Production of soluble eukaryotic recombinant proteins in E. coli is favoured in early log-phase cultures induced at low temperature

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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Rapid E. coli-based cloning and expression system for production of recombinant proteins

Journal of Biotechnology ◽

10.1016/j.jbiotec.2014.07.237 ◽

2014 ◽

Vol 185 ◽

pp. S70

Author(s):

Boguslaw Lupa ◽

Krzysztof Stawujak ◽

Igor Rozanski ◽

Justyna Stec-Niemczyk

Keyword(s):

Recombinant Proteins ◽

Expression System ◽

Cloning And Expression ◽

E Coli

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Changes in Aerobic Plate and Escherichia coli–Coliform Counts and in Populations of Inoculated Foodborne Pathogens on Inshell Walnuts during Storage

Journal of Food Protection ◽

10.4315/0362-028x.jfp-15-553 ◽

2016 ◽

Vol 79 (7) ◽

pp. 1143-1153 ◽

Cited By ~ 8

Author(s):

JOHN C. FRELKA ◽

GORDON R. DAVIDSON ◽

LINDA J. HARRIS

Keyword(s):

Escherichia Coli ◽

Listeria Monocytogenes ◽

Relative Humidity ◽

Low Temperature ◽

Foodborne Pathogens ◽

Limit Of Detection ◽

Sampling Time ◽

E Coli ◽

Plate Counts ◽

Forced Air

ABSTRACT After harvest, inshell walnuts are dried using low-temperature forced air and are then stored in bins or silos for up to 1 year. To better understand the survival of bacteria on inshell walnuts, aerobic plate counts (APCs) and Escherichia coli–coliform counts (ECCs) were evaluated during commercial storage (10 to 12°C and 63 to 65% relative humidity) over 9 months. APCs decreased by 1.4 to 2.0 log CFU per nut during the first 5 months of storage, and ECCs decreased by 1.3 to 2.2 log CFU per nut in the first month of storage. Through the remaining 4 to 8 months of storage, APCs and ECCs remained unchanged (P > 0.05) or decreased by <0.15 log CFU per nut per month. Similar trends were observed on kernels extracted from the inshell walnuts. APCs and ECCs were consistently and often significantly higher on kernels extracted from visibly broken inshell walnuts than on kernels extracted from visibly intact inshell walnuts. Parameters measured in this study were used to determine the survival of five-strain cocktails of E. coli O157:H7, Listeria monocytogenes, and Salmonella inoculated onto freshly hulled inshell walnuts (~8 log CFU/g) after simulated commercial drying (10 to 12 h; 40°C) and simulated commercial storage (12 months at 10°C and 65% relative humidity). Populations declined by 2.86, 5.01, and 4.40 log CFU per nut for E. coli O157:H7, L. monocytogenes, and Salmonella, respectively, after drying and during the first 8 days of storage. Salmonella populations changed at a rate of −0.33 log CFU per nut per month between days 8 and 360, to final levels of 2.83 ± 0.79 log CFU per nut. E. coli and L. monocytogenes populations changed by −0.17 log CFU per nut per month and −0.26 log CFU per nut per month between days 8 and 360, respectively. For some samples, E. coli or L. monocytogenes populations were below the limit of detection by plating (0.60 log CFU per nut) by day 183 or 148, respectively; at least one of the six samples was positive at each subsequent sampling time by either plating or by enrichment.

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Effect of Gamma Irradiation on the Survival of Pathogens in Kwamegi, a Traditional Korean Semidried Seafood

Journal of Food Protection ◽

10.4315/0362-028x-66.11.2093 ◽

2003 ◽

Vol 66 (11) ◽

pp. 2093-2096 ◽

Cited By ~ 25

Author(s):

S. P. CHAWLA ◽

D. H. KIM ◽

C. JO ◽

J. W. LEE ◽

H. P. SONG ◽

...

Keyword(s):

Low Temperature ◽

Salmonella Typhimurium ◽

Water Activity ◽

Pathogenic Bacteria ◽

Radiation Sensitivity ◽

E Coli ◽

Public Health Significance ◽

Irradiation Treatment ◽

Test Organisms ◽

Health Significance

Kwamegi (semidried raw Pacific saury) is traditional seafood available in Korea. It has water activity in the range of 0.90 to 0.95. Spoilage and the growth of most pathogenic bacteria is retarded because of low water activity, low temperature, and packaging. However, it is contaminated with bacteria of public health significance and poses a hazard to the consumer because it is consumed raw without any cooking. The effectiveness of these hurdles in preventing the growth of Staphylococcus aureus, Bacillus cereus, Salmonella Typhimurium, and Escherichia coli and the efficacy of irradiation treatment in eliminating these bacteria from kwamegi using inoculated pack studies was examined. Radiation sensitivity of S. aureus, B. cereus, Salmonella Typhimurium, and E. coli in kwamegi was investigated. D10-values of these organisms in kwamegi were 590 ± 13.6, 640 ± 14.9, 560 ± 45.4, and 550 ± 8.6 Gy, respectively. The growth of all four test organisms inoculated into these foods during 4 weeks of storage at an ambient winter temperature (ranging from −5°C to +5°C) was recorded. All four pathogens (inoculated at 106 CFU/g) were eliminated by irradiation at 4 kGy. These studies unequivocally demonstrate that irradiation, with a combination of low water activity and low temperature, results in microbiologically safe kwamegi.

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Induction of Viable but Nonculturable Escherichia coli O157:H7 in the Phyllosphere of Lettuce: a Food Safety Risk Factor

Applied and Environmental Microbiology ◽

10.1128/aem.05020-11 ◽

2011 ◽

Vol 77 (23) ◽

pp. 8295-8302 ◽

Cited By ~ 81

Author(s):

Laura-Dorina Dinu ◽

Susan Bach

Keyword(s):

Escherichia Coli ◽

Food Safety ◽

Low Temperature ◽

Cell Density ◽

Prolonged Storage ◽

Potential Hazard ◽

Vbnc State ◽

Viable But Nonculturable ◽

Content Type ◽

E Coli

ABSTRACTEscherichia coliO157:H7 continues to be an important human pathogen and has been increasingly linked to food-borne illness associated with fresh produce, particularly leafy greens. The aim of this work was to investigate the fate ofE. coliO157:H7 on the phyllosphere of lettuce under low temperature and to evaluate the potential hazard of viable but nonculturable (VBNC) cells induced under such stressful conditions. First, we studied the survival of six bacterial strains following prolonged storage in water at low temperature (4°C) and selected two strains with different nonculturable responses for the construction ofE. coliO157:H7 Tn7gfptransformants in order to quantitatively assess the occurrence of human pathogens on the plant surface. Under a suboptimal growth temperature (16°C), bothE. coliO157:H7 strains maintained culturability on lettuce leaves, but under more stressful conditions (8°C), the bacterial populations evolved toward the VBNC state. The strain-dependent nonculturable response was more evident in the experiments with different inoculum doses (109and 106E. coliO157:H7 bacteria per g of leaf) when strain BRMSID 188 lost culturability after 15 days and strain ATCC 43895 lost culturability within 7 days, regardless of the inoculum dose. However, the number of cells entering the VBNC state in high-cell-density inoculum (approximately 55%) was lower than in low-cell-density inoculum (approximately 70%). We recorded the presence of verotoxin for 3 days in samples that contained a VBNC population of 4 to 5 log10cells but did not detect culturable cells. These findings indicate thatE. coliO157:H7 VBNC cells are induced on lettuce plants, and this may have implications regarding food safety.

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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.

Biochemistry and Cell Biology ◽

10.1139/o07-017 ◽

2007 ◽

Vol 85 (2) ◽

pp. 203-208 ◽

Cited By ~ 2

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.

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Different expression levels of two KgmB-His fusion proteins

Journal of the Serbian Chemical Society ◽

10.2298/jsc0512401m ◽

2005 ◽

Vol 70 (12) ◽

pp. 1401-1407 ◽

Cited By ~ 3

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.

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