Designer Outer Membrane Protein Facilitates Uptake of Decoy Molecules into a Cytochrome P450BM3‐Based Whole‐Cell Biocatalyst

2021 ◽  
Author(s):  
Masayuki Karasawa ◽  
Kai Yonemura ◽  
Joshua Kyle Stanfield ◽  
Kazuto Suzuki ◽  
Osami Shoji
Keyword(s):  
Membrane Protein ◽  
Outer Membrane ◽  
Outer Membrane Protein ◽  
Whole Cell ◽  
Whole Cell Biocatalyst

scholarly journals Expression and Refolding of Truncated Recombinant Major Outer Membrane Protein Antigen (r56) of Orientia tsutsugamushi and Its Use in Enzyme-Linked Immunosorbent Assays

1998 ◽  
Vol 5 (4) ◽  
pp. 519-526 ◽  
Author(s):  
W.-M. Ching ◽  
H. Wang ◽  
C. Eamsila ◽  
D. J. Kelly ◽  
G. A. Dasch
Keyword(s):  
Membrane Protein ◽  
Outer Membrane ◽  
Outer Membrane Protein ◽  
Scrub Typhus ◽  
The United States ◽  
Major Outer Membrane Protein ◽  
Orientia Tsutsugamushi ◽  
Whole Cell ◽  
Whole Cell Lysate ◽  
Cell Lysate

ABSTRACT The variable 56-kDa major outer membrane protein of Orientia tsutsugamushi is the immunodominant antigen in human scrub typhus infections. The gene encoding this protein from Karp strain was cloned into the expression vector pET11a. The recombinant protein (r56) was expressed as a truncated nonfusion protein (amino acids 80 to 456 of the open reading frame) which formed an inclusion body when expressed in Escherichia coli BL21. Refolded r56 was purified and compared to purified whole-cell lysate of the Karp strain of O. tsutsugamushi by immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) for reactivity with rabbit sera prepared against eight antigenic prototypes of O. tsutsugamushi as well as several other species of Rickettsiales and nonrickettsial antigens. Refolded r56 exhibited broad reactivity with the rabbit antisera against the Orientia prototypes, and the ELISA reactions with the r56 and Karp whole-cell lysate antigens correlated well (r = 0.81, n = 22, sensitivity compared to that of standard ELISA of 91%). Refolded r56 did not react with most antisera against other rickettsial species or control antigens (specificity = 92%, n = 13) using a positive cutoff value determined with eight uninfected rabbit sera. Refolded r56 was evaluated further by ELISA, using 128 sera obtained from patients with suspected scrub typhus from Korat, Thailand, and 74 serum specimens from healthy Thai soldiers. By using the indirect immunoperoxidase assay as the reference assay, the recombinant antigen exhibited a sensitivity and specificity of 93% or greater for detection of both IgG and IgM in the ELISA at 1:400 serum dilution. These results strongly suggest that purified r56 is a suitable candidate for replacing the density gradient-purified, rickettsia-derived, whole-cell antigen currently used in the commercial dipstick assay available in the United States.

scholarly journals Immune response to Vi polysaccharide, heat-killed whole cells, and outer membrane protein of Salmonella typhi

2015 ◽  
Vol 9 (06) ◽  
pp. 642-649 ◽  
Author(s):  
Alaa El-Din Shawky Hosny ◽  
Mohamed Reda Diab ◽  
Rania Abdelmonem Khattab ◽  
Heba Osama Awad
Keyword(s):  
Immune Response ◽  
Membrane Protein ◽  
Outer Membrane ◽  
Outer Membrane Protein ◽  
Enzyme Linked Immunosorbent Assay ◽  
Whole Cell ◽  
Coating Antigen ◽  
Whole Cells ◽  
Cell Elisa

Introduction: Salmonella typhiVi capsular polysaccharide (ViCPS) is a licensed vaccine against typhoid fever in many countries; in Egypt, the killed whole-cell vaccine is still used. In this study, mice were used as an animal model to evaluate the immune response to ViCPS and other S. typhiantigens such as heat-killed whole cells and outer membrane protein (OMP). Methodology: The three antigens were laboratory prepared, injected into mice groups, and the humoral response was evaluated using the indirect whole-cell enzyme-linked immunosorbent assay (ELISA). The sensitivity of this assay was investigated using in situ or pre-heated whole cells as coating antigens. In addition, the effect of the immunization route for ViCPS was examined. Results: Immunizing doses of heat-killed whole cells as well as ViCPS, 2 and 4 µg given subcutaneously (SC) and 4 µg given intraperitoneally (IP), showed significant immune response compared to controls. However, the responses to these doses were not significantly different from each other. The OMP showed a higher significant response. The sensitivity of indirect whole-cell ELISA was enhanced significantly by in situ heat treatment of the coating antigen rather than the pre-heated coating antigen. Conclusions: The three antigens showed significant immune response. The immune response to OMP was higher. Though heat-killed whole cells and ViCPS are almost similar in immunizing level, ViCPS is recommended. The SC route was more immunizing than the IP one. Furthermore, the sensitivity of the indirect whole-cell ELISA technique could be enhanced by in situ heat inactivation of the coating cells.

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