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Microarray-based method for screening of immunogenic proteins from bacteria.

Hoppe S, Bier FF, von Nickisch-Rosenegk M - J Nanobiotechnology (2012)

Bottom Line: This enhances the specific binding of the proteins compared to nitrocellulose.Thus, it helps to reduce the number of false positives significantly.It enables us to screen for immunogenic proteins in a shorter time, with more samples and statistical reliability.

View Article: PubMed Central - HTML - PubMed

Affiliation: Fraunhofer Institute for Biomedical Engineering, Branch Potsdam, Am Mühlenberg 13, 14476 Potsdam, Germany. sebastian.hoppe@ibmt.fraunhofer.de

ABSTRACT

Background: Detection of immunogenic proteins remains an important task for life sciences as it nourishes the understanding of pathogenicity, illuminates new potential vaccine candidates and broadens the spectrum of biomarkers applicable in diagnostic tools. Traditionally, immunoscreenings of expression libraries via polyclonal sera on nitrocellulose membranes or screenings of whole proteome lysates in 2-D gel electrophoresis are performed. However, these methods feature some rather inconvenient disadvantages. Screening of expression libraries to expose novel antigens from bacteria often lead to an abundance of false positive signals owing to the high cross reactivity of polyclonal antibodies towards the proteins of the expression host. A method is presented that overcomes many disadvantages of the old procedures.

Results: Four proteins that have previously been described as immunogenic have successfully been assessed immunogenic abilities with our method. One protein with no known immunogenic behaviour before suggested potential immunogenicity.We incorporated a fusion tag prior to our genes of interest and attached the expressed fusion proteins covalently on microarrays. This enhances the specific binding of the proteins compared to nitrocellulose. Thus, it helps to reduce the number of false positives significantly. It enables us to screen for immunogenic proteins in a shorter time, with more samples and statistical reliability. We validated our method by employing several known genes from Campylobacter jejuni NCTC 11168.

Conclusions: The method presented offers a new approach for screening of bacterial expression libraries to illuminate novel proteins with immunogenic features. It could provide a powerful and attractive alternative to existing methods and help to detect and identify vaccine candidates, biomarkers and potential virulence-associated factors with immunogenic behaviour furthering the knowledge of virulence and pathogenicity of studied bacteria.

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Related in: MedlinePlus

SDS-PAGE of HaloTag® fusion proteins incubated with HaloTag® Alexa Fluor 488 ligand. M refers to PageRuler Plus prestained protein ladder (Fermentas) with fluorescent bands at 70 kDa and 25 kDa, The HaloTag® standard protein (HT-SP) was added as an additional size reference at 60 kDa. The bands match the expected sizes for each fusion protein. Additionally small fragments of only HaloTag® (34 kDa) are visible which might be due to early-terminated translation.
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Figure 4: SDS-PAGE of HaloTag® fusion proteins incubated with HaloTag® Alexa Fluor 488 ligand. M refers to PageRuler Plus prestained protein ladder (Fermentas) with fluorescent bands at 70 kDa and 25 kDa, The HaloTag® standard protein (HT-SP) was added as an additional size reference at 60 kDa. The bands match the expected sizes for each fusion protein. Additionally small fragments of only HaloTag® (34 kDa) are visible which might be due to early-terminated translation.

Mentions: Most of the fusion proteins investigated fall into a range between 61 and 73 kDa, namely HaloTag® fused to argC (73 kDa), pyrC (72 kDa), pseB (71 kDa), gapA (70 kDa), cjaA (65 kDa), peb1 (62 kDa), hisJ (62 kDa) and flaC (61 kDa). Outside of this size range, only HaloTag®-flaA (93 kDa) and the small HaloTag®-pal (52 kDa) are found. For each protein, bands with the correct size could be detected, see Figure 4. Additionally, bands of smaller size are visible (34 kDA) which might be due to untimely termination of translation, potentially comprising only the HaloTag®, which features the corresponding size.


Microarray-based method for screening of immunogenic proteins from bacteria.

Hoppe S, Bier FF, von Nickisch-Rosenegk M - J Nanobiotechnology (2012)

SDS-PAGE of HaloTag® fusion proteins incubated with HaloTag® Alexa Fluor 488 ligand. M refers to PageRuler Plus prestained protein ladder (Fermentas) with fluorescent bands at 70 kDa and 25 kDa, The HaloTag® standard protein (HT-SP) was added as an additional size reference at 60 kDa. The bands match the expected sizes for each fusion protein. Additionally small fragments of only HaloTag® (34 kDa) are visible which might be due to early-terminated translation.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3368735&req=5

Figure 4: SDS-PAGE of HaloTag® fusion proteins incubated with HaloTag® Alexa Fluor 488 ligand. M refers to PageRuler Plus prestained protein ladder (Fermentas) with fluorescent bands at 70 kDa and 25 kDa, The HaloTag® standard protein (HT-SP) was added as an additional size reference at 60 kDa. The bands match the expected sizes for each fusion protein. Additionally small fragments of only HaloTag® (34 kDa) are visible which might be due to early-terminated translation.
Mentions: Most of the fusion proteins investigated fall into a range between 61 and 73 kDa, namely HaloTag® fused to argC (73 kDa), pyrC (72 kDa), pseB (71 kDa), gapA (70 kDa), cjaA (65 kDa), peb1 (62 kDa), hisJ (62 kDa) and flaC (61 kDa). Outside of this size range, only HaloTag®-flaA (93 kDa) and the small HaloTag®-pal (52 kDa) are found. For each protein, bands with the correct size could be detected, see Figure 4. Additionally, bands of smaller size are visible (34 kDA) which might be due to untimely termination of translation, potentially comprising only the HaloTag®, which features the corresponding size.

Bottom Line: This enhances the specific binding of the proteins compared to nitrocellulose.Thus, it helps to reduce the number of false positives significantly.It enables us to screen for immunogenic proteins in a shorter time, with more samples and statistical reliability.

View Article: PubMed Central - HTML - PubMed

Affiliation: Fraunhofer Institute for Biomedical Engineering, Branch Potsdam, Am Mühlenberg 13, 14476 Potsdam, Germany. sebastian.hoppe@ibmt.fraunhofer.de

ABSTRACT

Background: Detection of immunogenic proteins remains an important task for life sciences as it nourishes the understanding of pathogenicity, illuminates new potential vaccine candidates and broadens the spectrum of biomarkers applicable in diagnostic tools. Traditionally, immunoscreenings of expression libraries via polyclonal sera on nitrocellulose membranes or screenings of whole proteome lysates in 2-D gel electrophoresis are performed. However, these methods feature some rather inconvenient disadvantages. Screening of expression libraries to expose novel antigens from bacteria often lead to an abundance of false positive signals owing to the high cross reactivity of polyclonal antibodies towards the proteins of the expression host. A method is presented that overcomes many disadvantages of the old procedures.

Results: Four proteins that have previously been described as immunogenic have successfully been assessed immunogenic abilities with our method. One protein with no known immunogenic behaviour before suggested potential immunogenicity.We incorporated a fusion tag prior to our genes of interest and attached the expressed fusion proteins covalently on microarrays. This enhances the specific binding of the proteins compared to nitrocellulose. Thus, it helps to reduce the number of false positives significantly. It enables us to screen for immunogenic proteins in a shorter time, with more samples and statistical reliability. We validated our method by employing several known genes from Campylobacter jejuni NCTC 11168.

Conclusions: The method presented offers a new approach for screening of bacterial expression libraries to illuminate novel proteins with immunogenic features. It could provide a powerful and attractive alternative to existing methods and help to detect and identify vaccine candidates, biomarkers and potential virulence-associated factors with immunogenic behaviour furthering the knowledge of virulence and pathogenicity of studied bacteria.

Show MeSH
Related in: MedlinePlus