Expression and rapid purification of recombinant biologically active ovine growth hormone with DsbA targeting to Escherichia coli inner membrane

Durrani, Faiza Gul; Gul, Roquyya; Sadaf, Saima; Akhtar, Muhammad

doi:10.1007/s00253-015-6751-6

Cited by 11 publications

(13 citation statements)

References 24 publications

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“…We hypothesized that a new strategy could hamper the overall translocation process to study the impact of mutations in the hydrophobic core of DsbAss, rather than finding DsbA inhibitors to observe its effects on translocation of rOGH. The rOGH is a 22-kDa protein consisting of two disulfide bonds, and has been successfully expressed in a soluble form in high yield and translocated across periplasm of E. coli by DsbAss and the T7 promoter system [27]. The SRP targeting mechanism in E. coli [19] constitutes a “fifty-four homolog” protein (Ffh, a homolog of eukaryotic SRP54 protein), necessary for viability, efficient protein export, and 4.5S RNA.…”

Section: Introductionmentioning

confidence: 99%

Mutagenesis of DsbAss is Crucial for the Signal Recognition Particle Mechanism in Escherichia coli: Insights from Molecular Dynamics Simulations

Durrani

Gul²,

Mirza

et al. 2019

Biomolecules

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The disulfide bond signal sequence (DsbAss) protein is characterized as an important virulence factor in gram-negative bacteria. This study aimed to analyze the “alanine” alteration in the hydrophobic (H) region of DsbAss and to understand the conformational DsbAss alteration(s) inside the fifty-four homolog (Ffh)-binding groove which were revealed to be crucial for translocation of ovine growth hormone (OGH) to the periplasmic space in Escherichia coli via the secretory (Sec) pathway. An experimental design was used to explore the hydrophobicity and alteration of alanine (Ala) to isoleucine (Ile) in the tripartite structure of DsbAss. As a result, two DsbAss mutants (Ala at positions -11 and -13) with same hydrophobicity of 1.539 led to the conflicting translocation of the active OGH gene. We performed molecular dynamics (MD) simulations and molecular mechanics generalized born surface area (MM-GBSA) binding free energy calculations to examine the interaction energetic and dynamic aspects of DsbAss/signal repetition particle 54 (SRP54) binding, which has a principle role in Escherichia coli Sec pathways. Although both DsbAss mutants retained helicity, the MD simulation analysis evidenced that altering Ala-13 changed the orientation of the signal peptide in the Ffh M binding domain groove, favored more stable interaction energies (MM-GBSA ΔGtotal = −140.62 kcal mol−1), and hampered the process of OGH translocation, while Ala-11 pointed outward due to unstable conformation and less binding energy (ΔGtotal = −124.24 kcal mol−1). Here we report the dynamic behavior of change of “alanine” in the H-domain of DsbAss which affects the process of translocation of OGH, where MD simulation and MM-GBSA can be useful initial tools to investigate the virulence of bacteria.

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Section: Introductionmentioning

confidence: 99%

Mutagenesis of DsbAss is Crucial for the Signal Recognition Particle Mechanism in Escherichia coli: Insights from Molecular Dynamics Simulations

Durrani

Gul²,

Mirza

et al. 2019

Biomolecules

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“…In Pakistan, at present in order to confirm the COVID-19 infection, only molecular based RT-qPCR is allowed. RT-PCR technique is very well established in all the research institutes of the country 17 . In particular case of COVID-19, It is performed with the kits available to the Govt.…”

Section: Diagnostic Tests For Covid-19 With Special Reference To Pakimentioning

confidence: 99%

An Approach to Screen COVID-19 Patients: With Special Reference to Pakistan

Durrani¹,

Zaheer²

2020

Preprint

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The World Health Organisation declared Covid-19 as a pandemic on 11th March 2020. The main approach to tackle Covid-19 worldwide is to screen and provide supportive care to patients. For screening purpose RT-qPCR based test are used as an initial detections assay. The test is expensive, time consuming and not suitable for mass scale screening/ confirmation requirement. A recent advancement is development of Immunoassay procedures (liquid Phase tests or bed side 10-20minute strip test). In order to help and accelerate bringing life to normal after lock down, Pakistan is in dire need to develop and adopt the immunoassay procedures for mass scale screening and confirmation of COVID-19 infection. It is cheap and easy to perform without a lab requirement.

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“…For SRP-dependent secretion, e.g. the ones from DsbA [106,116,[125][126][127], SfmC [127][128][129], TolB [116,127,128], and TorT [116,[127][128][129] can be chosen. Heterologous signal sequences include signal peptides from Bortadetella pertussis [130], G1 (Bacillus sp.)…”

Section: Secretion Signalsmentioning

confidence: 99%

Secretion of recombinant proteins from E. coli

Kleiner-Grote

Risse

Friehs

2018

Engineering in Life Sciences

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The microorganism Escherichia coli is commonly used for recombinant protein production. Despite several advantageous characteristics like fast growth and high protein yields, its inability to easily secrete recombinant proteins into the extracellular medium remains a drawback for industrial production processes. To overcome this limitation, a multitude of approaches to enhance the extracellular yield and the secretion efficiency of recombinant proteins have been developed in recent years. Here, a comprehensive overview of secretion mechanisms for recombinant proteins from E. coli is given and divided into three main sections. First, the structure of the E. coli cell envelope and the known natural secretion systems are described. Second, the use and optimization of different one‐ or two‐step secretion systems for recombinant protein production, as well as further permeabilization methods are discussed. Finally, the often‐overlooked role of cell lysis in secretion studies and its analysis are addressed. So far, effective approaches for increasing the extracellular protein concentration to more than 10 g/L and almost 100% secretion efficiency exist, however, the large range of optimization methods and their combinations suggests that the potential for secretory protein production from E. coli has not yet been fully realized.

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Expression and rapid purification of recombinant biologically active ovine growth hormone with DsbA targeting to Escherichia coli inner membrane

Cited by 11 publications

References 24 publications

Mutagenesis of DsbAss is Crucial for the Signal Recognition Particle Mechanism in Escherichia coli: Insights from Molecular Dynamics Simulations

Mutagenesis of DsbAss is Crucial for the Signal Recognition Particle Mechanism in Escherichia coli: Insights from Molecular Dynamics Simulations

An Approach to Screen COVID-19 Patients: With Special Reference to Pakistan

Secretion of recombinant proteins from E. coli

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