragment. It was demonstrated that the inclusion body proteins in the cell homogenate could be directly refolded
with higher yield. To verify the applicability of this method, we have tested with success three types of the starting materials,
i.e., rhGH monomer, inclusion bodies containing the fusion protein, and the E. coli cell homogenate. This direct refolding process could reduce the number of the renaturation steps required and allow the refolding
at a higher concentration, approximately 2 mg fusion protein per ml resin.
This revised version was published online in July 2006 with corrections to the Cover Date. 相似文献
3.
A simple and effective Escherichia coli (E. coli) bioprocess is demonstrated for the preparation of recombinant human alpha-fetoprotein (rhAFP), a pharmaceutically promising protein that has important immunomodulatory functions. The new rhAFP process employs only unit operations that are easy to scale and validate, and reduces the complexity embedded in existing inclusion body processing methods. A key requirement in the establishment of this process was the attainment of high purity rhAFP prior to protein refolding because (i) rhAFP binds easily to hydrophobic contaminants once refolded, and (ii) rhAFP aggregates during renaturation, in a contaminant- dependent way. In this work, direct protein extraction from cell suspension was coupled with a DNA precipitation-centrifugation step prior to purification using two simple chromatographic steps. Refolding was conducted using a single-step, redox-optimized dilution refolding protocol, with refolding success determined by reversed phase HPLC analysis, ELISA, and circular dichroism spectroscopy. Quantitation of DNA and protein contaminant loads after each unit operation showed that contaminant levels were reduced to levels comparable to traditional flowsheets. Protein microchemical modification due to carbamylation in this urea-based process was identified and minimized, yielding a final refolded and purified product that was significantly purified from carbamylated variants. Importantly, this work conclusively demonstrates, for the first time, that a chemical extraction process can substitute the more complex traditional inclusion body processing flowsheet, without compromising product purity and yield. This highly intensified and simplified process is expected to be of general utility for the preparation of other therapeutic candidates expressed as inclusion bodies. 相似文献
4.
Takayuki Y. Nara Hideaki Togashi Chisato Sekikawa Kengo Sakaguchi Fujio Mizukami Tatsuo Tsunoda 《Biotechnology progress》2009,25(4):1071-1077
We established a 96‐well‐plate‐based refolding screening system using zeolite. In this system, protein denatured and solubilized with 6 M guanidine hydrochloride is adsorbed onto zeolite placed in a 96‐well plate. The refolding conditions can be tested by incubating the samples with refolding buffers under various conditions of pH, salts, and additives. In this study, we chose green fluorescent protein as the model protein. Green fluorescent protein was expressed as inclusion bodies, and we tested the effects of four pH conditions and six additives on its refolding. The results demonstrate that green fluorescent protein was more efficiently refolded with zeolite than with the conventional dilution method. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009 相似文献
5.
Aims: To isolate, clone and express a novel phytase gene (phy) from Bacillus sp. in Escherichia coli; to recover the active enzyme from inclusion bodies; and to characterize the recombinant phytase. Methods and Results: The molecular weight of phytase was estimated as 40 kDa on SDS-polyacrylamide gel electrophoresis. A requirement of Ca2+ ions was found essential both for refolding and activity of the enzyme. Bacillus phytase exhibited a specific activity of 16 U mg−1 protein; it also revealed broad pH and temperature ranges of 5·0 to 8·0 and 25 to 70°C, respectively. The Km value of phytase for hydrolysis of sodium phytate has been determined as 0·392 mmol l−1. The activity of enzyme has been inhibited by EDTA. The enzyme exhibited ample thermostability upon exposure to high temperatures from 75 to 95°C. After 9 h of cultivation of transformed E. coli in the bioreactor, the cell biomass reached 26·81 g wet weight (ww) per l accounting for 4289 U enzyme activity compared with 1·978 g ww per l producing 256 U activity in shake-flask cultures. In silico analysis revealed a β-propeller structure of phytase. Conclusions: This is the first report of its kind on the purification and successful in vitro refolding of Bacillus phytase from the inclusion bodies formed in the transformed E. coli. Significance and Impact of the Study: Efficient and reproducible protocols for cloning, expression, purification and in vitro refolding of Bacillus phytase enzyme from the transformed E. coli have been developed. The novel phytase, with broad pH and temperature range, renaturation ability and substrate specificity, appears promising as an ideal feed supplement. Identification of site between 179th amino acid leucine and 180th amino acid asparagine offers scope for insertion of small peptides/domains for production of chimeric genes without altering enzyme activity. 相似文献
6.
Laima Baranauskaite Genovaite Gedminiene Zana Bumeliene Vladas-Algirdas Bumelis 《Biocatalysis and Biotransformation》2013,31(3-4):185-189
Escherichia. coli cells expressing porcine growth hormone were grown in a batch fermentation process. The expression level was estimated to be nearly 40% of the total cellular protein after 2–3 h of induction with 1?mM isopropyl β-d-thiogalactoside. Porcine growth hormone expressed as inclusion bodies was solubilized in 8 M urea. Refolding conditions following a dilution protocol in the presence of β-mercaptoethanol or using a glutathione pair were tested. Reverse phase-HPLC was applied to distinguish oxidized, misfolded and reduced forms of the hormone. A ratio of reduced to oxidized glutathione equal to 2/1 was chosen to avoid the formation of misfolded forms at high protein concentration. 相似文献
7.
Trésaugues L Collinet B Minard P Henckes G Aufrère R Blondeau K Liger D Zhou CZ Janin J Van Tilbeurgh H Quevillon-Cheruel S 《Journal of structural and functional genomics》2004,5(3):195-204
The South-Paris Yeast Structural Genomics Project aims at systematically expressing, purifying and determining the structure of S. cerevisiae proteins with no detectable homology to proteins of known structure. We brought 250 yeast ORFs to expression in E. coli, but 37% of them form inclusion bodies. This important fraction of proteins that are well expressed but lost for structural studies prompted us to test methodologies to recover these proteins. Three different strategies were explored in parallel on a set of 20 proteins: (1) refolding from solubilized inclusion bodies using an original and fast 96-well plates screening test, (2) co-expression of the targets in E. coli with DnaK-DnaJ-GrpE and GroEL-GroES chaperones, and (3) use of the cell-free expression system. Most of the tested proteins (17/20) could be resolubilized at least by one approach, but the subsequent purification proved to be difficult for most of them. 相似文献
8.
Gonzalez-Montalban N Natalello A García-Fruitós E Villaverde A Doglia SM 《Biotechnology and bioengineering》2008,100(4):797-802
Recent observations indicate that bacterial inclusion bodies formed in absence of the main chaperone DnaK result largely enriched in functional, properly folded recombinant proteins. Unfortunately, the molecular basis of this intriguing fact, with obvious biotechnological interest, remains unsolved. We have explored here two non-excluding physiological mechanisms that could account for this observation, namely selective removal of inactive polypeptides from inclusion bodies or in situ functional activation of the embedded proteins. By combining structural and functional analysis, we have not observed any preferential selection of inactive and misfolded protein species by the dissagregating machinery during inclusion body disintegration. Instead, our data strongly support that folding intermediates aggregated as inclusion bodies could complete their natural folding process once deposited in protein clusters, which conduces to significant functional activation. In addition, in situ folding and protein activation in inclusion bodies is negatively regulated by the chaperone DnaK. 相似文献
9.
Aggresomes, inclusion bodies and protein aggregation 总被引:34,自引:0,他引:34
Kopito RR 《Trends in cell biology》2000,10(12):173-530
Intracellular and extracellular accumulation of aggregated protein are linked to many diseases, including ageing-related neurodegeneration and systemic amyloidosis. Cells avoid accumulating potentially toxic aggregates by mechanisms including the suppression of aggregate formation by molecular chaperones and the degradation of misfolded proteins by proteasomes. Once formed, aggregates tend to be refractory to proteolysis and to accumulate in inclusion bodies. This accumulation has been assumed to be a diffusion-limited process, but recent studies suggest that, in animal cells, aggregated proteins are specifically delivered to inclusion bodies by dynein-dependent retrograde transport on microtubules. This microtubule-dependent inclusion body is called an aggresome. 相似文献
10.
Vitreoscilla hemoglobin (VHb), a homodimeric protein containing two heme groups in its native state, was used as a model to investigate inclusion body approtein solubilization, prosthetic group incorporation, and reactivation. High-level expression in recombinant Escherichia coli results in accumulation of a substantial portion of heme-free VHb in inclusion bodies. VHb can be solubilized from these inclusion bodies by relatively low concentrations of urea with the dissolution midpoint at approximately 3.2M urea. Dissolution in the presence of stoichiometric heme shifts the dissolution midpoint to approximately 4.5M urea without influencing the dissolution properties of contaminant proteins, suggesting the effect is specific for VHb. Denaturation of apoVHb and holoVHb obtained from purified native VHb has midpoints of 2.9M and 5.1M urea, respectively. VHb solubilized from inclusion bodies with urea at concentrations from 0 to 3.5M urea can be regenerated by heme addition without dilution of urea to yield active holoVHb. The fraction of solubilized VHb reconstituted upon heme addition is maximum at around 30% when solubilization and reconstitution is conducted in less than 1M urea. At these low urea concentrations, approximately 5% of inclusion body VHb is solubilized. These results show the utility of prosthetic group addition to reconstitute holoVHb in the presence of urea. Also, these findings suggest that some inclusion body protein has partially folded conformation and that a fractional dissolution and refolding process may be advantageous. 相似文献
11.
Lee SH Carpenter JF Chang BS Randolph TW Kim YS 《Protein science : a publication of the Protein Society》2006,15(2):304-313
High hydrostatic pressure (HHP)-mediated solubilization and refolding of five inclusion bodies (IBs) produced from bacteria, three gram-negative binding proteins (GNBP1, GNBP2, and GNBP3) from Drosophila, and two phosphatases from human were investigated in combination of a redox-shuffling agent (2 mM DTT and 6 mM GSSG) and various additives. HHP (200 MPa) combined with the redox-shuffling agent resulted in solubilization yields of approximately 42%-58% from 1 mg/mL of IBs. Addition of urea (1 and 2 M), 2.5 M glycerol, L-arginine (0.5 M), Tween 20 (0.1 mM), or Triton X-100 (0.5 mM) significantly enhanced the solubilization yield for all proteins. However, urea, glycerol, and nonionic surfactants populated more soluble oligomeric species than monomeric species, whereas arginine dominantly induced functional monomeric species (approximately 70%-100%) to achieve refolding yields of approximately 55%-78% from IBs (1 mg/mL). Our results suggest that the combination of HHP with arginine is most effective in enhancing the refolding yield by preventing aggregation of partially folded intermediates populated during the refolding. Using the refolded proteins, the binding specificity of GNBP2 and GNBP3 was newly identified the same as with that of GNBP1, and the enzymatic activities of the two phosphatases facilitates their further characterization. 相似文献
12.
Amber Haynes Fradkin Carl S. Boand Stephen P. Eisenberg Mary S. Rosendahl Theodore W. Randolph 《Biotechnology progress》2010,26(3):743-749
We expressed recombinant murine growth hormone (rmGH) in E. coli as a cost‐effective way to produce large quantities (gram scale) of the protein for use in murine studies of immunogenicity to therapeutic proteins. High hydrostatic pressure was used to achieve high solubility and high refolding yields of rmGH protein produced in E. coli inclusion bodies. A two‐step column purification protocol was used to produce 99% pure monomeric rmGH. Secondary and tertiary structures of purified rmGH were investigated using circular dichroism and 2D‐UV spectroscopy. The purified rmGH produced was found to be biologically active in hypophysectomized rats. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010 相似文献
13.
Raghava S Barua B Singh PK Das M Madan L Bhattacharyya S Bajaj K Gopal B Varadarajan R Gupta MN 《Protein science : a publication of the Protein Society》2008,17(11):1987-1997
Many recombinant eukaryotic proteins tend to form insoluble aggregates called inclusion bodies, especially when expressed in Escherichia coli. We report the first application of the technique of three-phase partitioning (TPP) to obtain correctly refolded active proteins from solubilized inclusion bodies. TPP was used for refolding 12 different proteins overexpressed in E. coli. In each case, the protein refolded by TPP gave either higher refolding yield than the earlier reported method or succeeded where earlier efforts have failed. TPP-refolded proteins were characterized and compared to conventionally purified proteins in terms of their spectral characteristics and/or biological activity. The methodology is scaleable and parallelizable and does not require subsequent concentration steps. This approach may serve as a useful complement to existing refolding strategies of diverse proteins from inclusion bodies. 相似文献
14.
Takeshi Sangawa Sanae Tabata Kei Suzuki Yasushi Saheki Keiji Tanaka Junichi Takagi 《Protein science : a publication of the Protein Society》2013,22(6):840-850
Expression and purification of aggregation‐prone and disulfide‐containing proteins in Escherichia coli remains as a major hurdle for structural and functional analyses of high‐value target proteins. Here, we present a novel gene‐fusion strategy that greatly simplifies purification and refolding procedure at very low cost using a unique hyperacidic module derived from the human amyloid precursor protein. Fusion with this polypeptide (dubbed FATT for Flag‐Acidic‐Target Tag) results in near‐complete soluble expression of variety of extracellular proteins, which can be directly refolded in the crude bacterial lysate and purified in one‐step by anion exchange chromatography. Application of this system enabled preparation of functionally active extracellular enzymes and antibody fragments without the need for condition optimization. 相似文献
15.
Protein folding in vivo and renaturation of recombinant proteins from inclusion bodies 总被引:13,自引:0,他引:13
Eukaryotic proteins expressed inEscherichia coli often accumulate within the cell as insoluble protein aggregates or inclusion bodies. The recovery of structure and activity
from inclusion bodies is a complex process, there are no general rules for efficient renaturation. Research into understanding
how proteins fold in vivo is giving rise to potentially new refolding methods, for example, using molecular chaperones. In
this article we review what is understood about the main three classes of chaperone: the Stress 60, Stress 70, and Stress
90 proteins. We also give an overview of current process strategies for renaturing inclusion bodies, and report the use of
novel developments that have enhanced refolding yields. 相似文献
16.
Sang Joong Park Kang Ryu Chang Woo Suh Young Gyu Chai Oh Byung Kwon Seung Kook Park Eun Kyu Lee 《Biotechnology and Bioprocess Engineering》2002,7(1):1-5
A fusion protein, consisting of a human epidermal growth factor (hEGF) as the recognition domain and human angiogenin as the
toxin domain, can be used as a targeted therapeutic against breast cancer cells among others. The fusion protein was expressed
as inclusion body in recombinantE. coli, and when the conventional, solution-phase refolding process was used the refolding yield was very low due to severe aggregation.
It was probably because of the opposite electric charge at a neutral pH resulting from the vastly different pI values of each
domain. The solidphase refolding process that exploited the ionic interactions between ionic exchanger surface and the fusion
protein was tried, but the adsorption yield was also very low, below 30%, regardless of the resins and pH conditions used.
Therefore, to provide a higher ionic affinity toward the solid matrix, six lysine residues were tagged to theN-terminus of the hEGF domain. When heparin-Sepharose was used as the matrix, the adsorption capacity increased 2.5–3 times
to about 88%. Besides the intrinsic affinity of angiogenin to heparin the poly-lysine tag provided additional ionic affinity.
And the subsequent refolding yield increased nearly 13-fold, fromc.a 4.8% in the conventional refolding of the untagged fusion protein to 63.6%. The process was highly reproducible. The refolded
protein in the column eluate retained R Nase bioactivity, of angiogenin. 相似文献
17.
A refolding strategy was described for on-column refolding of recombinant human interferon-gamma (rhIFN-gamma) inclusion bodies by expanded bed adsorption (EBA) chromatography. After the denatured rhIFN-gamma protein bound onto the cation exchanger of STREAMLINE SP, the refolding process was performed in expanded bed by gradually decreasing the concentration of urea in the buffer and the refolded rhIFN-gamma protein was recovered by the elution in packed bed mode. It was demonstrated that the denatured rhIFN-gamma protein could be efficiently refolded by this method with high yield. Under appropriate experimental conditions, the protein yield and specific activity of rhIFN-gamma was up to 52.7% and 8.18 x 10(6) IU/mg, respectively. 相似文献
18.
Prochymosin contains three disulfide bonds linking Cys45 to Cys50, Cys206 to Cys210, and Cys250 to Cys283. To analyze the disulfide bonding pattern between domain sequences in the recombinant prochymosin molecule solubilized from inclusion bodies by 8 M urea (designated as solubilized prochymosin), a simple peptide mapping method was established. This process consists of thiol alkylation, cleavage with cyanogen bromide, diagonal electrophoresis on polyacrylamide gel, and N-terminal sequencing. By using this procedure it was found that Cys45 and Cys50 located in the N-terminal domain are not mispaired with the cysteine residues, located in the C-terminal domain, in the solubilized wild-type prochymosin and its mutants. This result implies that Cys45 and Cys50, the partners of a native disulfide, are restricted in some ordered structures existing in inclusion bodies and remaining after solubilization. These native structural elements act as folding nuclei to initiate and facilitate correct refolding. The strategy of preserving the native-like structures including native disulfide in the solubilized inclusion bodies to enhance renaturation efficiency may be applicable to other recombinant proteins.Both authors contributed equally to this work 相似文献
19.
We have synthesized and optimized a high-yielding Escherichia coli expression system to produce trypsinogen from anchovy Engraulis japonicus and have developed conditions for its successful refolding. Recombinant anchovy trypsinogen precipitated in E. coli Rosetta (DE3) placI strain as inclusion bodies was denatured by 6 M guanidine-HCl followed by refolding with drop wise addition to a large excess of a folding buffer containing 0.5 M non-detergent sulfobetaine (NDSB-251) and a redox potential of oxidized and reduced glutathiones. The folded trypsinogen
was autocatalytically activated to its mature form, trypsin, and purified with a MonoQ ion-exchange column. NH2-terminal amino acid sequencings revealed that E. coli efficiently processed NH2-terminal methionine residue from the expressed trypsinogen and that trypsinogen was activated at the correct site to generate
active trypsin. The recombinant enzyme showed kinetic properties comparable to those of the native enzyme and demonstrated
a typical cleavage preference for arginine over lysine residue against a protein substrate. The optimized expression and folding
procedures yielded 12 mg of purified, active trypsin from 1 L of bacterial culture or 45 g wet weight cells, which is quite
enough for various analytical and semipreparative purposes. 相似文献
20.
Francis VG Majeed MA Gummadi SN 《Journal of industrial microbiology & biotechnology》2012,39(7):1041-1048
Human phospholipid scramblase (hPLSCR1) is a transmembrane protein involved in rapid bidirectional scrambling of phospholipids across the plasma membrane in response to elevated intracellular calcium (Ca(2+)) levels. Overexpression of recombinant hPLSCR1 in Escherichia coli BL21 (DE3) leads to its deposition in inclusion bodies (IBs). N-lauroyl sarcosine was used to solubilize IBs and to recover functionally active hPLSCR1 from them. Protein was purified to homogeneity by nickel-nitrilotriacetic acid (Ni(2+)-NTA) affinity chromatography and was >98% pure. Functional activity of the purified protein was validated by in vitro reconstitution studies, ~18% of 7-nitrobenz-2-oxa-1, 3-diazol-4-yl-phosphatidylcholine (NBD-PC) phospholipids was translocated across the lipid bilayer in the presence of Ca(2+) ions. Far ultraviolet circular dichroism (UV-CD) studies reveal that the secondary structure of protein is predominantly an α-helix, and under nondenaturing conditions, the protein exists as a monomer. Here we describe a method to purify recombinant membrane protein with higher yield than previously described methods involving renaturation techniques. 相似文献