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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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Western Blot of whole cell lysates. Four bands are visible in the KRX whole cell lysate with a more prominent band at 70 kDa and three weaker bands at 55 kDa, 28 kDa and roughly 18 kDa. These bands are most likely due to cross-reactive proteins within the E.coli lysates. All four proteins previously described as non-immunogenic (argC, gapA, pseB and pyrC) show band patterns highly similar to KRX. This underlines their lack of immunogenic potential. The five proteins previously described as immunogenic elsewhere show stronger bands at roughly 70 kDa for cjaA, hisJ, peb1a, flaC and 55 kDa for pal respectively. These sizes correspond closely to the expected sizes of the HaloTag® fusion proteins. However, the interference of the background bands from KRX - being in the same range as the target proteins under investigations - prevents a secure identification of immunogenic proteins.
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Figure 10: Western Blot of whole cell lysates. Four bands are visible in the KRX whole cell lysate with a more prominent band at 70 kDa and three weaker bands at 55 kDa, 28 kDa and roughly 18 kDa. These bands are most likely due to cross-reactive proteins within the E.coli lysates. All four proteins previously described as non-immunogenic (argC, gapA, pseB and pyrC) show band patterns highly similar to KRX. This underlines their lack of immunogenic potential. The five proteins previously described as immunogenic elsewhere show stronger bands at roughly 70 kDa for cjaA, hisJ, peb1a, flaC and 55 kDa for pal respectively. These sizes correspond closely to the expected sizes of the HaloTag® fusion proteins. However, the interference of the background bands from KRX - being in the same range as the target proteins under investigations - prevents a secure identification of immunogenic proteins.

Mentions: In contrast, Figure 10 shows a western blot performed directly with whole lysates after recombinant expression without further purification. At least four bands are visible in all the samples with the most prominent band at 70 kDa. Bands of lower intensity appear at approximately 55 kDa, 28 kDa and 18 kDa. The first five lanes, corresponding to the known immunogenic proteins, cjaA, hisJ, peb1a, flaC and pal, show bands of higher intensity than the remaining five lanes. However, as the investigated fusion proteins fall either into the 70 kDa or in case of HT-pal into the 55 kDa range, a clear differentiation between positive bands and background caused by KRX cross-reactive proteins is hardly possible.


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

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

Western Blot of whole cell lysates. Four bands are visible in the KRX whole cell lysate with a more prominent band at 70 kDa and three weaker bands at 55 kDa, 28 kDa and roughly 18 kDa. These bands are most likely due to cross-reactive proteins within the E.coli lysates. All four proteins previously described as non-immunogenic (argC, gapA, pseB and pyrC) show band patterns highly similar to KRX. This underlines their lack of immunogenic potential. The five proteins previously described as immunogenic elsewhere show stronger bands at roughly 70 kDa for cjaA, hisJ, peb1a, flaC and 55 kDa for pal respectively. These sizes correspond closely to the expected sizes of the HaloTag® fusion proteins. However, the interference of the background bands from KRX - being in the same range as the target proteins under investigations - prevents a secure identification of immunogenic proteins.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 10: Western Blot of whole cell lysates. Four bands are visible in the KRX whole cell lysate with a more prominent band at 70 kDa and three weaker bands at 55 kDa, 28 kDa and roughly 18 kDa. These bands are most likely due to cross-reactive proteins within the E.coli lysates. All four proteins previously described as non-immunogenic (argC, gapA, pseB and pyrC) show band patterns highly similar to KRX. This underlines their lack of immunogenic potential. The five proteins previously described as immunogenic elsewhere show stronger bands at roughly 70 kDa for cjaA, hisJ, peb1a, flaC and 55 kDa for pal respectively. These sizes correspond closely to the expected sizes of the HaloTag® fusion proteins. However, the interference of the background bands from KRX - being in the same range as the target proteins under investigations - prevents a secure identification of immunogenic proteins.
Mentions: In contrast, Figure 10 shows a western blot performed directly with whole lysates after recombinant expression without further purification. At least four bands are visible in all the samples with the most prominent band at 70 kDa. Bands of lower intensity appear at approximately 55 kDa, 28 kDa and 18 kDa. The first five lanes, corresponding to the known immunogenic proteins, cjaA, hisJ, peb1a, flaC and pal, show bands of higher intensity than the remaining five lanes. However, as the investigated fusion proteins fall either into the 70 kDa or in case of HT-pal into the 55 kDa range, a clear differentiation between positive bands and background caused by KRX cross-reactive proteins is hardly possible.

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