Optimization of phosphate-limited autoinduction broth for two-stage heterologous protein expression in Escherichia coli

Autoinducible, two-stage protein expression leveraging phosphate-inducible promoters has been recently shown to enable not only high protein titers but also consistent performance across scales from screening systems (microtiter plates) to instrumented bioreactors. However, to date, small-scale production using microtiter plates and shake flasks relies on a complex autoinduction broth (AB) that requires making numerous media components, not all amenable to autoclaving. In this report, the authors develop a simpler media formulation (AB-2) with just a few autoclavable components. AB-2 is robust to small changes in its composition and performs equally, if not better, than AB across different scales. AB-2 will facilitate the adoption of phosphate-limited two-stage protein expression protocols.

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Optimization of Phosphate Limited Autoinduction Broth for 2-Stage Heterologous Protein Expression in E. coli

10.1101/2021.06.08.447502 ◽

2021 ◽

Author(s):

Romel Menacho-Melgar ◽

Jennifer N Hennigan ◽

Michael D Lynch

Keyword(s):

Protein Expression ◽

Small Scale ◽

Heterologous Protein Expression ◽

Scale Production ◽

Inducible Promoters ◽

E Coli ◽

Media Formulation ◽

Microtiter Plates ◽

Shake Flasks ◽

Consistent Performance

Autoinducible, 2-stage protein expression leveraging phosphate inducible promoters has been recently shown to enable not only high protein titers but also consistent performance across scales from screening systems (microtiter plates) to instrumented bioreactors. However, to date small scale production using microtiter plates and shake flasks rely on a complex autoinduction broth (AB) that requires making numerous media components, not all amenable to autoclaving. In this report, we develop a simpler media formulation (AB-2) with just a few autoclavable components. We show that AB-2 is robust to small changes in its composition and performs equally, if not better, than AB across different scales. AB-2 will facilitate adoption of phosphate limited 2-stage protein expression protocols.

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Improved, two-stage protein expression and purification via autoinduction of both autolysis and auto DNA/RNA hydrolysis conferred by phage lysozyme and DNA/RNA endonuclease

10.1101/2020.01.09.900753 ◽

2020 ◽

Cited By ~ 2

Author(s):

Romel Menacho-Melgar ◽

Eirik A. Moreb ◽

John P. Efromson ◽

Michael D. Lynch

Keyword(s):

Protein Expression ◽

Membrane Integrity ◽

Heterologous Protein Expression ◽

List Type ◽

Two Stage ◽

Microtiter Plates ◽

Thaw Cycle ◽

Protein Expression And Purification ◽

Phosphate Depletion ◽

Rna Endonuclease

AbstractWe report improved release of recombinant proteins in E. coli, which relies on combined cellular autolysis and DNA/RNA autohydrolysis, conferred by the tightly controlled autoinduction of both phage lysozyme and the non specific DNA/RNA endonuclease from S. marcescens. Autoinduction occurs in a two-stage process wherein heterologous protein expression and autolysis enzymes are induced upon entry into stationary phase by phosphate depletion. Cytoplasmic lysozyme and periplasmic endonuclease are kept from inducing lysis until membrane integrity is disrupted. Post cell harvest, the addition of detergent (0.1% Triton-X100) and a single 30 minutes freezer thaw cycle results in > 90% release of protein (GFP). This cellular lysis is accompanied by complete oligonucleotide hydrolysis. The approach has been validated for shake flask cultures, high throughput cultivation in microtiter plates and larger scale stirred-tank bioreactors. This tightly controlled system enables robust growth and resistance to lysis in routine media when cells are propagated and autolysis/hydrolysis genes are only induced upon phosphate depletion.HighlightsAutoinduction of both cell lysis and nucleotide hydrolysis>90 % lysis and DNA degradationStrains are stable to lysis in the absence of phosphate depletion.

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Two-stage Dynamic Deregulation of Metabolism Improves Process Robustness & Scalability in Engineered E. coli

10.1101/2020.08.30.274290 ◽

2020 ◽

Cited By ~ 2

Author(s):

Zhixia Ye ◽

Shuai Li ◽

Jennifer N. Hennigan ◽

Juliana Lebeau ◽

Eirik A. Moreb ◽

...

Keyword(s):

High Throughput ◽

Dynamic Control ◽

Pilot Scale ◽

Small Scale ◽

Scale Production ◽

Industrial Chemicals ◽

Two Stage ◽

E Coli ◽

Process Robustness ◽

Microtiter Plates

AbstractWe report improved strain and bioprocess robustness as a result of the dynamic deregulation of central metabolism using two-stage dynamic control. Dynamic control is implemented using combinations of CRISPR interference and controlled proteolysis to reduce levels of central metabolic enzymes in the context of a standardized two-stage bioprocesses. Reducing the levels of key enzymes alters metabolite pools resulting in deregulation of the metabolic network. The deregulated network is more robust to environmental conditions improving process robustness, which in turn leads to predictable scalability from high throughput small scale screens to fully instrumented bioreactors as well as to pilot scale production. Additionally, as these two-stage bioprocesses are standardized, a need for traditional process optimization is minimized. Predictive high throughput approaches that translate to larger scales are critical for metabolic engineering programs to truly take advantage of the rapidly increasing throughput and decreasing costs of synthetic biology. In this work we demonstrate that the improved robustness of E. coli strains engineered for the improved scalability of the important industrial chemicals alanine, citramalate and xylitol, from microtiter plates to pilot reactors.

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Cold-Inducible Promoters for Heterologous Protein Expression

E. coli Gene Expression Protocols ◽

10.1385/1-59259-301-1:01 ◽

2003 ◽

pp. 01-18

Author(s):

François Baneyx ◽

Mirna Mujacic

Keyword(s):

Protein Expression ◽

Heterologous Protein ◽

Heterologous Protein Expression ◽

Inducible Promoters

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Spontaneous excision of BAC vector sequences from bacmid-derived baculovirus expression vectors upon passage in insect cells

Journal of General Virology ◽

10.1099/vir.0.19438-0 ◽

2003 ◽

Vol 84 (10) ◽

pp. 2669-2678 ◽

Cited By ~ 65

Author(s):

Gorben P. Pijlman ◽

Jessica E. van Schijndel ◽

Just M. Vlak

Keyword(s):

Protein Expression ◽

Viral Genome ◽

Large Scale ◽

Heterologous Protein ◽

Insect Cells ◽

Expression Cassette ◽

Expression Vectors ◽

Green Fluorescent Protein Gene ◽

Heterologous Protein Expression ◽

Scale Production

Repeated baculovirus infections in cultured insect cells lead to the generation of defective interfering viruses (DIs), which accumulate at the expense of the intact helper virus and compromise heterologous protein expression. In particular, Autographa californica multicapsid nucleopolyhedovirus (AcMNPV) DIs are enriched in an origin of viral DNA replication (ori) not associated with the homologous regions (hrs). This non-hr ori is located within the coding sequence of the non-essential p94 gene. We investigated the effect of a deletion of the AcMNPV non-hr ori on the heterologous protein expression levels following serial passage in Sf21 insect cells. Using homologous ET recombination in E. coli, deletions within the p94 gene were made in a bacterial artificial chromosome (BAC) containing the entire AcMNPV genome (bacmid). All bacmids were equipped with an expression cassette containing the green fluorescent protein gene and a gene encoding the classical swine fever virus E2 glycoprotein (CSFV-E2). For the parental (intact) bacmid only, a strong accumulation of DIs with reiterated non-hr oris was observed. This was not observed for the mutants, indicating that removal of the non-hr ori enhanced the genetic stability of the viral genome upon passaging. However, for all passaged viruses it was found that the entire BAC vector including the expression cassette was spontaneously deleted from the viral genome, leading to a rapid decrease in GFP and CSFV-E2 production. The rationale for the (intrinsic) genetic instability of the BAC vector in insect cells and the implications with respect to large-scale production of proteins with bacmid-derived baculoviruses are discussed.

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AGRICULTURAL PRODUCTION OF CA DONG PEOPLE IN RESETTLEMENT AREA OF TRANH RIVER HYDROPOWER NUMBER 2: CURRENT SITUATION AND IMPACT FACTORS

Tạp chí Nghiên cứu dân tộc ◽

10.25073/0866-773x/428 ◽

2020 ◽

Vol 9 (2) ◽

Author(s):

Bùi Thị Bích Lan

Keyword(s):

Social Relations ◽

Agricultural Production ◽

Large Scale ◽

Transition Process ◽

Impact Factors ◽

Small Scale ◽

Scale Production ◽

Technical Application ◽

Production Models ◽

Role Of The State

In Vietnam, the construction of hydropower projects has contributed significantly in the cause of industrialization and modernization of the country. The place where hydropower projects are built is mostly inhabited by ethnic minorities - communities that rely primarily on land, a very important source of livelihood security. In the context of the lack of common productive land in resettlement areas, the orientation for agricultural production is to promote indigenous knowledge combined with increasing scientific and technical application; shifting from small-scale production practices to large-scale commodity production. However, the research results of this article show that many obstacles in the transition process are being posed such as limitations on natural resources, traditional production thinking or the suitability and effectiveness of scientific - technical application models. When agricultural production does not ensure food security, a number of implications for people’s lives are increasingly evident, such as poverty, preserving cultural identity, social relations and resource protection. Since then, it has set the role of the State in researching and building appropriate agricultural production models to exploit local strengths and ensure sustainability.

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Study of Diabetic Mellitus and Knowledge of Lotion Foot Care on the Community

International Journal of Pharmaceutical Quality Assurance ◽

10.25258/ijpqa.v9i3.13659 ◽

2018 ◽

Vol 9 (3) ◽

Author(s):

Hariyadi DM ◽

Athiyah U ◽

Hendradi E ◽

Rosita N ◽

Erawati T ◽

...

Keyword(s):

Blood Glucose ◽

T Test ◽

Small Scale ◽

Scale Production ◽

Foot Care ◽

Diabetic Mellitus ◽

Glucose Levels ◽

Blood Glucose Levels ◽

Before And After ◽

History Of

The prevention of Diabetic Mellitus (DM) and its complications is the main aim of this study, in addition to the training of lotion foot care application and the development of small scale industry. The research team delivered knowledge in the form of training on Diabetic Mellitus, healthy food, treatment and prevention of complications, and small-scale production of cosmetic products. The aim of this study was to determine the correlation between training on diabetic and lotion foot care application as preventive measures against diabetic complications on the patient's blood glucose levels in the community of residents in Banyuurip Jaya, Surabaya. It was expected from this training that the knowledge of the residents increases and people living with diabetic undergo lifestyle changes and therefore blood sugar levels can be controlled. The parameters measured in this research were blood glucose levels, the anti diabetic drug types consumed, and compliance on diabetics. This study used the data taken from 60 patients with DM over a period of one month. Questionnaires and log books was used to retrieve data and changes in blood glucose levels in diabetic patients. The results showed the demographic data of patients with type 2 diabetic of 85% female and 15% male, with the range of patients aged of 61-70 years of 46.67% and had history of diabetic (90%). The history of drugs consumed by respondents was anti diabetic drugs such as metformin (40%), glimepiride (33.37%) and insulin (6.67%). In addition, the increased knowledge of DM patients after being given the training compared to before training was shown in several questions in the questionnaire. A statistical analysis using t-test analyzed a correlation between training provided in order to enhance understanding of the patient, as well as correlation with blood glucose levels. A paired T-test showed that there was a relationship between the knowledge of trainees before and after training (p less than 0.05). An interesting result was that there was no relationship between blood glucose levels before and after training provided (p> 0.05).

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