Showing 4 results for Chaperone
F. Vahdani, H. Ghafoori , S. Sarikhan,
Volume 10, Issue 2 (7-2019)
Abstract
Hsp70 family members are central components of the cellular network of molecular chaperones and folding catalysts. The gene encoding a protein related to Hsp70 or DnaK in the domain bacteria is called dnaK. DnaK proteins are involved in de novo protein folding, formation, and disassembly of protein complexes and degradation of misfolded proteins. The gene dnaJ which codes for Hsp40 in bacteria, modulate the activities of DnaK by acting as co-chaperone. In the present study, we cloned and expressed DnaK from Bacillus halodurans Guj1 were identified. The dnaK gene of B. halodurans was successfully expressed in E. coli BL21 (DE3) using pET-28a+ expression system. The open reading frame of the cloned gene contained 1839bp and encoded 612 amino acid residues. Calculated molecular weight and pI of the protein were 66.18kDa and 4.55 respectively. The deduced amino acid sequence of B. halodurans Guj1 showed about 60% identity with the E. coli counterpart. The 3D structure of dnaK from B. halodurans was constructed using the crystal structure of human HSP70 chaperone BiP as the template, which showed an identity of 88.8% together. Partially purified recombinant DnaK by heat treatment showed a band at approximately 70kDa on SDS-PAGE. Our findings showed that the recombinant DnaK improved the refolding efficiency of the carbonic anhydrase by 27% after 60min at 54°C. According to the results obtained, DnaK from B. halodurans can potentially be used for improving the functional properties of enzymes and proteins in various applications.
Nasrin Kardan, Bahareh Dabirmanesh, Khosro Khajeh,
Volume 12, Issue 1 (12-2020)
Abstract
Protein deposition due to the process of accumulation inside or outside cells causes many neurological diseases such as Alzheimerchr('39')s, Huntingtonchr('39')s or Parkinsonchr('39')s seizures. Parkinsonchr('39')s disease is the second most common neurological disease after Alzheimerchr('39')s, in which patients develop disorders due to the accumulation of leprosy and the destruction of dopamine neurons. Alpha-synuclein protein contains 140 amino acids, the main protein known in lewy body accumulations. During the aggregation process, alpha-synuclein protein monomers bind together as oligomers and eventually become amyloid filaments. So far, there is no drug to stop or delay the progression of Parkinsonchr('39')s, but studies on the molecular mechanism of amyloid formation and the identification of inhibitors are increasing. For this purpose, in this study, the effect of BRICHOS domain resulting from BRI2, which can have various functions, including antimicrobial properties, on the process of alpha-synuclein accumulation as a model protein was investigated.The gene was first optimized and synthesized and then multiplied by PCR. The product was digested by enzymes Xho I and Nde1 and entered the expression vector pET28 a, which was transformed into E. coli bacteria.Finally, the peptide was purified by nickel chromatography. The alpha-synuclein gene was also expressed separately and purified.The anti-cumulative effect of BRICHOS domain on alpha-synuclein fibrillation was investigated using Toflavin T fluorescence method and TEM technique.
Seyedeh Mahdieh Sadadt, Zahra Hajihassan, Mohammad Barshan-Tashnizi,
Volume 12, Issue 3 (9-2021)
Abstract
Nerve growth factor (NGF) is a neurotrophic factor that is functional in survival, maintenance and differentiation of peripheral and central nervous system cells. This protein has three subunits that its beta subunit has main activity. According to scientific studies, it can be used as a therapeutic agent in treatment of many diseases such as peripheral neuropathy associated with diabetes, Alzheimer's disease, Parkinson's disease, skin disease and so on. Prokaryotic expression of recombinant NGF should be done in the periplasmic space because of its oxidative envoronment. It is worth noting that co-expression of cytoplasmic molecular chaperones can facilitate the secretion of the recombinant proteins to the periplasmic space and also enhance the protein solubility.
In this study, the effect of cytoplasmic chaperones of GroEL / GroES, DnaK / DnaJ, GrpE, Trigger Factor (TF) on the periplasmic production of recombinant NGF protein was studied. For this purpose, β-NGF subunit was expressed in pET39b(+) expression vector simultaneously with chaperone plasmids pG-Tf2, pTf16, pGro7, pKJE7 and pG-KJE8 in E. coli DE3 strain.
The results showed that in the presence of TF chaperone (pTf16 plasmid), the total protein and periplasmic production increased. Also, the DnaK/DnaJ and GroEL/GroES chaperones (pG-KJE8 plasmid) have also increased the production to some extent.; while the expression of GroEL/ GroES (pGro7) or DnaK / DnaJ (pKJE7) had no effect on protein expression. Also treatment of PC12 cell line with recombinant β-NGF showed differentiation to nerve cells which indicates that the produced protein is fully functional.
Volume 16, Issue 7 (9-2016)
Abstract
Alzheimer is the most common form of dementia. Amyloid beta peptides play a key role in the pathology of Alzheimer and the recent surveys have demonstrated that amyloid beta oligomers are the most toxic component of them. Among oligomers, considering the high durability of dimer in comparison to other kinds, it has more toxic effects. Prefoldin is a molecular chaperone which prevents accumulation of misfolded proteins. Prefoldin is demonstrated that it can also operate as a nano actuator. In this article, we investigate the interaction between the prefoldin nano actuator and dimeric pathogenic nano cargo in molecular dimensions, hence the all-atom molecular dynamic simulation in explicit solvent were performed at physiological temperature. Visualizing the results and investigating the atomic distance between nano actuator and pathogenic nano cargo revealed that two arms of six arms of prefoldin nano actuator have been able to capture cargo and during the simulation they have made hydrogen bonds. Furthermore, investigating the hydrophobic effects between the hydrophobic amino acids in the cargo and nano actuator revealed that these effects have positively affected the stability of the binding between arms and the cargo. This article introduces the prefoldin as an inhibitor factor for dimeric oligomer from amyloid beta.
