Bacterial expression of the recombinant functionally active N-terminal module of the B. taurus tyrosyl-tRNA synthetase

The nucleotide sequence coding N-terminal module of Bos taurus tyrosyl-tRNA synthetase (mini TyrRS) was cloned into the bacterial expression vector pET23d(+). Bacterial expression of the recombinant protein mini TyrRS was performed in E. coli BL21 (DE3)pLysE cells with the use of the constructed vector pET-23d(+)39YRS for subsequent physical and chemical protein studies. The catalytic activity of the recombinant mini TyrRS has been studied in the aminoacylation reaction of homologous tRNATyr.

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Specific T-cell Responses to CFP10, an Secreted Antigens of Mycobacterium Tuberculosis Protein, in Chinese HIV Positive Individuals

Infection International ◽

10.1515/ii-2017-0034 ◽

2013 ◽

Vol 2 (1) ◽

pp. 6-12

Author(s):

Bin Zhang ◽

Wen-hui Lun ◽

Xing-wang Li ◽

Qi Wang ◽

Jun Cheng ◽

...

Keyword(s):

T Cell ◽

Recombinant Protein ◽

Recombinant Proteins ◽

Prokaryotic Expression ◽

Expression Vector ◽

T Cell Responses ◽

Hiv Positive ◽

E Coli ◽

Cell Responses ◽

Prokaryotic Expression Vector

Abstract Objective To construct prokaryotic expression vector of CFP-10 gene, and obtain recombinant protein, and the recombinant CFP-10 protein was taken as stimulus to detect specific T cell responses, to set up a method to faciliate to detect potential TB infection in China. Methods CFP-10 was cloned into inducible prokaryotic expression vector pET-32a (+) and transfected into E. coli BL21 (DE3). After IPTG induction, the product were verified with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot hybridization were carried out to verify the antigenicity; the recombinant CFP-10 protein was taken as stimulus to detect specific T cell responses in HIV (+) persons with or without clinical manifestation of TB diseases, and HIV (-) controls with or without TB diseases. Results The CFP-10 recombinant protein exsited in the form of inclusion body and accounted for 94% in total bacterial protein of E. coli and the molecular weight is 31 kD; Western blot confirmed the recombinant proteins had high antigenicity; our in-house ELISpot-IFN-γ assay with recombinant antigen derived from CFP-10 proteins showed significant higher frequencies in TB patients with or without HIV infection than that in the healthy controls and only HIV (+) group. Conclusions The recombinant CFP-10 genes can be expressed successfully in prokaryotic expression system of E. coli and recombinant proteins with high antigenicity were obtained, which will set foundation for further study on their immunogenicity and bioinformatics. Our results proved that it is indeed true that some HIV positive patient have high frequencies of TB specific T cell responses, which maybe a clue to find latent TB infection in this population.

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Bacterial expression systems for recombinant protein production: E. coli and beyond

Biotechnology Advances ◽

10.1016/j.biotechadv.2011.09.013 ◽

2012 ◽

Vol 30 (5) ◽

pp. 1102-1107 ◽

Cited By ~ 168

Author(s):

Rachel Chen

Keyword(s):

Recombinant Protein ◽

Recombinant Protein Production ◽

Protein Production ◽

Bacterial Expression ◽

Expression Systems ◽

E Coli ◽

Bacterial Expression Systems

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The relation between catalytic activity and gene regulation in the case of E coli threonyl-tRNA synthetase

Biochimie ◽

10.1016/0300-9084(90)90072-o ◽

1990 ◽

Vol 72 (6-7) ◽

pp. 485-494 ◽

Cited By ~ 3

Author(s):

P. Romby ◽

H. Moine ◽

P. Lesage ◽

M. Graffe ◽

J. Dondon ◽

...

Keyword(s):

Gene Regulation ◽

Catalytic Activity ◽

Trna Synthetase ◽

E Coli

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Soluble Expression and Purification of Q59L Mutant L-asparaginase in the Presence of Chaperones in SHuffle™ T7 strain

International Journal of Medical Laboratory ◽

10.18502/ijml.v8i2.6278 ◽

2021 ◽

Author(s):

Elham Biglari Goliloo ◽

Abdolnabi Tollabi ◽

Hossein Zarei Jaliani

Keyword(s):

Recombinant Protein ◽

Expression Vector ◽

Trigger Factor ◽

Lower Side ◽

Expression And Purification ◽

E Coli ◽

Nickel Affinity Chromatography ◽

Potential Applications ◽

Soluble State ◽

Soluble Recombinant Protein

Background and Aims: Q59L mutant of L-asparaginase enzyme from Escherichia coli (E. coli) has been introduced with lower side effects. This version of the enzyme might have potential applications in the treatment of leukemia patients. We utilized SHuffle T7 strain of E. coli, to produce the mutant enzyme in the presence of chaperone molecules. Materials and Methods: Q59LAsp gene was cloned into pET28a expression vector, and two strains of E. coli (BL21 DE3 and SHuffle T7 strains) were used to produce recombinant protein. In parallel, PG-Tf2 plasmid was cloned into the same strains, and the effect of trigger factor chaperone and groELS chaperonines was studied. The his-tagged recombinant protein was expressed and purified using nickel affinity chromatography. The amount of recombinant protein which is produced in each condition was determined and compared. Results: The amount of soluble recombinant protein was enhanced in the presence of chaperones in both strains of E. coli. SHuffle T7 strain produced more soluble recombinant protein in the soluble state than BL21 DE3 strain. So the best condition for the production of soluble recombinant Q59L mutant protein was the use of PG-Tf2 plasmid in the SHuffle T7 strain. Conclusion: Application of the new strain SHuffle T7, with chaperones simultaneously showed better results in the production of Q59L mutant version of L-Asparaginase.

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Cloning and Expression of PirA gene of Vibrio parahaemolyticus Strain K5 Causing Acute Hepatopancreatic Necrosis Disease in Whiteleg Shrimp in E. coli Host Cell

10.20944/preprints202201.0086.v1 ◽

2022 ◽

Author(s):

Nguyen Quang Linh ◽

Khanh Van Nguyen ◽

Dung Quoc Tran ◽

Van Khanh Tran Quang

Keyword(s):

Recombinant Protein ◽

Vibrio Parahaemolyticus ◽

Expression Vector ◽

Cloning And Expression ◽

Economic Losses ◽

Bacterial Disease ◽

Parahaemolyticus Strain ◽

E Coli ◽

Whiteleg Shrimp ◽

Acute Hepatopancreatic Necrosis Disease

Background: Acute hepatopancreatic necrosis disease (AHPND), is a bacterial disease of whiteleg shrimp, which has a high mortality rate (100%) and incurs economic losses. Our objective was to identify the genes which lead to cell and organ damage and investigate bioproducts to prevent and treat. Methods: Litopenaeus vannamei shrimp in Thua Thien Hue province, Vietnam were collected from an infected pond and analysed at the Institute of Biotechnology, Hue University. The PirA gene of Vibrio parahaemolyticus strain K5 was isolated and analyzed for nucleotide sequence and paired with the expression vector pQE30. The expression vector was transformed into E. coli strain M15, the PirA recombinant protein was expressed in the form of 6xHis-PirA fusion protein of about 15 kDa. PirA recombinant protein was purified and determined the PirAvp binding ratio, cloning and sequencing of PirA gene from Vibrio parahaemolyticus strain K5 causing AHPND by PCR method with specific primers and molecular weights of PirAvp and the PirAvp complex. Results: PirA gene from Vibrio parahaemolyticus strain K5 was cloned into pGEM-T easy vector (Promega, USA) and screened E. coli TOP10 colonies containing pGEM T easy/PirA recombinant plasmid on LB agar/ampicillin/IPTG/X-Gal medium. PCR showing a band of about 347 bp, matching the size of PirA gene and two nucleotide sequences (BamHI and HindIII). The results showed that PirA gene has a length of 336 bp and similar to PirA gene on GenBank (Code: KU556825.1). The results of protein extracted from E. coli M15 recombinant cells and 6xHis-PirA target protein was collected in elution fractions from EF2 to EF6, showed that the concentration of 6xHis-PirA protein and EF3 elution fraction collected a highest protein concentration (1,586.54 µg/ml). Conclusions: The purified PirA recombinant protein will provide materials for development research to create biological products to prevent and treat AHPND.

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High resolution electron microscopy of lanthanum phosphate crystals

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100108519 ◽

1978 ◽

Vol 36 (1) ◽

pp. 274-275

Author(s):

J. C. Wheatley ◽

J. M. Cowley

Keyword(s):

Electron Microscopy ◽

Catalytic Activity ◽

High Resolution ◽

Petroleum Industry ◽

High Resolution Electron Microscopy ◽

Lanthanum Phosphate ◽

Good Catalyst ◽

Resolution Electron ◽

Good Catalytic Activity ◽

Physical And Chemical

Rare-earth phosphates are of particular interest because of their catalytic properties associated with the hydrolysis of many aromatic chlorides in the petroleum industry. Lanthanum phosphates (LaPO4) which have been doped with small amounts of copper have shown increased catalytic activity (1). However the physical and chemical characteristics of the samples leading to good catalytic activity are not known.Many catalysts are amorphous and thus do not easily lend themselves to methods of investigation which would include electron microscopy. However, the LaPO4, crystals are quite suitable samples for high resolution techniques.The samples used were obtained from William L. Kehl of Gulf Research and Development Company. The electron microscopy was carried out on a JEOL JEM-100B which had been modified for high resolution microscopy (2). Standard high resolution techniques were employed. Three different sample types were observed: 669A-1-5-7 (poor catalyst), H-L-2 (good catalyst) and 27-011 (good catalyst).

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Ultrafiltration Process in Disinfection and Advanced Treatment of Tertiary Treated Wastewater

Membranes ◽

10.3390/membranes11030221 ◽

2021 ◽

Vol 11 (3) ◽

pp. 221

Author(s):

Rafał Tytus Bray ◽

Katarzyna Jankowska ◽

Eliza Kulbat ◽

Aneta Łuczkiewicz ◽

Aleksandra Sokołowska

Keyword(s):

Wastewater Treatment ◽

Wastewater Treatment Plants ◽

Microscopic Analysis ◽

Fecal Coliforms ◽

Treated Wastewater ◽

Chemical Parameters ◽

Virus Particles ◽

E Coli ◽

The One ◽

Physical And Chemical

The paper presents the results of research on the use of ultrafiltration, using membranes of 200 and 400 kDa separation, for disinfection of municipal treated wastewater. The research was conducted on a fractional technical scale using real municipal treated wastewater from two large wastewater treatment plants treating most of the wastewater over the one-million polycentric Gdańsk agglomeration (1.2 million inhabitants). UF 200 kDa and UF 400 kDa processes enabled further improvement of the physical and chemical parameters of treated wastewater. Total phosphorus (to below 0.2 mg/L–UF 200 kDa, 0.13 mg/L–UF 400 kDa) and turbid substances (to below 0.2 mg/L, both membranes) were removed in the highest degree. COD was reduced efficiently (to below 25.6 mgO2/L–UF 200 kDa, 26.8 mgO2/L–UF 400 kDa), while total nitrogen was removed to a small extent (to 7.12 mg/L–UF 200 kDa and 5.7 mg/L–UF 400 kDa. Based on the reduction of indicator bacteria; fecal coliforms including E. coli (FC) and fecal enterococci (FE) it was found that the ultrafiltration is an effective method of disinfection. Not much indicator bacterial were observed in the permeate after processes (UF 200 kDa; FC—5 CFU/L; FE—1 CFU/L and UF 400 kDa; FC—70 CFU/L; FE—10 CFU/L. However, microscopic analysis of prokaryotic cells and virus particles showed their presence after the application of both membrane types; TCN 3.0 × 102 cells/mL–UF 200 kDa, 5.0 × 103 cells/mL–UF 400 kDa, VP 1.0 × 105/mL. The presence of potentially pathogenic, highly infectious virus particles means that ultrafiltration cannot be considered a sufficient disinfection method for treated wastewater diverted for reuse or discharged from high load wastewater treatment plants to recreational areas. For full microbiological safety it would be advisable to apply an additional disinfection method (e.g., ozonation).

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Lysyl-tRNA synthetase from Escherichia coli K12. Chromatographic heterogeneity and the lysU-gene product

Biochemical Journal ◽

10.1042/bj2480043 ◽

1987 ◽

Vol 248 (1) ◽

pp. 43-51 ◽

Cited By ~ 17

Author(s):

J Charlier ◽

R Sanchez

Keyword(s):

Escherichia Coli ◽

Gene Product ◽

Activity Ratio ◽

Deae Cellulose ◽

Trna Synthetase ◽

Trna Synthetases ◽

E Coli ◽

Aminoacyl Trna Synthetases

In contrast with most aminoacyl-tRNA synthetases, the lysyl-tRNA synthetase of Escherichia coli is coded for by two genes, the normal lysS gene and the inducible lysU gene. During its purification from E. coli K12, lysyl-tRNA synthetase was monitored by its aminoacylation and adenosine(5′)tetraphospho(5′)adenosine (Ap4A) synthesis activities. Ap4A synthesis was measured by a new assay using DEAE-cellulose filters. The heterogeneity of lysyl-tRNA synthetase (LysRS) was revealed on hydroxyapatite; we focused on the first peak, LysRS1, because of its higher Ap4A/lysyl-tRNA activity ratio at that stage. Additional differences between LysRS1 and LysRS2 (major peak on hydroxyapatite) were collected. LysRS1 was eluted from phosphocellulose in the presence of the substrates, whereas LysRS2 was not. Phosphocellulose chromatography was used to show the increase of LysRS1 in cells submitted to heat shock. Also, the Mg2+ optimum in the Ap4A-synthesis reaction is much higher for LysRS1. LysRS1 showed a higher thermostability, which was specifically enhanced by Zn2+. These results in vivo and in vitro strongly suggest that LysRS1 is the heat-inducible lysU-gene product.

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Multiple Microfermentor Battery: a Versatile Tool for Use with Automated Parallel Cultures of Microorganisms Producing Recombinant Proteins and for Optimization of Cultivation Protocols

Applied and Environmental Microbiology ◽

10.1128/aem.00239-06 ◽

2006 ◽

Vol 72 (8) ◽

pp. 5225-5231 ◽

Cited By ~ 21

Author(s):

Emmanuel Frachon ◽

Vincent Bondet ◽

Hélène Munier-Lehmann ◽

Jacques Bellalou

Keyword(s):

Recombinant Protein ◽

Recombinant Proteins ◽

Protein Production ◽

Batch Cultures ◽

E Coli ◽

Working Volume ◽

Versatile Tool ◽

Labview Program ◽

Medium Formulation ◽

Conventional Medium

ABSTRACT A multiple microfermentor battery was designed for high-throughput recombinant protein production in Escherichia coli. This novel system comprises eight aerated glass reactors with a working volume of 80 ml and a moving external optical sensor for measuring optical densities at 600 nm (OD600) ranging from 0.05 to 100 online. Each reactor can be fitted with miniature probes to monitor temperature, dissolved oxygen (DO), and pH. Independent temperature regulation for each vessel is obtained with heating/cooling Peltier devices. Data from pH, DO, and turbidity sensors are collected on a FieldPoint (National Instruments) I/O interface and are processed and recorded by a LabVIEW program on a personal computer, which enables feedback control of the culture parameters. A high-density medium formulation was designed, which enabled us to grow E. coli to OD600 up to 100 in batch cultures with oxygen-enriched aeration. Accordingly, the biomass and the amount of recombinant protein produced in a 70-ml culture were at least equivalent to the biomass and the amount of recombinant protein obtained in a Fernbach flask with 1 liter of conventional medium. Thus, the microfermentor battery appears to be well suited for automated parallel cultures and process optimization, such as that needed for structural genomics projects.

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