scholarly journals Different expression levels of two KgmB-His fusion proteins

2005 ◽  
Vol 70 (12) ◽  
pp. 1401-1407 ◽  
Author(s):  
Sandra Markovic ◽  
Sandra Vojnovic ◽  
Milija Jovanovic ◽  
Branka Vasiljevic
Keyword(s):  
Recombinant Proteins ◽  
Fusion Proteins ◽  
Kanamycin Resistance ◽  
Expression Vectors ◽  
E Coli ◽  
C Terminus ◽  
N Terminus ◽  
Different Levels ◽  
Streptomyces Tenebrarius

The KgmB methylase from Streptomyces tenebrarius was expressed and purified using the QIAexpress System. Two expression vectors were made: pQEK-N, which places a (His)6 tag at the N-terminus, and pQEK-C, which places a (His)6 tag at the C-terminus of the recombinant KgmB protein. Kanamycin resistance of the E. coli cells containing either the pQEK-N or the pQEK-C recombinant plasmids confirmed the functionality of both KgmB-His fusion proteins in vivo. Interestingly, different levels of expression were observed between these two recombinant proteins. Namely, KgmB methylase with the (His)6 tag at the N-terminus showed a higher level of expression. Purification of the (His)6-tagged proteins using Ni-NTA affinity chromatography was performed under native conditions and the KgmB methylase with (His)6 tag at the N-terminus was purified to homogeneity >95 %. The recombinant KgmB protein was detected on a Western blot using anti-Sgm antibodies.

scholarly journals Expression of Overlapping PreS1 Fragment Recombinant Proteins for the Determination of Immunogenic Domains in HBsAg PreS1 Region

2004 ◽  
Vol 36 (6) ◽  
pp. 397-404 ◽  
Author(s):  
Wei-Guo Hu ◽  
Jun Wei ◽  
Xin-Xiu Yang ◽  
Heng-Chuan Xia ◽  
Feng Li ◽  
...  
Keyword(s):  
Recombinant Proteins ◽  
Fusion Proteins ◽  
Competitive Elisa ◽  
Soluble Form ◽  
Immunological Property ◽  
E Coli ◽  
C Terminus ◽  
New Vaccines ◽  
Gst Fusion Proteins

Abstract In this study, eight preS1 fragments overlapped in preS1 (21–119) region of HBV adr subtype, i.e. preS1 (21–47), preS1 (34–59), preS1 (48–70), preS1 (60–85), preS1 (71–94), preS1 (86–109), preS1 (95–119) and preS1 (21–119), were cloned by PCR, and expressed as GST fusion proteins. These GST-preS1 fusion proteins were highly expressed in soluble form in E. coli, and about 50 to 90 mg soluble fusion proteins were purified from 1 L culture. Using these fusion proteins, the immunogenic domains in preS1 (21–119) region were identified by Western blot analysis and competitive ELISA. The results showed that the immunogenic domains mainly existed in preS1 (21–59) in N-terminus and preS1 (95–109) in C-terminus, and more importantly, a major immunogenic domain preS1 (34–59), which has much stronger immunogenicity, was identified. It was also supported by the predictions of secondary structure and immunological property in the preS1 (21–119) region. The results here would be helpful for the design of new vaccines against HBV.

scholarly journals Cotranslational assembly imposes evolutionary constraints on homomeric proteins

2016 ◽  
Author(s):  
Eviatar Natan ◽  
Tamaki Endoh ◽  
Liora Haim-Vilmovsky ◽  
Guilhem Chalancon ◽  
Tilman Flock ◽  
...  
Keyword(s):  
Protein Function ◽  
Protein Complexes ◽  
Evolutionary Constraints ◽  
E Coli ◽  
Protein Library ◽  
C Terminus ◽  
N Terminus ◽  
Polypeptide Chains

AbstractThere is increasing evidence that some proteins fold during translation, i.e. cotranslationally, which implies that partial protein function, including interactions with other molecules, could potentially be unleashed early on during translation. Although little is known about cotranslational assembly mechanisms, for homomeric protein complexes, translation by the ribosome, folding and assembly, should be well-coordinated to avoid misassembly in the context of polysomes. We analysed 3D structures of homomers and identified a statistically significant trend conserved across evolution that supports this hypothesis: namely that homomeric contacts tend to be localized towards the C-terminus rather than N-terminus of homomeric polypeptide chains. To probe this in more detail, we expressed a GFP-based library of 611 homomeric E. coli genes, and analyzing their folding and assembly in vivo. Consistent with our hypothesis, interface residues tend to be located near the N-terminus in cotranslationally aggregating homomers. In order to dissect the mechanisms of folding and assembly under controlled conditions, we engineered a protein library with three variable components: (i) the position and type homomerization domain, (ii) the reporter domain and (iii) the linker length that connects the two. By analyzing the misassembly rates of these engineered constructs in vivo, in vitro and in silico, we confirmed our hypothesis that C-terminal homomerization is favorable to N-terminal homomerization. More generally, these results provide a set of spatiotemporal constraints within polypeptide chains that favor efficient assembly, with implications for protein evolution and design.

Expression and bioactivity of recombinant segments of human perforinThis paper is one of a selection of papers in this Special Issue, entitled International Symposium on Recent Advances in Molecular, Clinical, and Social Medicine, and has undergone the Journal's usual peer-review process.

2007 ◽  
Vol 85 (2) ◽  
pp. 203-208 ◽  
Author(s):  
Hongmei Dong ◽  
Xiaohu Xu ◽  
Mohong Deng ◽  
Xiaojun Yu ◽  
Hu Zhao ◽  
...  
Keyword(s):  
Recombinant Proteins ◽  
Fusion Proteins ◽  
Target Genes ◽  
Review Process ◽  
Peer Review Process ◽  
Nitrilotriacetic Acid ◽  
E Coli ◽  
Recombinant Plasmids ◽  
Expression Plasmids ◽  
Selection Of

The aim of the study was to prepare an active recombinant human perforin by comparing 5 candidate segments of human perforin. Full-length perforin, MAC1 (28–349 aa), MAC2 (166–369 aa), C-100, and N-60 of human perforin were selected as candidate active segments and designated, respectively, HP1, HP2, HP3, HP4, and HP5. The target genes were amplified by PCR and the products were individually subcloned into pGEM-T. The genes for HP1, HP2, HP3, and HP5 were subcloned into pET-DsbA, whereas pET-41a (+) was used as the expression vector of HP4. The fusion proteins were expressed in Escherichia coli BL21pLysS(DE3) and purified using nickel nitrilotriacetic acid (NTA) agarose affinity chromatography. The hemolysis microassay was used as an activity assay of fusion protein. From this study, we obtained the recombinant plasmids pGEM-T-HP1, -HP2, -HP3, -HP4 and -HP5, consisting of 1600, 960, 600, 300bp, and 180, respectively. From these recombinant plasmids, expression plasmids were successfully constructed and expressed in E. coli BL21pLysS(DE3). The resultant fusion proteins, affinity purified using Ni–NTA, were ~80, 58, 45, 44, and 30 kDa, respectively. The recombinant proteins were assayed for activity on hemolysis. HP2 and HP5 were the only recombinant proteins that were active in hemolysis, and the hemolytic function was concentration dependent. These results demonstrate that active recombinant forms of perforin can be synthesized in a prokaryote model. The recombinant N-60 and MAC1 (28–349 aa) of human perforin have the function of forming pores. Our study provides the experimental basis for further investigation on the application of perforin.

A fusion protein with improved thrombolytic effect and low bleeding risk

2009 ◽  
Vol 102 (12) ◽  
pp. 1194-1203 ◽  
Author(s):  
Lisheng Wang ◽  
Qinglin Zhang ◽  
Yide Qin ◽  
Chutse Wu ◽  
Xiudong Wang ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Anticoagulant Activity ◽  
Bleeding Risk ◽  
Fibrin Clot ◽  
Clot Lysis ◽  
Recognition Sequence ◽  
Linker Peptide ◽  
C Terminus ◽  
N Terminus

SummaryTo resolve the therapeutic dilemma between efficacy of thrombolysis and bleeding risk associated with the use of a combination of thrombolytic and anticoagulant treatments, we created a fusion protein. Staphylokinase was fused to the N-terminus of hirudin using thrombin recognition sequence as linker peptide, resulting in a fusion protein STH.We hypothesised that STH would be cleaved by thrombin at the thrombus site, releasing staphylokinase and hirudin to perform bifunctionally, and attenuating bleeding risk. SDS-PAGE andWestern blot analyses indicated that the linker peptide could be specially recognised and cleaved by thrombin. Amidolytic and thromboelastogram assays showed that the N-terminus of hirudin in STH was blocked by staphylokinase and linker peptide, impeding hirudin’s anticoagulant activity. Once cleaved, STH displayed 35.7% of the anticoagulant activity of equimolar hirudin and exhibited anticoagulant effects in the fibrin clot lysis assay.Thrombin-binding and fibrin clot lysis assays showed that the C-terminus of hirudin retained its high affinity for thrombin. Moreover, STH showed improved thrombolytic effects and a lower bleeding risk in animals. Thus, STH may have the capacity to perform bifunctionally and release anticoagulant activity in a thrombus-targeted manner in vivo, which may reduce the bleeding risk that often accompanies high thrombolytic efficacy in the treatment of thromboembolic diseases.

Cdc18p can block mitosis by two independent mechanisms

1998 ◽  
Vol 111 (20) ◽  
pp. 3101-3108 ◽  
Author(s):  
E. Greenwood ◽  
H. Nishitani ◽  
P. Nurse
Keyword(s):  
Dna Replication ◽  
S Phase ◽  
Checkpoint Control ◽  
Replication Checkpoint ◽  
Mitotic Kinase ◽  
C Terminus ◽  
N Terminus ◽  
Dna Replication Checkpoint ◽  
Checkpoint Genes

The DNA replication checkpoint is required to maintain the integrity of the genome, inhibiting mitosis until S phase has been successfully completed. The checkpoint preventing premature mitosis in Schizosaccharomyces pombe relies on phosphorylation of the tyrosine-15 residue on cdc2p to prevent its activation and hence mitosis. The cdc18 gene is essential for both generating the DNA replication checkpoint and the initiation of S phase, thus providing a key role for the overall control and coordination of the cell cycle. We show that the C terminus of the protein is capable of both initiating DNA replication and the checkpoint function of cdc18p. The C terminus of cdc18p acts upstream of the DNA replication checkpoint genes rad1, rad3, rad9, rad17, hus1 and cut5 and requires the wee1p/mik1p tyrosine kinases to block mitosis. The N terminus of cdc18p can also block mitosis but does so in the absence of the DNA replication checkpoint genes and the wee1p/mik1p kinases therefore acting downstream of these genes. Because the N terminus of cdc18p associates with cdc2p in vivo, we suggest that by binding the cdc2p/cdc13p mitotic kinase directly, it exerts an effect independently of the normal checkpoint control, probably in an unphysiological manner.

scholarly journals Insight from TonB Hybrid Proteins into the Mechanism of Iron Transport through the Outer Membrane

2008 ◽  
Vol 190 (11) ◽  
pp. 4001-4016 ◽  
Author(s):  
Wallace A. Kaserer ◽  
Xiaoxu Jiang ◽  
Qiaobin Xiao ◽  
Daniel C. Scott ◽  
Matthew Bauler ◽  
...  
Keyword(s):  
Amino Acids ◽  
Outer Membrane ◽  
Cell Envelope ◽  
Outer Membrane Proteins ◽  
Binding Motif ◽  
Inner Membrane ◽  
E Coli ◽  
Hybrid Proteins ◽  
C Terminus ◽  
N Terminus

ABSTRACT We created hybrid proteins to study the functions of TonB. We first fused the portion of Escherichia coli tonB that encodes the C-terminal 69 amino acids (amino acids 170 to 239) of TonB downstream from E. coli malE (MalE-TonB69C). Production of MalE-TonB69C in tonB + bacteria inhibited siderophore transport. After overexpression and purification of the fusion protein on an amylose column, we proteolytically released the TonB C terminus and characterized it. Fluorescence spectra positioned its sole tryptophan (W213) in a weakly polar site in the protein interior, shielded from quenchers. Affinity chromatography showed the binding of the TonB C-domain to other proteins: immobilized TonB-dependent (FepA and colicin B) and TonB-independent (FepAΔ3-17, OmpA, and lysozyme) proteins adsorbed MalE-TonB69C, revealing a general affinity of the C terminus for other proteins. Additional constructions fused full-length TonB upstream or downstream of green fluorescent protein (GFP). TonB-GFP constructs had partial functionality but no fluorescence; GFP-TonB fusion proteins were functional and fluorescent. The activity of the latter constructs, which localized GFP in the cytoplasm and TonB in the cell envelope, indicate that the TonB N terminus remains in the inner membrane during its biological function. Finally, sequence analyses revealed homology in the TonB C terminus to E. coli YcfS, a proline-rich protein that contains the lysin (LysM) peptidoglycan-binding motif. LysM structural mimicry occurs in two positions of the dimeric TonB C-domain, and experiments confirmed that it physically binds to the murein sacculus. Together, these findings infer that the TonB N terminus remains associated with the inner membrane, while the downstream region bridges the cell envelope from the affinity of the C terminus for peptidoglycan. This architecture suggests a membrane surveillance model of action, in which TonB finds occupied receptor proteins by surveying the underside of peptidoglycan-associated outer membrane proteins.

scholarly journals Overexpression of Trigger Factor Prevents Aggregation of Recombinant Proteins in Escherichia coli

2000 ◽  
Vol 66 (3) ◽  
pp. 884-889 ◽  
Author(s):  
Kazuyo Nishihara ◽  
Masaaki Kanemori ◽  
Hideki Yanagi ◽  
Takashi Yura
Keyword(s):  
Escherichia Coli ◽  
Recombinant Proteins ◽  
Protein Production ◽  
Trigger Factor ◽  
Human Lysozyme ◽  
E Coli ◽  
Constructed Plasmids ◽  
Expression Plasmids

ABSTRACT To examine the effects of overexpression of trigger factor (TF) on recombinant proteins produced in Escherichia coli, we constructed plasmids that permitted controlled expression of TF alone or together with the GroEL-GroES chaperones. The following three proteins that are prone to aggregation were tested as targets: mouse endostatin, human oxygen-regulated protein ORP150, and human lysozyme. The results revealed that TF overexpression had marked effects on the production of these proteins in soluble forms, presumably through facilitating correct folding. Whereas overexpression of TF alone was sufficient to prevent aggregation of endostatin, overexpression of TF together with GroEL-GroES was more effective for ORP150 and lysozyme, suggesting that TF and GroEL-GroES play synergistic roles in vivo. Although coexpression of the DnaK-DnaJ-GrpE chaperones was also effective for endostatin and ORP150, coexpression of TF and GroEL-GroES was more effective for lysozyme. These results attest to the usefulness of the present expression plasmids for improving protein production inE. coli.

scholarly journals Localization of Functional Domains of the Mitogenic Toxin of Pasteurella multocida

2001 ◽  
Vol 69 (12) ◽  
pp. 7839-7850 ◽  
Author(s):  
Gillian D. Pullinger ◽  
R. Sowdhamini ◽  
Alistair J. Lax
Keyword(s):  
Amino Acids ◽  
Fusion Proteins ◽  
Pasteurella Multocida ◽  
Transmembrane Domain ◽  
Polyclonal Antibodies ◽  
Full Length ◽  
Cell Binding ◽  
Terminal Fragment ◽  
C Terminus ◽  
N Terminus

ABSTRACT The locations of the catalytic and receptor-binding domains of thePasteurella multocida toxin (PMT) were investigated. N- and C-terminal fragments of PMT were cloned and expressed as fusion proteins with affinity tags. Purified fusion proteins were assessed in suitable assays for catalytic activity and cell-binding ability. A C-terminal fragment (amino acids 681 to 1285) was catalytically active. When microinjected into quiescent Swiss 3T3 cells, it induced changes in cell morphology typical of toxin-treated cells and stimulated DNA synthesis. An N-terminal fragment with a His tag at the C terminus (amino acids 1 to 506) competed with full-length toxin for binding to surface receptors and therefore contains the cell-binding domain. The inactive mutant containing a mutation near the C terminus (C1165S) also bound to cells in this assay. Polyclonal antibodies raised to the N-terminal PMT region bound efficiently to full-length native toxin, suggesting that the N terminus is surface located. Antibodies to the C terminus of PMT were microinjected into cells and inhibited the activity of toxin added subsequently to the medium, confirming that the C terminus contains the active site. Analysis of the PMT sequence predicted a putative transmembrane domain with predicted hydrophobic and amphipathic helices near the N terminus over the region of homology to the cytotoxic necrotizing factors. The C-terminal end of PMT was predicted to be a mixed α/β domain, a structure commonly found in catalytic domains. Homology to proteins of known structure and threading calculations supported these assignments.

scholarly journals The C-terminus of cytochrome b5 confers endoplasmic reticulum specificity by preventing spontaneous insertion into membranes

2007 ◽  
Vol 401 (3) ◽  
pp. 701-709 ◽  
Author(s):  
Matthew P. A. Henderson ◽  
Yeen Ting Hwang ◽  
John M. Dyer ◽  
Robert T. Mullen ◽  
David W. Andrews
Keyword(s):  
Endoplasmic Reticulum ◽  
Subcellular Localization ◽  
Lipid Bilayers ◽  
Fusion Proteins ◽  
Molecular Mechanisms ◽  
Cytochrome B5 ◽  
Terminal Sequence ◽  
C Terminus

The molecular mechanisms that determine the correct subcellular localization of proteins targeted to membranes by tail-anchor sequences are poorly defined. Previously, we showed that two isoforms of the tung oil tree [Vernicia (Aleurites) fordii] tail-anchored Cb5 (cytochrome b5) target specifically to ER (endoplasmic reticulum) membranes both in vivo and in vitro [Hwang, Pelitire, Henderson, Andrews, Dyer and Mullen (2004) Plant Cell 16, 3002–3019]. In the present study, we examine the targeting of various tung Cb5 fusion proteins and truncation mutants to purified intracellular membranes in vitro in order to assess the importance of the charged CTS (C-terminal sequence) in targeting to specific membranes. Removal of the CTS from tung Cb5 proteins resulted in efficient binding to both ER and mitochondria. Results from organelle competition, liposome-binding and membrane proteolysis experiments demonstrated that removal of the CTS results in spontaneous insertion of tung Cb5 proteins into lipid bilayers. Our results indicate that the CTSs from plant Cb5 proteins provide ER specificity by preventing spontaneous insertion into incorrect subcellular membranes.

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