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1.
A system for the production of soluble interferon (IFN)-λ2 was developed by fusing the IFN-λ2, NusA protein, polyhistidine
and S peptide genes and then expressing the fused product (Nus-His-S-tagged IFN-λ2) in Escherichia coli. The expressed fusion protein was purified by Ni-NTA affinity chromatography. The fusion tag was removed from recombinant
IFN-λ2 by cleavage with enterokinase. N-Terminal sequencing confirmed the identity of the purified protein. When compared with commercial IFN-α2b, IFN-λ2 had similar
antiviral activity. The production and characterization of biologically active IFN-λ2 will be beneficial for its potential
role in clinical applications. 相似文献
2.
Enhanced protease cleavage efficiency on the glucagon-fused interleukin-2 by the addition of synthetic oligopeptides 总被引:1,自引:1,他引:0
Kim SW Kim JB Lee WS Jung WH Ryu JM Jang HW Jo YB Jung JK Kim JH 《Protein expression and purification》2007,55(1):159-165
Human interleukin-2 (hIL-2) was produced as a recombinant fusion protein (G3.IL-2/HF) consisting of three tandem-arranged human glucagon molecules (G3) and hIL-2. For the recovery of hIL-2, a factor Xa (FXa) cleavage sequence was introduced next to the N-terminus of hIL-2. Cleavage efficiency on this recombinant protein construct was very low because its recognition sequence was sterically hindered within the G3.IL-2/HF molecule and hence FXa access to the cleavage site was insufficient. We therefore introduced various synthetic oligopeptides upstream from the FXa cleavage site as a means to change substrate conformation and thereby increase cleavage efficiency. Among these oligopeptides, acidic or nucleophilic constructs were the most effective for the FXa-mediated cleavage of the fusion protein. In addition, insertion of various oligopeptides into the G3.IL-2/HF molecule varied the solubility of each construct depending on their physical properties. Consequently, the G3.IL-2/DF construct showed the highest final hIL-2 yields via FXa-mediated removal of the fusion partner. Lastly, we confirmed that cleavage efficiency was greatly increased but native hIL-2 was cleaved internally by non-specific cleavage when the acidic oligopeptide D4 (DDDD) was introduced upstream of the EK cleavage site within G3.IL-2/HE molecule. The G3.IL-2/HE molecule was shown to be an inefficient substrate to EK in a previous report (Biotechnol. Bioprocess Eng. (2000) 5, 13-16). 相似文献
3.
4.
Chang Soo Kang Seung-Yeol Son In Seok Bang 《Journal of microbiology (Seoul, Korea)》2008,46(6):656-661
The cabbage butterfly (Artogeia rapae) antimicrobial peptide hinnavinII as a member of cecropin family is synthesized as 37 residues in size with an amidated lysine
at C-terminus and shows the humoral immune response to a bacterial invasion. In this work, a synthetic gene for hinnavinII-38-Asn
(HIN) with an additional amino acid asparagine residue containing amide group at C-terminus was cloned into pET-32a(+) vector
to allow expression of HIN as a Trx fusion protein in Escherichia coli strain BL21 (DE3) pLysS. The resulting expression level of the fusion protein Trx-HIN could reach 15–20% of the total cell
proteins and more than 70% of the target proteins were in soluble form. The fusion protein could be purified successfully
by HiTrap Chelating HP column and a high yield of 15 mg purified fusion protein was obtained from 80 ml E. coli culture. Recombinant HIN was readily obtained by enterokinase cleavage of the fusion protein followed by FPLC chromatography,
and 3.18 mg pure active recombinant HIN was obtained from 80 ml culture. The molecular mass of recombinant HIN determined
by MALDI-TOF mass spectrometer is 4252.084 Da which matches the theoretical mass (4252.0 Da) of HIN. Comparing the antimicrobial
activities of the recombinant hinnavinII with C-amidated terminus to that without an amidated C-terminus, we found that the
amide of asparagine at C-terminus of hinnavinII improved its potency on certain microorganism such as E. coli, Enterobacter cloacae, Bacillus megaterium, and Staphylococcus aureus. 相似文献
5.
C. S. Shin M. S. Hong D. Y. Kim H. C. Shin J. Lee 《Applied microbiology and biotechnology》1998,49(4):364-370
Synthesis of two recombinant proteins (human glucagon and human growth hormone) was investigated in fed-batch cultures at
high cell concentrations of recombinant Escherichia coli. The glucose-limited growth was achieved without accumulation of metabolic by-products and hence the cellular environment
is presumed invariable during growth and recombinant protein synthesis. Via exponential feeding in the two-phase fed-batch
operation, the specific cell growth rate was successfully controlled at the desired rates and the fed-batch mode employed
is considered appropriate for examining the correlation between the specific growth rate and the efficiency of recombinant
product formation in the recombinant E. coli strains. The two recombinant proteins were expressed as fusion proteins and the concentration in the culture broth was increased
to 15 g fusion growth hormone l−1 and 7 g fusion glucagon l−1. The fusion growth hormone was initially expressed as soluble protein but seemed to be gradually aggregated into inclusion
bodies as the expression level increased, whereas the synthesized fusion glucagon existed as a cytoplasmic soluble protein
during the whole induction period. The stressful conditions of cultivation employed (i.e. high-cell-density cultivation at
low growth rate) may induce the increased production of various host-derived chaperones and thereby enhance the folding efficiency
of synthesized heterologous proteins. The synthesis of the recombinant fusion proteins was strongly growth-dependent and more
efficient at a higher specific growth rate. The mechanism linking specific growth rate with recombinant protein productivity
is likely to be related to the change in cellular ribosomal content.
Received: 27 May 1997 / Received last revision: 31 October 1997 / Accepted: 21 November 1997 相似文献
6.
Dae-Young Kim Nam-Kyu Shin Seung-Gu Chang Hang-Cheol Shin 《Biotechnology Techniques》1996,10(9):669-672
Summary A novel approach to the production of a human glucagon in E. coli is described. The 29 amino acids of human glucagon and pentapeptide linker containing enzyme processing site were fused at the amino terminus to a 57 residue N-terminal portion of the human tumor necrosis factor-alpha (hTNF-). The fusion protein was expressed in the E. coli cytoplasm at levels up to 30% of the total cell protein. Precipitation of the fusion protein near its isoelectric point, specific enterokinase cleavage at the linker site and subsequent HPLC purification makes this approach suitable for the production of glucagon as well as other relatively small peptides with therapeutic interests. 相似文献
7.
Kazumasa Yoshikawa Hiroshige Tsuzuki Masafumi Fujimoto Masahiro Tohkin Takashi Matsubara Hiroshi Yonezawa Hiroyuki Okamoto Hiroshi Teraoka Nobuo Yoshida 《Journal of Protein Chemistry》1992,11(5):517-525
Recombinant glucagon was expressed inEscherichia coli as a fusion protein including the glucagon sequence therein as previously reported [Ishizakiet al. (1992).Appl. Microbiol. Biotechnol.36, 483–486]. We developed a large-scale method for the isolation and purification of recombinant glucagon. After cell disruption, the resultant pellets were solubilized with 2 M guanidine-HCl, to whichStaphylococcus aureus V8 protease had been added, and were digested into intermediates composed of 53- and 60-residue peptides containing the glucagon moiety. After the digestion came to an end, the solution was desalted, and the remaining V8 protease was allowed to resume digestion of the intermediates into glucagon, followed by partial purification by S-Sepharose and Sephacryl S-100 chromatographies. The glucagon obtained was found to be not less than 99.5% pure by analytical HPLC. One liter of culture produced about 180 mg of pure glucagon. The amino acid composition and the sequence agreed well with the theoretical values. Radioreceptor assay gave an affinity constant similar to that of pancreatic glucagon, and similar activities in cAMP production and glycogenolysis were also observed. Thus, the recombinant glucagon was confirmed to be biochemically identical with pancreatic glucagon. 相似文献
8.
A simple method for the purification of an antimicrobial peptide in recombinant Escherichia coli 总被引:2,自引:0,他引:2
Hwang SW Lee JH Park HB Pyo SH So JE Lee HS Hong SS Kim JH 《Molecular biotechnology》2001,18(3):193-198
A magainin derivative, designated MSI-344, was produced in Escherichia coli as fusion protein, by utilizing a truncated amidophsphoribosyltransferase of E. coli as a fusion partner. Bacterial cells transformed with the gene encoding the fusion protein were grown to a high cell density
and induced with isopropyl-1-thio-b-D-galatoside (IPTG) to initiate product expression. The fusion protein was accumulated
into cytoplasmic inclusion body and recombinant MSI-344 was released from the fusion partner by hydroxylamine treatment. Following
cleavage of the fusion protein with hydroxylamine, the released MSI-344 was purified to homogeneity by cationic exchange chromatography.
The final purity was at least 95% by reversed-phase high performance liquid chromatography (RP-HPLC). Purified recombinant
MSI-344 was found to be indistinguishable from the synthetic peptide determined by amino acid sequences and antimicrobial
activity assay. 相似文献
9.
Jianying Luo Julie Choulet James C. Samuelson 《Protein science : a publication of the Protein Society》2009,18(8):1735-1744
We have designed a novel protein fusion partner (P8CBD) to utilize the co‐translational SRP pathway in order to target heterologous proteins to the E. coli inner membrane. SRP‐dependence was demonstrated by analyzing the membrane translocation of P8CBD‐PhoA fusion proteins in wt and SRP‐ffh77 mutant cells. We also demonstrate that the P8CBD N‐terminal fusion partner promotes over‐expression of a Thermotoga maritima polytopic membrane protein by replacement of the native signal anchor sequence. Furthermore, the yeast mitochondrial inner membrane protein Oxa1p was expressed as a P8CBD fusion and shown to function within the E. coli inner membrane. In this example, the mitochondrial targeting peptide was replaced by P8CBD. Several practical features were incorporated into the P8CBD expression system to aid in protein detection, purification, and optional in vitro processing by enterokinase. The basis of membrane protein over‐expression toxicity is discussed and solutions to this problem are presented. We anticipate that this optimized expression system will aid in the isolation and study of various recombinant forms of membrane‐associated protein. 相似文献
10.
Lei Huang Susanna Su Jan Leong Rongrong Jiang 《Applied microbiology and biotechnology》2009,84(2):301-308
This study reports the first successful recombinant expression of cationic antimicrobial peptides human beta-defensin-26 and
human beta-defensin-27 in Escherichia coli. HBD26 and HBD27 genes were synthesized through codon optimization, and each gene was then cloned into the expression vector
pET32, which feature fusion protein thioredoxin at the N-terminal. The recombinant plasmids were then transformed into E. coli BL21 (DE3) and cultured in MBL medium, which gave yields of HBD26 and HBD27 fusion proteins of up to 1.38 and 1.29 g l−1, respectively. Affinity chromatography was used to purify the soluble fusion proteins, and the N-terminal TrxA tags were
cleaved off by enterokinase. Pure HBD26 and HBD27 were then obtained by cationic exchange chromatography. The overall recovery
of HBD26 was 38% and that of HBD27 reached 36%. Both variants showed salt-sensitive antimicrobial activity against gram-negative
E. coli but not against gram-positive Staphylococcus aureus and Saccharomyces cerevisiae. 相似文献
11.
Rohinton Edalji Tami J. Pilot-Matias Steven D. Pratt David A. Egan Jean M. Severin Earl G. Gubbins Andrew M. Petros Stephen W. Fesik Neal S. Burres Thomas F. Holzman 《Journal of Protein Chemistry》1992,11(3):213-223
The human peptidyl-prolyl isomerase FK-binding protein (FKBP) was cloned as a fusion partner with CMP-KDO synthetase (CKS), and the resultant construct was characterized as an improved high-expression source for FKBP. The CKS-FKBP fusion was expressed as a soluble protein at levels approaching 1 gm/L inEscherichia coli fermentations. The fusion protein was purified to near homogeneity by a one-step ammonium sulfate fractionation of whole cell lysate. After selective cleavage, the fusion precursor produced yields approaching 300 mg of purified FKBP per liter of harvested culture, a 30 to 60-fold increase over that observed for a nonfusion construct. Selective cleavage of the fusion partners was accomplished using either hydroxylamine or specific, limited proteolysis. Once separated from the CKS fusion partner, the FKBP was isolated in a single step by either reversed-phase HPLC or chromatography on Q-Sepharose. For comparison of physical and chemical properties, a nonfusion construct of recombinant human FKBP was expressed inE. coli and isolated. The purified FKBPs exhibited expected SDS-PAGE molecular weights and N-terminal sequences. The proteins had similar proton NMR spectra and binding to [3H]FK-506. The fusion construct, CKS-FKBP, was also found to bind [3H]FK-506. These data indicate that FKBP fused to the C-terminus of CKS folds independently of the fusion partner and suggests the fused FKBP adopts a conformation resembling that of the native protein. 相似文献
12.
The fusion protein of green fluorescent protein (GFP) and human interleukin-2 (hIL-2) was produced in insect Trichoplusia ni larvae infected with recombinant baculovirus derived from the Autographa californica nuclear polyhedrosis virus (AcNPV). This fusion protein was composed of a metal ion binding site (His)6 for rapid one-step purification using immobilized metal affinity chromatography (IMAC), UV-optimized GFP (GFPuv), enterokinase cleavage site for recovering hIL-2 from purified fusion protein, and hIL-2 protein. The additional histidine residues on fusion protein enabled the efficient purification of fusion protein based on immobilized metal affinity chromatography. In addition to advantages of GFP as a fusion marker, GFP was able to be used as a selectable purification marker; we easily determined the correct purified fusion protein sample fraction by simply detecting GFP fluorescence. 相似文献
13.
An evaluation of different enzymatic cleavage methods for recombinant fusion proteins, applied on des(1–3)insulin-like growth factor I 总被引:4,自引:0,他引:4
G?ran Forsberg Barbro Baastrup Helena Rondahl Erik Holmgren Gunnar Pohl Maris Hartmanis Mats Lake 《Journal of Protein Chemistry》1992,11(2):201-211
Different enzymatic methods for cleavage of recombinant fusion proteins were compared. To find an efficient cleavage method, five different fusion proteins were produced. The fusion proteins differed only in the linker region between the fusion partner and the desired product, human des(1–3)insulin-like growth factor I. A cleavage study was performed with enterokinase, plasmin, thrombin, urokinase, and recombinant H64A subtilisin. Significant cleavage was obtained using thrombin, H64A subtilisin, and enterokinase. Thrombin cleavage was studied on a larger scale and des(1–3)IGF-I was recovered at a final yield of 3 mg/L growth medium. Thrombin and enterokinase were also studied as immobilized proteases and they cleaved the fusion proteins with retained activity. To further improve thrombin cleavage, a continuous reactor was constructed, consisting of a closed system with a thrombin column and an ion exchange column in series. Here, the fusion protein circulated while free des(1–3)IGF-I was bound to the ion exchange column after release from the fusion protein. In the reactor, thrombin was as efficient as the free enzyme but gave a diminished rate of product degradation. 相似文献
14.
To prevent in vivo degradation, small peptides are usually expressed in fusion proteins from which target peptides can be
released by proteolytic or chemical reagents. In this report, small ubiquitin-related modifier (SUMO) linked with a hexa-histidine
tag was used as a fusion partner for the production of recombinant human urodilatin, a hormone for the treatment of acute
decompensated heart failure. The fusion protein, which was overexpressed mainly as inclusion bodies in Escherichia coli, constituted about 25% of the total cell proteins. After purification by Ni-sepharose affinity chromatography and renaturation
in refolding buffer, the fusion protein was cleaved with SUMO protease 1. Urodilatin was separated from the fusion partner
by the subtractive chromatography using Ni-sepharose once again, and then further purified with reverse-phase high performance
liquid chromatography. In vitro activity assay demonstrated that the recombinant urodilatin had a potent vasodilatory effect
on rabbit aortic strips with an EC50 of 1.77 ± 0.53 μg/ml, which was similar to that of the synthetic urodilatin standard. The expression strategy presented in
this study allows convenient high yield and easy purification of small recombinant peptides with native sequences.
Z. Sun and Z. Xia contribute equally to the work. 相似文献
15.
Hiroyuki Okamoto Hideo Iwamoto Hiroshige Tsuzuki Hiroshi Teraoka Nobuo Yoshida 《Journal of Protein Chemistry》1995,14(7):521-526
Glucagon was expressed inEscherichia coli as a fusion protein including the glucagon sequence [Ishizakiet al. (1992),Appl. Microbiol. Biotechnol.36, 483–486]. The high-level expression of a protein inE. coli often results in an insoluble aggregate called an inclusion body containing a fusion protein. In our previous report [Yoshikawaet al. (1992),J. Protein Chem.
11, 517–525], we solubilized this inclusion body by using guanidinium chloride. However, the existence of denaturant caused problems such as a low proteolytic activity for transforming the fusion protein into glucagon and complicated purification methods. We tried to improve the method to enable large-scale purification. At alkaline pH, the inclusion body could be solubilized to a high concentration and cleaved by amino acid-specific endopeptidases. By utilizing isoelectric precipitations as a new economical purification method for glucagon from intermediates, the glucagon obtained was shown to be over 99.5% pure by analytical RP-HPLC. The yield was almost equal that of our previous method, and the glucagon produced was chemically and biochemically equivalent to natural glucagon. 相似文献
16.
A cDNA coding mutated cecropin CMIV fromBombyx mori was synthesized according to its amino acid sequence usingE. coli biased codons. The gene was cloned into the fusion expression vector pEZZ318 and was expressed inE. coli HB101. The fusion protein produced was purified by affinity chromatography to yield 26 mg/L fusion product. The anti-bacterial
activities of recombinant cecropin CMIV were recovered after cleavage by chemical method. 相似文献
17.
Heinz Döbeli Herbert Andres Nicola Breyer Nicholas Draeger Dorothea Sizmann Maria Tomás Zuber Brian Weinert Beat Wipf 《Protein expression and purification》1998,12(3):404-414
We report the biotechnical production of peptides of approximately 35–50 amino acids in length containing one intramolecular disulfide bridge, using a recombinant fusion tail approach. This method fills the technological gap when either (a) chemical synthesis fails due to known problematic peptide sequences or (b) if simple recombinant expression is unsuccessful due to degradation. The fusion tail described here serves several purposes: (i) it enables high expression levels inEscherichia colito be achieved; (ii) it renders the fusion protein fairly soluble; (iii) it contains a histidine affinity tag for easy purification on Ni-chelate resins, which also serves as a catalyst for the oxygen-dependent formation of the disulfide bridge; and (iv) it suppresses the formation of concatamers during the oxidation process through steric hindrance. The purified fusion protein is then immobilized on a reversed phase column for two purposes: (i) chemical cleavage of the fusion tail by cyanogen bromide and (ii) subsequent purification of the peptide. A very hydrophilic fusion partner is required so that immobilization on the reversed phase column always occurs due to the peptide. Sensitive hydrophobic residues are thereby protected from the cleavage reagent while the cleaved hydrophilic fusion tail is easily separated from the hydrophobic peptide. The method is exemplified by eight peptides representing an immunodominant epitope of the human immunodeficiency virus, but may be useful for a significant variety of similar peptides. 相似文献
18.
重组牛肠激酶轻链基因在毕赤酵母中的表达与纯化 总被引:1,自引:0,他引:1
目的:构建重组牛肠激酶轻链的基因工程菌,并进行表达和纯化,以获得高纯度和高活性的重组牛肠激酶轻链蛋白。方法:以GenBank公共数据库中的牛肠激酶轻链基因序列(AccessionNo.NM174439)设计引物,利用RT-PCR合成牛肠激酶轻链基因片段,并克隆进pPIC9K载体,同时在基因N端插进6个组氨酸标签,转化毕赤酵母GS115,进行筛选和诱导表达。产物经镍离子螯和层析和Q-SepharoseFF柱纯化,并酶切融合蛋白检测其活性。结果:培养液中重组牛肠激酶轻链蛋白表达量为3.0mg/L。对含有肠激酶酶切位点的IL-11/MBP融合蛋白进行酶切,结果表明,酶解率可达到90%以上。结论:表达并获得了高纯度的重组肠激酶轻链蛋白,为大规模生产打下了基础。 相似文献
19.
Xue-mei Lu Xiao-bao Jin Jia-yong Zhu Han-fang Mei Yan Ma Fu-jiang Chu Yan Wang Xiao-bo Li 《Applied microbiology and biotechnology》2010,87(6):2169-2176
Lysozyme is an abundant, cationic antimicrobial protein that plays an important role in host defense. It targets the β (1–4)
glycosidic bond between N-acetylglucosamine and N-acetylmuramic residues that make up peptidoglycan, making lysozyme highly active against Gram-positive bacteria. However,
lysozyme alone is inactive against Gram-negative bacteria because it cannot reach the peptidoglycan layer. Cecropins are cationic
molecules with a wide range of antimicrobial activities. The main target for these peptides is the cytoplasmic membrane. We
resume that cecopin may disrupt the outer membrane, giving the enzyme access to the peptidoglycan in cell wall. So in the
present study, novel hybrid protein combining Musca domestica cecropin (Mdc) with human lysozyme (Hly) was designed. The DNA sequence encoding recombination fusion protein Mdc–hly was
cloned into the pET-32a vector for protein expression in Escherichia coli strain BL21 (DE3). The protein was expressed as a His-tagged fusion protein, and the Mdc–hly was released from the fusion
by enterokinase cleavage and separated from the carrier thioredoxin. Antimicrobial activity assays showed that the recombinant
fusion protein Mdc–hly has improved in vitro antimicrobial activity and action spectrum compared to Mdc and hly. Mdc–hly may
have important potential application as a future safely administered human drug and food additive. 相似文献
20.
A fusion protein of human interleukin-2 (hIL-2) and green fluorescent protein (GFP) was expressed in insect Sf-9 cells infected with recombinant baculovirus derived from the Autographa californica nuclear polyhedrosis virus (AcNPV). This fusion protein was comprised of a histidine affinity ligand for simplified purification using immobilized metal affinity chromatography (IMAC), UV-optimized GFP (GFPuv) as a marker, an enterokinase cleavage site for recovery of hIL-2 from the fusion, and the model product hIL-2. Successful production of hIL-2 as a fusion protein (approximately 52,000 Da) with GFPuv was obtained. GFPuv enabled rapid monitoring and quantification of the hIL-2 by simply checking the fluorescence, obviating the need for Western blot and/or ELISA assays during infection and production stages. There was no increased 'metabolic burden' due to the presence of GFPuv in the fusion product. The additional histidine residues at the N-terminus enabled efficient one-step purification of the fusion protein using IMAC. Additional advantages of GFP as a fusion marker were seen, particularly during separation and purification in that hIL-2 containing fractions were identified simply by illumination with UV light. Our results demonstrated that GFP was an effective non-invasive on-line marker for the expression and purification of heterologous protein in the suspended insect cell/baculovirus expression system. 相似文献