Expression of Recombinant Antigens in Escherichia coli: Application on Immunochemical Studies of Schistosoma mansoni Tegumental Antigens

Abath, Frederico G. C.; Xavier, Edeneide Maria; Silva, Joanne D'Arc B; Morais, Marcos Antônio de; Montenegro, Sílvia Maria Lucena

doi:10.1590/s0074-02761997000500014

Cited by 6 publications

(5 citation statements)

References 12 publications

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“…Recombinant eukaryotic polypeptides synthesized in the Escherichia coli expression system can dier from their authentic counterparts (Marston 1986) because several eukaryotic post-translational modi®cations are not performed in E. coli, such as glycosylation, acetylation and amidation. The limitations of the use of eukaryotic genes expressed in bacteria in immunology studies have been discussed elsewhere (Chappel and Holder 1993;Abath et al 1997). Nonetheless, the antibodies raised in rabbits against MBP-rSm13 recognized Sm13 in S. mansoni, indicating that at least some of the relevant epitopes are not dependent on post-translational modi®cations.…”

Section: Discussionmentioning

confidence: 99%

Characterization of Sm13, a tegumental antigen of Schistosoma mansoni

Abath¹,

Xavier²,

Allen³

et al. 2000

Parasitol Res

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Sm13, a 13-kDa Schistosoma mansoni tegumental antigen, is one of the principal polypeptides recognized by antibodies from mice protectively vaccinated with adult-worm tegumental membranes. To obtain the complete gene encoding Sm13 we subcloned and sequenced a cDNA and a fragment of a genomic clone. The collated sequence contains 1,088 nucleotides and represents the full-length open reading frame of the gene, encoding a protein of 104 amino acids with a calculated molecular mass of 11,923 Da, compatible with the native protein identified in the tegumental membranes. The sequence derived from genomic DNA contains a 45-nucleotide intron. The analysis of the predicted protein suggests the presence of both N- and C-terminal hydrophobic membrane-spanning segments, and the coding region contains no homology in the currently available data bases. Additionally, the coding region is preceded by putative CCAAT and TATA boxes that may be involved in the control of expression. Western-blot analysis and indirect immunofluorescence resulted in the identification of a 13-kDa protein (Sm13) in the tegument of adult worms. The present study reveals that Sm13 behaves as an integral membrane protein upon partitioning in Triton X-1 14 and that it is present in worms of 3 weeks or older but not in schistosomula or miracidia. Moreover, it is also specifically recognized by sera from some schistosomiasis patients in enzyme-linked immunosorbent assay and Western-blot analysis, suggesting that it is immunogenic in human schistosomiasis.

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Section: Discussionmentioning

confidence: 99%

Characterization of Sm13, a tegumental antigen of Schistosoma mansoni

Abath¹,

Xavier²,

Allen³

et al. 2000

Parasitol Res

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“…In addition, antigens synthesized by E.coli host cells are suitable materials for antibody production (Rancour, Backues, & Bednarek, 2010). Numerous studies have been performed to optimize the expression conditions of recombinant proteins in E. coli (Abath, Xavier, Silva, Junior, & Montenegro, 1997;Arévalo-Herrera et al, 2015;Hu et al, 2004;Vaz, et al, 2011;Zhao et al, 2011).…”

Section: Pira Recombinant Antigen Purificationmentioning

confidence: 99%

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

Linh¹,

Nguyen²,

Tran³

et al. 2022

Preprint

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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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“…After IPTG induction, a 55 kDa recombinant MPB-Sm13 fusion protein was produced. Details about Sm13, a tegumental antigen of S. mansoni can be found elsewhere (Abath et al, 1997).…”

Section: Recombinant Strainmentioning

confidence: 99%

Liquid-liquid extraction of a Schistosoma mansoni recombinant protein from an Escherichia coli extract in aqueous two-phase system

Chaves¹,

Lucena‐Silva

Abath

et al. 2000

Bioprocess Engineering

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This work describes the partition of a Schistosoma mansoni tegumental antigen produced by a recombinant Escherichia coli strain using an aqueous two-phase system composed of polyethylene glycol (PEG) and potassium phosphate. The effects of the polymer molecular weight, tie line length and pH on antigen partitioning were investigated. The detection of the antigen in both phases were determined by ELISA. The system composed of PEG 3550 (19.7% w/w) and potassium phosphate (17.7% w/w) led to a yield of 59% and an antigen puri®cation factor of 3 in the PEG-rich phase. It was observed that the antigen partition in ATPS was strongly affected by the pH value and tie line length. In addition, it was possible in a single step, to remove the cell debris, that precipitated at the interface of the system.

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Expression of Recombinant Antigens in Escherichia coli: Application on Immunochemical Studies of Schistosoma mansoni Tegumental Antigens

Cited by 6 publications

References 12 publications

Characterization of Sm13, a tegumental antigen of Schistosoma mansoni

Characterization of Sm13, a tegumental antigen of Schistosoma mansoni

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

Liquid-liquid extraction of a Schistosoma mansoni recombinant protein from an Escherichia coli extract in aqueous two-phase system

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