scholarly journals Characterization and Analysis of Sip1Aa Protein Expressed by cry1Ac Promoter

2021 ◽  
Author(s):  
Jing Wang ◽  
Mingyue Ding ◽  
Lin Wang ◽  
Jun Cui ◽  
Haitao Li ◽  
...  
Keyword(s):  
Recombinant Proteins ◽  
Insecticidal Activity ◽  
Inclusion Bodies ◽  
Functional Verification ◽  
T7 Promoter ◽  
E Coli ◽  
Significant Difference ◽  
New Ideas ◽  
Pcr Technology ◽  
New Research

Abstract In this study, based on the pUC19 vector and using overlapping PCR technology, the researchers constructed an expression vector of the Sip1Aa protein guided by a cry1Ac promoter. The expression situation, insecticidal activity and solubility were researched, and the expression of the Sip1Aa protein, as guided by a T7 promoter, was compared. Additionally, the fermentation conditions were explored on a preliminary basis and a histidine label was added to the recombinant plasmid via reverse PCR for subsequent purification of recombinant proteins. The results showed that both the cry1Ac and T7 promoters could guide Sip1Aa to express as a soluble protein of 37.6 kDa, and there was no significant difference in insecticidal activity against Colaphellus bowringi Baly, with LC50 values of 1.637 mg/mL and 1.683 μg/mL, respectively. The soluble component of the Sip1Aa protein, when guided by the cry1Ac promoter, was significantly higher than when it was guided by the T7 promoter. The expression of the cry1Ac guider-promoted Sip1Aa protein was more suitable at 37℃ and 16 h. The recombinant protein was purified after an exogenous histidine sequence was added. This provides a new research method and idea to solve the problem of the Sip1Aa protein usually producing a large number of inclusion bodies when expressed in E. coli, and provides new ideas for the study of sip gene rapid expression, functional verification and insecticidal mechanisms.

Expression, solubilization, refolding and final purification of recombinant proteins as expressed in the form of "classical inclusion bodies" in E. coli

2020 ◽  
Vol 27 ◽  
Author(s):  
Mohammad Sadegh Hashemzadeh ◽  
Mozafar Mohammadi ◽  
Hadi Esmaeili Gouvarchin Ghaleh ◽  
Mojtaba Sharti ◽  
Ali Choopani ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Recombinant Proteins ◽  
Inclusion Bodies ◽  
Native Conformation ◽  
Native Form ◽  
Over Expression ◽  
E Coli ◽  
Mild Conditions ◽  
Final Purification

: Escherichia coli has been most widely used for production of the recombinant proteins. Over-expression of the recombinant proteins is the mainspring of the inclusion bodies formation. The refolding of these proteins into bioactive forms is cumbersome and partly time-consuming. In the present study, we reviewed and discussed most issues regarding the recovery of "classical inclusion bodies" by focusing on our previous experiences. Performing proper methods of expression, solubilization, refolding and final purification of these proteins, would make it possible to recover higher amounts of pro-teins into the native form with appropriate conformation. Generally, providing mild conditions and proper refolding buffers, would lead to recover more than 40% of inclusion bodies into bioactive and native conformation.

scholarly journals Expression and purification of four truncated 56 kDa antigens from Orientia tsutsugamushi in Escherichia coli

2018 ◽  
Vol 16 (3) ◽  
pp. 533-541
Author(s):  
Le Thi Lan Anh ◽  
Trinh Van Toan ◽  
Pham Thi Ha Giang ◽  
Bui Thi Thanh Nga ◽  
Vo Viet Cuong ◽  
...  
Keyword(s):  
Recombinant Proteins ◽  
Inclusion Bodies ◽  
Scrub Typhus ◽  
Febrile Illness ◽  
Affinity Column ◽  
Orientia Tsutsugamushi ◽  
Expression And Purification ◽  
E Coli ◽  
Clinical Presentations ◽  
Insoluble Form

Scrub typhus is an acute febrile illness caused by Orientia tsutsugamushi, transmitted to humans by the bite of the larva of trombiculid mites. Diagnosis of scrub typhus is normally based on the clinical presentations. However, it is difficult to differentiate scrub typhus from other acute febrile illnesses, such as dengue fever, malaria and leptospirosis due to similar symptoms. For differential diagnosis of scrub typhus from other acute febrile diseases, a rapid and reliable serological diagnosis is important. In order to produce an ELISA kit for detection of antibodies against O. tsutsugamushi in Vietnam, four truncated 56 kDa antigenic genes of O. tsutsugamushi including Karp (HT-09), Gilliam (HT-11), TA763 (HT-49), and Kato (YB-50) íolated from the most prevalent cases in Vietnam were cloned and expressed in E. coli Rosetta 1 cells. The recombinant proteins formed inclusion bodies when expressed in E. coli. The recombinant 56 kDa proteins in insoluble form were solubilized in 6M urea and were successfully purified by Ni2+affinity column. The purity of four recombinant proteins,HT-09, HT-11, HT-49 and YB-50,reached more than 95% and their concentrations are 12,57 mg/ml; 11,6 mg/ml; 8,98 mg/ml và 8,02 mg/ml, respectively.

scholarly journals Challenges Associated With the Formation of Recombinant Protein Inclusion Bodies in Escherichia coli and Strategies to Address Them for Industrial Applications

2021 ◽  
Vol 9 ◽  
Author(s):  
Arshpreet Bhatwa ◽  
Weijun Wang ◽  
Yousef I. Hassan ◽  
Nadine Abraham ◽  
Xiu-Zhen Li ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Recombinant Proteins ◽  
Inclusion Bodies ◽  
Growth Conditions ◽  
Industrial Applications ◽  
Expression Vectors ◽  
Bacterial Host ◽  
New Developments ◽  
E Coli ◽  
Host Strains

Recombinant proteins are becoming increasingly important for industrial applications, where Escherichia coli is the most widely used bacterial host for their production. However, the formation of inclusion bodies is a frequently encountered challenge for producing soluble and functional recombinant proteins. To overcome this hurdle, different strategies have been developed through adjusting growth conditions, engineering host strains of E. coli, altering expression vectors, and modifying the proteins of interest. These approaches will be comprehensively highlighted with some of the new developments in this review. Additionally, the unique features of protein inclusion bodies, the mechanism and influencing factors of their formation, and their potential advantages will also be discussed.

scholarly journals Chemical Assistance in Refolding of Bacterial Inclusion Bodies

2011 ◽  
Vol 2011 ◽  
pp. 1-6 ◽  
Author(s):  
Mona Alibolandi ◽  
Hasan Mirzahoseini
Keyword(s):  
Escherichia Coli ◽  
Recombinant Proteins ◽  
Inclusion Bodies ◽  
Industrial Processes ◽  
Small Scale ◽  
Heterologous Proteins ◽  
E Coli ◽  
Major Bottleneck ◽  
In Vitro Refolding

Escherichia coliis one of the most widely used hosts for the production of recombinant proteins but insoluble expression of heterologous proteins is a major bottleneck in production of recombinant proteins inE. coli.In vitrorefolding of inclusion body into proteins with native conformations is a solution for this problem but there is a need for optimization of condition for each protein specifically. Several approaches have been described for in vitro refolding; most of them involve the use of additives for assisting correct folding. Cosolutes play a major role in refolding process and can be classified according to their function as aggregation suppressors and folding enhancers. This paper presents a review of additives that are used in refolding process of insoluble recombinant proteins in small scale and industrial processes.

scholarly journals A novel in vitro urethra model to demonstrate bacterial displacement during urinary catheter insertion

2020 ◽  
Vol 2 (7A) ◽  
Author(s):  
Yvonne J. Cortese ◽  
Victoria E. Wagner ◽  
Morgan Tierney ◽  
David Scully ◽  
Declan M. Devine ◽  
...  
Keyword(s):  
Bacterial Species ◽  
Growth Control ◽  
Catheter Insertion ◽  
E Coli ◽  
Significant Difference ◽  
New Research ◽  
Development And Validation ◽  
Control Introduction ◽  
Urethral Model

Background: There is currently no standard established in vitro model to test the efficacy of intermittent catheters to prevent or control introduction/movement of bacteria into the urethra during device insertion. This study aimed to address this issue by developing a reproducible agar based in vitro urethral model. Method: A novel in vitro model and testing method was developed to quantify the displacement of bacterial growth after intermittent catheter insertion.The urethral model consists primarily of a preformed channel within a specifically formulated agar based matrix. The urethra model was inoculated at one side of the channel to act as the urethral meatus, a catheter was then inserted. After incubation the bacteria within the urethra channel was quantified. Results: Once optimised, the model produced reliable and reproducible results with both E. coli and S. aureus (P≥0.265). The model was used to test three different intermittent catheter types. When compared to the growth control there was a significant difference in bacterial distribution when inserting an uncoated (P≤0.001) or hydrophilic coated (P≤0.009) catheter; there was no significant difference when a prototype catheter was inserted with either bacterial species used (P≥0.423). Conclusion: These findings support the hypothesis that a single catheter insertion can initiate a catheter-associated urinary tract infection. The in vitro urethra model and associated methodology provide a new research tool for the development and validation of emerging technologies in urological healthcare.

Immunogold labeling of CMP-KDO synthetase produced by recombinant E. Coli cells

1987 ◽  
Vol 45 ◽  
pp. 924-925
Author(s):  
F. A. Durum ◽  
R. G. Goldman ◽  
T. J. Bolling ◽  
M. F. Miller
Keyword(s):  
Inclusion Bodies ◽  
Large Scale ◽  
Recombinant Dna ◽  
Test System ◽  
Cell Protein ◽  
Gram Negative Bacteria ◽  
Cytoplasmic Inclusions ◽  
Isolation And Purification ◽  
E Coli ◽  
Low Concentrations

CMP-KDO synthetase (CKS) is an enzyme which plays a key role in the synthesis of LPS, an outer membrane component unique to gram negative bacteria. CKS activates KDO to CMP-KDO for incorporation into LPS. The enzyme is normally present in low concentrations (0.02% of total cell protein) which makes it difficult to perform large scale isolation and purification. Recently, the gene for CKS from E. coli was cloned and various recombinant DNA constructs overproducing CKS several thousandfold (unpublished data) were derived. Interestingly, no cytoplasmic inclusions of overproduced CKS were observed by EM (Fig. 1) which is in contrast to other reports of large proteinaceous inclusion bodies in various overproducing recombinant strains. The present immunocytochemical study was undertaken to localize CKS in these cells.Immune labeling conditions were first optimized using a previously described cell-free test system. Briefly, this involves soaking small blocks of polymerized bovine serum albumin in purified CKS antigen and subjecting them to various fixation, embedding and immunochemical conditions.

Suitable Signal Peptides for Secretory Production of Recombinant Granulocyte Colony Stimulating Factor in Escherichia coli

2020 ◽  
Vol 14 (4) ◽  
pp. 269-282
Author(s):  
Sadra S. Tehrani ◽  
Golnaz Goodarzi ◽  
Mohsen Naghizadeh ◽  
Seyyed H. Khatami ◽  
Ahmad Movahedpour ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Signal Peptide ◽  
Fusion Proteins ◽  
Inclusion Bodies ◽  
Clinical Aspects ◽  
Granulocyte Colony Stimulating Factor ◽  
Colony Stimulating Factor ◽  
E Coli ◽  
Secretory Production ◽  
Stimulating Factor

Background: Granulocyte colony-stimulating factor (G-CSF) expressed in engineered Escherichia coli (E. coli) as a recombinant protein is utilized as an adjunct to chemotherapy for improving neutropenia. Recombinant proteins overexpression may lead to the creation of inclusion bodies whose recovery is a tedious and costly process. To overcome the problem of inclusion bodies, secretory production might be used. To achieve a mature secretory protein product, suitable signal peptide (SP) selection is a vital step. Objective: In the present study, we aimed at in silico evaluation of proper SPs for secretory production of recombinant G-CSF in E. coli. Methods: Signal peptide website and UniProt were used to collect the SPs and G-CSF sequences. Then, SignalP were utilized in order to predict the SPs and location of their cleavage site. Physicochemical features and solubility were investigated by ProtParam and Protein-sol tools. Fusion proteins sub-cellular localization was predicted by ProtCompB. Results: LPP, ELBP, TSH, HST3, ELBH, AIDA and PET were excluded according to SignalP. The highest aliphatic index belonged to OMPC, TORT and THIB and PPA. Also, the highest GRAVY belonged to OMPC, ELAP, TORT, BLAT, THIB, and PSPE. Furthermore, G-CSF fused with all SPs were predicted as soluble fusion proteins except three SPs. Finally, we found OMPT, OMPF, PHOE, LAMB, SAT, and OMPP can translocate G-CSF into extracellular space. Conclusion: Six SPs were suitable for translocating G-CSF into the extracellular media. Although growing data indicate that the bioinformatics approaches can improve the precision and accuracy of studies, further experimental investigations and recent patents explaining several inventions associated to the clinical aspects of SPs for secretory production of recombinant GCSF in E. coli are required for final validation.

scholarly journals Isolation of a Gene Responsible for the Oxidation oftrans-Anethole topara-Anisaldehyde by Pseudomonas putida JYR-1 and Its Expression in Escherichia coli

2012 ◽  
Vol 78 (15) ◽  
pp. 5238-5246 ◽  
Author(s):  
Dongfei Han ◽  
Ji-Young Ryu ◽  
Robert A. Kanaly ◽  
Hor-Gil Hur
Keyword(s):  
Escherichia Coli ◽  
Pseudomonas Putida ◽  
Hypothetical Protein ◽  
Aldehyde Group ◽  
Agrobacterium Vitis ◽  
T7 Promoter ◽  
Content Type ◽  
E Coli ◽  
Cell Extracts ◽  
Isoeugenol Monooxygenase

ABSTRACTA plasmid, pTA163, inEscherichia colicontained an approximately 34-kb gene fragment fromPseudomonas putidaJYR-1 that included the genes responsible for the metabolism oftrans-anethole to protocatechuic acid. Three Tn5-disrupted open reading frame 10 (ORF 10) mutants of plasmid pTA163 lost their abilities to catalyzetrans-anethole. Heterologously expressed ORF 10 (1,047 nucleotides [nt]) under a T7 promoter inE. colicatalyzed oxidative cleavage of a propenyl group oftrans-anethole to an aldehyde group, resulting in the production ofpara-anisaldehyde, and this gene was designatedtao(trans-anetholeoxygenase). The deduced amino acid sequence of TAO had the highest identity (34%) to a hypothetical protein ofAgrobacterium vitisS4 and likely contained a flavin-binding site. Preferred incorporation of an oxygen molecule from water intop-anisaldehyde using18O-labeling experiments indicated stereo preference of TAO for hydrolysis of the epoxide group. Interestingly, unlike the narrow substrate range of isoeugenol monooxygenase fromPseudomonas putidaIE27 andPseudomonas nitroreducensJin1, TAO fromP. putidaJYR-1 catalyzed isoeugenol,O-methyl isoeugenol, and isosafrole, all of which contain the 2-propenyl functional group on the aromatic ring structure. Addition of NAD(P)H to the ultrafiltered cell extracts ofE. coli(pTA163) increased the activity of TAO. Due to the relaxed substrate range of TAO, it may be utilized for the production of various fragrance compounds from plant phenylpropanoids in the future.

scholarly journals Transformation and functional verification of Cry5Aa in cotton

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Shihao Zhao ◽  
Feng Wang ◽  
Qiuping Zhang ◽  
Jiayi Zou ◽  
Zhangshu Xie ◽  
...  
Keyword(s):  
Resistance Gene ◽  
Insect Resistance ◽  
Expression Patterns ◽  
Instar Larva ◽  
Cotton Bollworm ◽  
Pcr Analysis ◽  
Functional Verification ◽  
Study Results ◽  
Significant Difference ◽  
Transgenic Strains

AbstractMost of the cotton bollworm-resistant genes applied in cotton are more than 20 years and they all belong to Cry1Ab/c family, but the insect-resistant effects of Cry5Aa on cotton were rarely reported. The possible risk of resistance is increasing. The study synthesized a novel bollworm-resistant gene Cry5Aa artificially based on preferences of cotton codon. The new gene was transferred to cotton through the method of pollen tube pathway. The transgenic strains were identified by kanamycin test in field and laboratory PCR analysis. Meanwhile, an insect resistance test was conducted by artificial bollworm feeding with transgenic leaves and GK19 was used as a control in this study. Results showed that rate of positive transgenic strains with kanamycin resistance in the first generation (T1), the second generation (T2) and the third generation (T3) respectively were 7.76%, 73.1% and 95.5%. However, PCR analysis showed that the positive strain rate in T1, T2 and T3 were 2.35%, 55.8% and 94.5%, respectively. The resistant assay of cotton bollworm showed that the mortality rate of the second, third and fourth instar larva feed by the transgenic cotton leaves, were 85.42%, 73.35% and 62.79%, respectively. There was a significant difference between transgenic plant of Cry5Aa and GK19 in insect resistance. Finally, we also conducted the further analysis of gene expression patterns, gene flow and the effect on non-target pest in the study. The results showed that Cry5Aa gene had less environmental impact, and Cry5Aa has been transferred successfully and expressed stably in cotton. Therefore, the novel bollworm resistance gene can partially replace the current insect-resistance gene of Lepidoptera insects.

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