Over-Expression of a Core Repeat from an Insect Silk Protein that forms Intramolecular Disulfide Bonds

1992 ◽  
Vol 292 ◽  
Author(s):  
Stanley V. Smith ◽  
Steven T. Case
Keyword(s):  
Recombinant Protein ◽  
Electrophoretic Mobility ◽  
Disulfide Bonds ◽  
Protein A ◽  
Reducing Agents ◽  
Silk Protein ◽  
Chironomus Tentans ◽  
Bacterial Cells ◽  
Gene Encoding ◽  
Over Expression

AbstractA gene encoding one complete [C+SR] core repeat from spIa, a 1000-kDa silk protein from Chironomus tentans, was synthesized and its recombinant protein expressed to high levels in bacterial cells. We observed that reducing agents significantly alter the electrophoretic mobility of this protein. A variety of data indicate that the purified recombinant protein is folded and its structure stabilized by two intramolecular disulfide bonds.

Disulfide Bonds in Recombinant Repeat Units From an Aquatic Insect's Silk Protein

1993 ◽  
Vol 330 ◽  
Author(s):  
Steven T. Case ◽  
Stanley V. Smith ◽  
Melyssa R. Bratton
Keyword(s):  
Amino Acid ◽  
Electrospray Ionization ◽  
Disulfide Bonds ◽  
Silk Protein ◽  
Ionization Mass ◽  
Chironomus Tentans ◽  
Wild Type ◽  
Amino Acid Sequencing ◽  
Repeat Units ◽  
Spectroscopic Analyses

ABSTRACTrCAS is a recombinant Constant And Subrepeat (rCAS) protein modelled after tandem core repeats found in a 1000-kDa silk protein synthesized by larvae of the midge, Chironomus tentans. rCAS is encoded by a synthetic gene (synCAS) which is expressed in bacteria. Purified rCAS has four cysteine residues that participate in formation of two intramolecular disulfide bonds. Here we report the results of amino acid sequencing and electrospray ionization mass spectroscopic analyses of tryptic fragments of native and reduced rCAS which suggest that these disulfide bonds are heterogeneous. To assist in studying the formation of disulfide bonds in reduced and refolded rCAS, a series of synCAS mutants were constructed with cysteine to alanine substitutions. In comparison to wild-type rCAS and a Cys79→Ala mutant, the refolding of Cys9→Ala appears to be partially inhibited.

scholarly journals Evolution of Bacterial Global Modulators: Role of a Novel H-NS Paralogue in the EnteroaggregativeEscherichia coliStrain 042

mSystems ◽  
2018 ◽  
Vol 3 (3) ◽  
Author(s):  
A. Prieto ◽  
M. Bernabeu ◽  
S. Aznar ◽  
S. Ruiz-Cruz ◽  
A. Bravo ◽  
...  
Keyword(s):  
Protein A ◽  
Bacterial Cells ◽  
Gene Encoding ◽  
Specific Expression ◽  
Protein Targets ◽  
Global Regulators ◽  
E Coli ◽  
The Family ◽  
Regulatory Properties

ABSTRACTBacterial genomes sometimes contain genes that code for homologues of global regulators, the function of which is unclear. In members of the familyEnterobacteriaceae, cells express the global regulator H-NS and its paralogue StpA. InEscherichia coli, out of providing a molecular backup for H-NS, the role of StpA is poorly characterized. The enteroaggregativeE. colistrain 042 carries, in addition to thehnsandstpAgenes, a third gene encoding anhnsparalogue (hns2). We present in this paper information about its biological function. Transcriptomic analysis has shown that the H-NS2 protein targets a subset of the genes targeted by H-NS. Genes targeted by H-NS2 correspond mainly with horizontally transferred (HGT) genes and are also targeted by the Hha protein, a fine-tuner of H-NS activity. Compared with H-NS, H-NS2 expression levels are lower. In addition, H-NS2 expression exhibits specific features: it is sensitive to the growth temperature and to the nature of the culture medium. This novel H-NS paralogue is widespread within theEnterobacteriaceae.IMPORTANCEGlobal regulators such as H-NS play key relevant roles enabling bacterial cells to adapt to a changing environment. H-NS modulates both core and horizontally transferred (HGT) genes, but the mechanism by which H-NS can differentially regulate these genes remains to be elucidated. There are several instances of bacterial cells carrying genes that encode homologues of the global regulators. The question is what the roles of these proteins are. We noticed that the enteroaggregativeE. colistrain 042 carries a new hitherto uncharacterized copy of thehnsgene. We decided to investigate why this pathogenicE. colistrain requires an extra H-NS paralogue, termed H-NS2. In our work, we show that H-NS2 displays specific expression and regulatory properties. H-NS2 targets a subset of H-NS-specific genes and may help to differentially modulate core and HGT genes by the H-NS cellular pool.

scholarly journals Disulfide bonds in a recombinant protein modeled after a core repeat in an aquatic insect's silk protein

2008 ◽  
Vol 4 (5) ◽  
pp. 945-954 ◽  
Author(s):  
Stanley V. Smith ◽  
John J. Correia ◽  
Steven T. Case
Keyword(s):  
Recombinant Protein ◽  
Disulfide Bonds ◽  
Silk Protein

scholarly journals Pengaruh Jumlah Inokulum Sel Inang Bakteri E.coli BL21 (DE3) pLysS dan Waktu Overekspresi pada Produksi Protein Rekombinan Fim-C Salmonella typhi

2018 ◽  
Vol 4 (2) ◽  
pp. 98-106
Author(s):  
Muktiningsih - Nurjayadi ◽  
Izzatul Ilma Chairinnisa ◽  
Geta Putri Mentari ◽  
Dudi Hardianto ◽  
Asri Sulfianti ◽  
...  
Keyword(s):  
Recombinant Protein ◽  
Large Scale ◽  
Salmonella Typhi ◽  
Cell Number ◽  
Bacterial Cells ◽  
Typhoid Vaccine ◽  
Over Expression ◽  
Bacterial Cultures ◽  
Sds Page

Recombinant protein Fim-C S.typhi is a potential protein that can be used as an alternative typhoid vaccine and produced on a large scale. However, before entering into a large-scale stage, laboratory optimum data on the factors that affect the production process of Fim-C Salmonella typhi proteins are required. This study aims to obtain information regarding the effect of host cell number E.coli BL21 (DE3) pLysS and overexpression time on production of recombinant protein Fim-C Salmonella typhi as the basis for developing candidate of typhoid vaccine. The optimization process of overexpression was done by adding 0.5 mM IPTG (Isopropyl-β-D-thiogalactopyranoside) inducer into bacterial cultures of 2%, 4%, and 6% with 4, 5, and 6 hours over expression. The measurement of the concentration of Fim-C recombinant protein extracted samples were done by a spectrophotometric method used BCA Kit Assay Thermo ScientificTM with wavelength 590nm. The characterization of those protein extracts was performed using SDS-PAGE method. The results from the study concluded that the number of 4% E.coli bacterial cells and four-hour overexpression time are the optimum condition of Fim- C Salmonella typhi characterized by the presence of high-intensity bands at ± 31 kDa.  

Mechanism of Activation of Human Plasma Prekallikrein

1979 ◽  
Author(s):  
Bonno N. Bouma ◽  
Riek A.A. Vlooswijk
Keyword(s):  
Human Plasma ◽  
Electrophoretic Mobility ◽  
Gel Electrophoresis ◽  
Disulfide Bonds ◽  
Polyacrylamide Gel Electrophoresis ◽  
Reducing Agents ◽  
Dextran Sulphate ◽  
Antigen Level ◽  
Normal Plasma ◽  
Kallikrein Activity

Human prekallikrein (PK) is in plasma complexed to High Moleculair Weight Kininogen (HMWK). The formation of a complex of PK with HMWK was demonstrated by cross-immunoelectrophoresis. Purified PK alone did not migrate upon electrophoresis, however when mixed with HMWK or Fletcher plasma, PK migrated with the same mobility as PK present in normal plasma. Also addition of PK to HMWK resulted in a reduction of the electrophoretic mobility of HMWK on cross-immunoelectrophoresis using anti-HMWK. Fitzgerald plasma which is deficient in HMWK has a PK clotting activity of 26% but no detectable PK-antigen level. Reconstitution with HMWK yielded PK-antigen levels corresponding to the PK-clotting activity. Addition of HMWK to normal plasma did not increase the PK-antigen levels.PK was activated by purified human (β-XIIa (28, 000 mol.wt) and by bovine α-XIIa (80, 000 mol.wt). Activation by αXIIa but not by by β-XII was enhanced by the presence of HMWK and dextran sulphate. After complete activation by α-XIIa in the presence of HMWK kallikrein remained complexed to HMWK. The generation of kallikrein activity paralelled the extent of proteolytic cleavage. SDS-polyacrylamide gel electrophoresis in the presence of reducing agents showed that both α-XIIa and β-XIIa generated fragments with apparent mol.wt’s of 52,000, 42,000 and 37,000. These fragments are initially held together by disulfide bonds, a second relatively slow cleavage generates fragments with similar mol. wt’s but observed on non reduced SDS-gels. This cleavage is not accompanied by a decrease in kallikrein activity.

scholarly journals Serodiagnosis of Hepatitis B Virus Using PreS Antigen from Pakistani Isolate SBS001

2021 ◽  
pp. 246-255
Author(s):  
Saima Iftikhar ◽  
Farheen Aslam ◽  
Muhammad Akhtar
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Recombinant Protein ◽  
High Titer ◽  
Protein A ◽  
Gene Encoding ◽  
Pakistani Population ◽  
Enhanced Sensitivity ◽  
Escherchia Coli ◽  
B Virus

Background: An estimated 325 million people worldwide live with hepatitis B and/or C and approximately, 5 million people are affected with hepatitis B. in Pakistan.This study aimed at developing PreS protein from Hepatitis B Virus Pakistani isolate (SBS001) with enhanced sensitivity to detect antibodies in serum as a diagnostic method. Methods: Gene encoding PreS region from hepatitis B Virus was cloned and expressed in Escherchia coli. The recombinant protein preS-His was purified by Ni-IDA affinity chromatography. Antibodies were raised in rabbit. This protein was screened for detection of antibodies in HBV patients’ sera through ELISA. This ELISA procedure was compared with commercially available Kit used for diagnosis of HBV infection. Results: Single band purified recombinant PreS protein was obtained with high titer of antibodies raised in rabbits. This recombinant protein was used in ELISA as antigen coated on the plate. That efficiently detected antibodies present in HBV patients. It was concluded that preS-His antigen/ protein A HRP-conjugate ELISA method was more sensitive than the commercial kit for detecting the antibodies present in HBV patient sera. Conclusion: It was concluded that SBS001 PreS recombinant protein can be used in ELISA kits for detection of HBV in Pakistani population.

Effectiveness of zona pellucida protein ZPB as an immunocontraceptive antigen

Reproduction ◽  
2000 ◽  
pp. 19-32 ◽  
Author(s):  
ML Martinez ◽  
JD Harris
Keyword(s):  
Menstrual Cycle ◽  
Recombinant Protein ◽  
Antibody Titre ◽  
Zona Pellucida ◽  
Protein A ◽  
Papio Cynocephalus ◽  
Cynomolgus Monkeys ◽  
Menstrual Cycles ◽  
Normal Menstrual Cycle

Immunization of female mammals with native zona pellucida (ZP) proteins is known to cause infertility. Since each human ZP protein is now available as a purified recombinant protein, is it possible to compare the immunocontraceptive potential of each ZP protein. A breeding study was conducted in cynomolgus monkeys (Macaca fasicularis) after immunization with recombinant human ZP (rhZP) proteins (ZPA, ZPB, ZPC) separately and in combinations. This study demonstrated that immunization with recombinant human ZPB (rhZPB) protein caused cynomolgus monkeys to become infertile for 9-35 months. A second study was conducted in baboons (Papio cynocephalus), which yielded a similar result. The baboons immunized with rhZPB became infertile for 9 to > 20 months. During the time of maximum antibody titre, some animals experienced disruption of the menstrual cycle, but eventually all of the animals resumed normal menstrual cycles. Control animals and animals immunized with other rhZP proteins all became pregnant before any of the rhZPB-treated animals. This is the first study in which a recombinant ZP protein has consistently induced infertility in a primate without permanent disruption of the normal menstrual cycle.

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