scholarly journals Expression byStreptomyces lividansof the RatαIntegrin CD11b A-Domain as a Secreted and Soluble Recombinant Protein

2007 ◽  
Vol 2007 ◽  
pp. 1-6 ◽  
Author(s):  
Dorra Zouari Ayadi ◽  
Hichem Chouayekh ◽  
Sonda Mhiri ◽  
Khaled Zerria ◽  
Dahmani M. Fathallah ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Recombinant Protein ◽  
Dna Sequence ◽  
Recombinant Proteins ◽  
Signal Peptide ◽  
Shuttle Vector ◽  
Host Strain ◽  
Blocking Antibody ◽  
Synthetic Signal ◽  
Soluble Recombinant Protein

We already reported the use of a long synthetic signal peptide (LSSP) to secrete theStreptomycessp. TO1 amylase byStreptomyces lividansstrain. We herein report the expression and secretion of the rat CD11b A-domain using the same LSSP andS. lividansas host strain. We have used theEscherichia coli/Streptomycesshuttle vector pIJ699 for the cloning of the A-domain DNA sequence downstream of LSSP and under the control of the constitutiveermE-uppromoter ofStreptomyces erythraeus. Using this construct andS. lividansas a host strain, we achieved the expression of 8 mg/L of soluble secreted recombinant form of the A-domain of the rat leukocyteβ2 integrin CD11/CD18 alpha M subunit (CD11b). This secreted recombinant CD11b A-domain reacted with a function blocking antibody showing that this protein is properly folded and probably functional. These data support the capability ofStreptomycesto produce heterologous recombinant proteins as soluble secreted form using the “LSSP” synthetic signal peptide.

6. Genetic engineering

2016 ◽  
Author(s):  
Aysha Divan ◽  
Janice A. Royds
Keyword(s):  
Gene Therapy ◽  
Genetic Engineering ◽  
Recombinant Protein ◽  
Gene Cloning ◽  
Dna Sequence ◽  
Recombinant Proteins ◽  
Host Cells ◽  
Therapeutic Antibodies ◽  
P Gene ◽  
Genetically Engineer

Gene-cloning processes enable us to produce large amounts of a DNA sequence so that its function can be studied. These technologies can also be applied in medicine and agriculture to genetically engineer production of biological proteins or whole organisms with new or modified traits. At the heart of these applications is the capability to produce recombinant proteins from cloned genes in host cells. ‘Genetic engineering’ outlines some of these applications: recombinant pharmaceuticals, monoclonal therapeutic antibodies, recombinant protein vaccines, gene therapy, and genetically modified foods. It also considers some of the concerns with these applications.

Recombinant Protein Expression in Escherichia coli (E.coli): What We Need to Know

2018 ◽  
Vol 24 (6) ◽  
pp. 718-725 ◽  
Author(s):  
Seyed Mohammad Gheibi Hayat ◽  
Najmeh Farahani ◽  
Behrouz Golichenari ◽  
Amirhossein Sahebkar
Keyword(s):  
Escherichia Coli ◽  
Recombinant Protein ◽  
Protein Expression ◽  
Recombinant Proteins ◽  
Recombinant Protein Expression ◽  
Culture Conditions ◽  
Strain Vector ◽  
Factors Affecting ◽  
Expression In Escherichia Coli ◽  
Pros And Cons

Background: Host, vector, and culture conditions (including cultivation media) are considered among the three main elements contributing to a successful production of recombinant proteins. Accordingly, one of the most common hosts to produce recombinant therapeutic proteins is Escherichia coli. Methodology: A comprehensive literature review was performed to identify important factors affecting production of recombinant proteins in Escherichia coli. Results: Escherichia coli is taken into account as the easiest, quickest, and cheapest host with a fully known genome. Thus, numerous modifications have been carried out on Escherichia coli to optimize it as a good candidate for protein expression and; as a result, several engineered strains of Escherichia coli have been designed. In general; host strain, vector, and cultivation parameters are recognized as crucial ones determining success of recombinant protein expression in Escherichia coli. In this review, the role of host, vector, and culture conditions along with current pros and cons of different types of these factors leading to success of recombinant protein expression in Escherichia coli were discussed. Conclusion: Successful protein expression in Escherichia coli necessitates a broad knowledge about physicochemical properties of recombinant proteins, selection among common strains of Escherichia coli and vectors, as well as factors related to media including time, temperature, and inducer.

scholarly journals Species-Specific Antibody Responses to the Recombinant 53-Kilodalton Excretory and Secretory Proteins in Mice Infected with Trichinella spp.

2008 ◽  
Vol 15 (3) ◽  
pp. 468-473 ◽  
Author(s):  
Isao Nagano ◽  
Zhiliang Wu ◽  
Yuzo Takahashi
Keyword(s):  
Escherichia Coli ◽  
Recombinant Protein ◽  
Recombinant Proteins ◽  
Specific Antibody ◽  
Expression System ◽  
Amino Acid Sequences ◽  
Antibody Responses ◽  
Secretory Proteins ◽  
Muscle Larvae ◽  
Species Specific

ABSTRACT The 53-kDa proteins in larval excretory and secretory (E-S) products were expressed from five Trichinella species (T. spiralis, T. britovi, T. nativa, T. pseudospiralis, and T. papuae), using the Escherichia coli expression system, and the antibody responses to the 53-kDa recombinant proteins in mice infected with Trichinella spp. were analyzed by Western blotting. The 53-kDa protein is conserved among the five Trichinella species, with >60% similarity in amino acid sequences. The 53-kDa recombinant proteins of T. spiralis and T. pseudospiralis reacted to sera from mice infected with T. spiralis and T. pseudospiralis at 8 days postinfection (p.i.), respectively. An antibody against the 53-kDa recombinant protein of T. spiralis recognized the 53-kDa protein in the crude extracts from adult worms and 30-day p.i. muscle larvae and E-S products from muscle larvae of T. spiralis but did not recognize any proteins from T. pseudospiralis. The sera from the mice infected with T. spiralis strongly reacted with the 53-kDa recombinant protein of T. spiralis but did not react with the 53-kDa recombinant proteins of T. britovi, T. nativa, T. pseudospiralis, and T. papuae. Similarly, the sera from mice infected with T. britovi, T. nativa, T. pseudospiralis, or T. papuae strongly reacted with the 53-kDa recombinant proteins of T. britovi, T. nativa, T. pseudospiralis, or T. papuae, respectively. These results showed that the 53-kDa recombinant proteins provide early and species-specific antibody responses in mice infected with Trichinella spp.

scholarly journals Nucleic Acids In Inclusion Bodies Obtained From E. Coli Cells Expressing Human Interferon-Gamma

2020 ◽  
Author(s):  
Elena Krachmarova ◽  
Ivan Ivanov ◽  
Genoveva Nacheva
Keyword(s):  
Escherichia Coli ◽  
Recombinant Protein ◽  
Nucleic Acids ◽  
Interferon Gamma ◽  
Recombinant Proteins ◽  
Gel Electrophoresis ◽  
Inclusion Bodies ◽  
Protein Aggregates ◽  
Human Interferon ◽  
Molecular Heterogeneity

Abstract BackgroundInclusion bodies (IBs) are protein aggregates in recombinant bacterial cells containing mainly the target recombinant protein. Although it has been shown that IBs contain functional proteins along with protein aggregates, their direct application as pharmaceuticals is hindered by their heterogeneity and hazardous contaminants with bacterial origin. Therefore, together with the production of soluble species, IBs remain the main source for manufacture of recombinant proteins with medical application. The quality and composition of the IBs affect the refolding yield and further purification of the recombinant protein. The knowledge whether nucleic acids are genuine components or concomitant impurities of the IBs is a prerequisite for the understanding of the IBs formation and for development of optimized protocols for recombinant protein refolding and purification. IBs isolated from Escherichia coli overexpressing human interferon-gamma (hIFNγ), a protein with therapeutic application, were used as a model. ResultsIBs were isolated from Escherichia coli LE392 cells transformed with a hIFNγ expressing plasmid under standard conditions and further purified by centrifugation on a sucrose cushion, followed by several steps of sonication and washings with non-denaturing concentrations of urea. The efficiency of the purification was estimated by SDS-PAGE gel electrophoresis and parallel microbiological testing for the presence of residual intact bacteria. Phenol/chloroform extraction showed that the highly purified IBs contain both DNA and RNA. The latter were studied by UV spectroscopy and agarose gel electrophoresis combined with enzymatic treatment and hybridization. DNA was observed as a diffuse fraction mainly in the range of 250 to 1000 bp. RNA isolated by TRIzol® also demonstrated a substantial molecular heterogeneity. Hybridization with 32P-labelled oligonucleotides showed that the IBs contain rRNA and are enriched of hIFNγ mRNA.ConclusionsThe results presented in this study indicate that the nucleic acids might be intrinsic components rather than co-precipitated impurities in the IBs. We assume that the nucleic acids are active participants in the aggregation of recombinant proteins and formation of the IBs that originate from the transcription and translation machinery of the microbial cell factory. Further studies are needed to ascertain this notion.

scholarly journals Impact of the Expression System on Recombinant Protein Production in Escherichia coli BL21

2021 ◽  
Vol 12 ◽  
Author(s):  
Gema Lozano Terol ◽  
Julia Gallego-Jara ◽  
Rosa Alba Sola Martínez ◽  
Adrián Martínez Vivancos ◽  
Manuel Cánovas Díaz ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Recombinant Protein ◽  
Protein Expression ◽  
Recombinant Proteins ◽  
Copy Number ◽  
Recombinant Protein Production ◽  
Protein Production ◽  
Recombinant Protein Expression ◽  
Expression System ◽  
E Coli

Recombinant protein production for medical, academic, or industrial applications is essential for our current life. Recombinant proteins are obtained mainly through microbial fermentation, with Escherichia coli being the host most used. In spite of that, some problems are associated with the production of recombinant proteins in E. coli, such as the formation of inclusion bodies, the metabolic burden, or the inefficient translocation/transport system of expressed proteins. Optimizing transcription of heterologous genes is essential to avoid these drawbacks and develop competitive biotechnological processes. Here, expression of YFP reporter protein is evaluated under the control of four promoters of different strength (PT7lac, Ptrc, Ptac, and PBAD) and two different replication origins (high copy number pMB1′ and low copy number p15A). In addition, the study has been carried out with the E. coli BL21 wt and the ackA mutant strain growing in a rich medium with glucose or glycerol as carbon sources. Results showed that metabolic burden associated with transcription and translation of foreign genes involves a decrease in recombinant protein expression. It is necessary to find a balance between plasmid copy number and promoter strength to maximize soluble recombinant protein expression. The results obtained represent an important advance on the most suitable expression system to improve both the quantity and quality of recombinant proteins in bioproduction engineering.

scholarly journals Nucleic Acids in Inclusion Bodies obtained from E. coli Cells Expressing Human Interferon-Gamma

2020 ◽  
Author(s):  
Elena Krachmarova ◽  
Ivan Ivanov ◽  
Genoveva Nacheva
Keyword(s):  
Escherichia Coli ◽  
Recombinant Protein ◽  
Nucleic Acids ◽  
Interferon Gamma ◽  
Recombinant Proteins ◽  
Gel Electrophoresis ◽  
Inclusion Bodies ◽  
Protein Aggregates ◽  
Human Interferon ◽  
Molecular Heterogeneity

Abstract Background Inclusion bodies (IBs) are protein aggregates in recombinant bacterial cells containing mainly the target recombinant protein. Although it has been shown that IBs contain functional proteins along with protein aggregates, their direct application as pharmaceuticals is hindered by their heterogeneity and hazardous contaminants with bacterial origin. Therefore, together with the production of soluble species, IBs remain the main source for manufacture of recombinant proteins with medical application. The quality and composition of the IBs affect the refolding yield and further purification of the recombinant protein. The knowledge whether nucleic acids are genuine components or concomitant impurities of the IBs is a prerequisite for the understanding of the IBs formation and for development of optimized protocols for recombinant protein refolding and purification. IBs isolated from Escherichia coli overexpressing human interferon-gamma (hIFNγ), a protein with therapeutic application, were used as a model. Results IBs were isolated from Escherichia coli LE392 cells transformed with a hIFNγ expressing plasmid under standard conditions and further purified by centrifugation on a sucrose cushion, followed by several steps of sonication and washings with non-denaturing concentrations of urea. The efficiency of the purification was estimated by SDS-PAGE gel electrophoresis and parallel microbiological testing for the presence of residual intact bacteria. Phenol/chloroform extraction showed that the highly purified IBs contain both DNA and RNA. The latter were studied by UV spectroscopy and agarose gel electrophoresis combined with enzymatic treatment and hybridization. DNA was observed as a diffuse fraction mainly in the range of 250 to 1000 bp. RNA isolated by TRIzol® also demonstrated a substantial molecular heterogeneity. Hybridization with 32 P-labelled oligonucleotides showed that the IBs contain rRNA and are enriched of hIFNγ mRNA. Conclusions The results presented in this study indicate that the nucleic acids are intrinsic components rather than co-precipitated impurities in the IBs. We assume that the nucleic acids are active participants in the aggregation of recombinant proteins and formation of the IBs that originate from the transcription and translation machinery of the microbial cell factory.

Complete signal peptide of Tk1884, an α-amylase from Thermococcus kodakarensis, is not necessary for extracellular secretion of the enzyme by Escherichia coli

Amylase ◽  
2017 ◽  
Vol 1 (1) ◽  
Author(s):  
Majida A. Muhammad ◽  
Samia Falak ◽  
Naeem Rashid ◽  
Nasir Ahmed ◽  
Qurra-Tul-Ann A. Gardner ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Recombinant Protein ◽  
Signal Peptide ◽  
Amylase Activity ◽  
Signal Sequence ◽  
Ionization Mass ◽  
Extracellular Medium ◽  
E Coli ◽  
Thermococcus Kodakarensis ◽  
Extracellular Secretion

AbstractIn order to elucidate if Escherichia coli secretion system recognizes the N-terminally truncated signal sequence of an archaeal α-amylase from Thermococcus kodakarensis (Tk1884) and secretes the recombinant protein to the extracellular medium, we have cloned Tk1884 with the deletion of the sixteen N-terminal amino acids and produced the recombinant protein Tk1884Δ16 in E. coli. Analysis of the intracellular, membranous and extracellular fractions demonstrated the presence of Tk1884Δ16 in all the three fractions. The intracellular α-amylase activity, similar to the membranous fraction, increased with the passage of time till 8 h of induction and then decreased. In contrast, the extracellular α-amylase activity slowly increased with the passage of time after induction. The extracellular amylase activity was purified and determination of the molecular mass by electrospray ionization mass spectrometry demonstrated that Tk1884Δ16 was secreted to the extracellular medium without cleavage of the signal peptide. To the best of our knowledge, this is the first report on recognition of N-terminally truncated signal peptide of archaeal origin by E. coli.

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