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High quality protein microarray using in situ protein purification.

Kwon K, Grose C, Pieper R, Pandya GA, Fleischmann RD, Peterson SN - BMC Biotechnol. (2009)

Bottom Line: An optimized wash buffer (PBST) composed of 10 mM phosphate buffer, 2.7 mM KCl, 140 mM NaCl and 0.05% Tween 20, pH 7.4, further improved protein purity levels.The methodology is compatible with higher throughput formats of in vivo protein expression, eliminates the need for resin-based purification and circumvents protein solubility and denaturation problems caused by buffer exchange steps and freeze-thaw cycles, which are associated with resin-based purification, intermittent protein storage and deposition on microarrays.In the context of immunoproteomics, such protein microarrays are complimentary to approaches using non-recombinant methods to discover and characterize bacterial antigens.

View Article: PubMed Central - HTML - PubMed

Affiliation: Pathogen Functional Genomics Resource Center, J. Craig Venter Institute, 9704 Medical Center Drive, Rockville, Maryland 20850, USA. kkwon@jcvi.org

ABSTRACT

Background: In the postgenomic era, high throughput protein expression and protein microarray technologies have progressed markedly permitting screening of therapeutic reagents and discovery of novel protein functions. Hexa-histidine is one of the most commonly used fusion tags for protein expression due to its small size and convenient purification via immobilized metal ion affinity chromatography (IMAC). This purification process has been adapted to the protein microarray format, but the quality of in situ His-tagged protein purification on slides has not been systematically evaluated. We established methods to determine the level of purification of such proteins on metal chelate-modified slide surfaces. Optimized in situ purification of His-tagged recombinant proteins has the potential to become the new gold standard for cost-effective generation of high-quality and high-density protein microarrays.

Results: Two slide surfaces were examined, chelated Cu2+ slides suspended on a polyethylene glycol (PEG) coating and chelated Ni2+ slides immobilized on a support without PEG coating. Using PEG-coated chelated Cu2+ slides, consistently higher purities of recombinant proteins were measured. An optimized wash buffer (PBST) composed of 10 mM phosphate buffer, 2.7 mM KCl, 140 mM NaCl and 0.05% Tween 20, pH 7.4, further improved protein purity levels. Using Escherichia coli cell lysates expressing 90 recombinant Streptococcus pneumoniae proteins, 73 proteins were successfully immobilized, and 66 proteins were in situ purified with greater than 90% purity. We identified several antigens among the in situ-purified proteins via assays with anti-S. pneumoniae rabbit antibodies and a human patient antiserum, as a demonstration project of large scale microarray-based immunoproteomics profiling. The methodology is compatible with higher throughput formats of in vivo protein expression, eliminates the need for resin-based purification and circumvents protein solubility and denaturation problems caused by buffer exchange steps and freeze-thaw cycles, which are associated with resin-based purification, intermittent protein storage and deposition on microarrays.

Conclusion: An optimized platform for in situ protein purification on microarray slides using His-tagged recombinant proteins is a desirable tool for the screening of novel protein functions and protein-protein interactions. In the context of immunoproteomics, such protein microarrays are complimentary to approaches using non-recombinant methods to discover and characterize bacterial antigens.

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Optimization of in situ protein purification. (A) His-tagged FolE and (B) His-tagged Lac repressor. Relative purities of in situ purified proteins were determined using the ratio of fluorescence signals of recombinant proteins versus the fluorescence signals of total protein, as shown in Equation 1 (see text). White and gray bars represent Cu2+/IDA/PEG slides and non-PEG coated Ni2+/NTA slides, respectively. A series of spotting buffers were examined: PBST [10 mM phosphate buffer, 2.7 mM KCl, 140 mM NaCl, 0.05% Tween 20, pH 7.4], PBS [10 mM phosphate buffer, 154 mM NaCl, pH 7.4], TS [50 mM Tris, 300 mM NaCl, 10 mM imidazole, pH 7.8] and SB [100 mM sodium borate, pH 7.8].
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Figure 2: Optimization of in situ protein purification. (A) His-tagged FolE and (B) His-tagged Lac repressor. Relative purities of in situ purified proteins were determined using the ratio of fluorescence signals of recombinant proteins versus the fluorescence signals of total protein, as shown in Equation 1 (see text). White and gray bars represent Cu2+/IDA/PEG slides and non-PEG coated Ni2+/NTA slides, respectively. A series of spotting buffers were examined: PBST [10 mM phosphate buffer, 2.7 mM KCl, 140 mM NaCl, 0.05% Tween 20, pH 7.4], PBS [10 mM phosphate buffer, 154 mM NaCl, pH 7.4], TS [50 mM Tris, 300 mM NaCl, 10 mM imidazole, pH 7.8] and SB [100 mM sodium borate, pH 7.8].

Mentions: The calculated relative purities were used as criteria to define optimal conditions for in situ purification. Instead of using two-channel detection method, one-channel detection method for His-tagged proteins and E. coli endogenous protein contaminants on a 2-pad slide system was used to eliminate the mutual inferences of two antibody interactions to the immobilized proteins and to keep the same background in scanning. Considering the fluorescence brightness of Alexa Fluor 555 and ATTO 550, we estimate that the true level of purity of the immobilized recombinant proteins is greater than the relative purity measured according to Equation 1. The fluorescence brightness, which is a product of an extinction coefficient and a quantum yield of the fluorophore, of Alexa Fluor 555 and ATTO 550 are 15,000 and 96,000, respectively [37]. Using GenePix 4000B image scanner and identical experimental conditions, the brightness of ATTO 550 is at least as high as to that of Alexa Fluor 555. Combinations of four spotting buffers, PBS, PBST, TS and SB and three washing buffers, PBS, PBST and SB were examined for in situ purification of FolE and LacI. Based upon the estimation of the relative purity levels of FolE and LacI, PBST delivered the best results as a spotting and wash buffer for both Cu2+/IDA/PEG and Ni2+/NTA slides. In Figure 2, relative purities of FolE and LacI, printed with four spotting buffers on both slide surfaces and washed with PBST, were compared. The relative purities of FolE with two spotting buffers, PBS and PBST were similar but the relative purity of Lac I with PBST spotting buffer was nearly 20% higher than that with PBS. Apparently, the nonionic detergent Tween-20 mediates partial denaturation of recombinant proteins and enhances the exposure of His-tags to the Cu2+ or Ni2+ on the slides. This effect may also increase competitive binding of recombinant proteins to histidine-rich contaminant E. coli proteins.


High quality protein microarray using in situ protein purification.

Kwon K, Grose C, Pieper R, Pandya GA, Fleischmann RD, Peterson SN - BMC Biotechnol. (2009)

Optimization of in situ protein purification. (A) His-tagged FolE and (B) His-tagged Lac repressor. Relative purities of in situ purified proteins were determined using the ratio of fluorescence signals of recombinant proteins versus the fluorescence signals of total protein, as shown in Equation 1 (see text). White and gray bars represent Cu2+/IDA/PEG slides and non-PEG coated Ni2+/NTA slides, respectively. A series of spotting buffers were examined: PBST [10 mM phosphate buffer, 2.7 mM KCl, 140 mM NaCl, 0.05% Tween 20, pH 7.4], PBS [10 mM phosphate buffer, 154 mM NaCl, pH 7.4], TS [50 mM Tris, 300 mM NaCl, 10 mM imidazole, pH 7.8] and SB [100 mM sodium borate, pH 7.8].
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2746808&req=5

Figure 2: Optimization of in situ protein purification. (A) His-tagged FolE and (B) His-tagged Lac repressor. Relative purities of in situ purified proteins were determined using the ratio of fluorescence signals of recombinant proteins versus the fluorescence signals of total protein, as shown in Equation 1 (see text). White and gray bars represent Cu2+/IDA/PEG slides and non-PEG coated Ni2+/NTA slides, respectively. A series of spotting buffers were examined: PBST [10 mM phosphate buffer, 2.7 mM KCl, 140 mM NaCl, 0.05% Tween 20, pH 7.4], PBS [10 mM phosphate buffer, 154 mM NaCl, pH 7.4], TS [50 mM Tris, 300 mM NaCl, 10 mM imidazole, pH 7.8] and SB [100 mM sodium borate, pH 7.8].
Mentions: The calculated relative purities were used as criteria to define optimal conditions for in situ purification. Instead of using two-channel detection method, one-channel detection method for His-tagged proteins and E. coli endogenous protein contaminants on a 2-pad slide system was used to eliminate the mutual inferences of two antibody interactions to the immobilized proteins and to keep the same background in scanning. Considering the fluorescence brightness of Alexa Fluor 555 and ATTO 550, we estimate that the true level of purity of the immobilized recombinant proteins is greater than the relative purity measured according to Equation 1. The fluorescence brightness, which is a product of an extinction coefficient and a quantum yield of the fluorophore, of Alexa Fluor 555 and ATTO 550 are 15,000 and 96,000, respectively [37]. Using GenePix 4000B image scanner and identical experimental conditions, the brightness of ATTO 550 is at least as high as to that of Alexa Fluor 555. Combinations of four spotting buffers, PBS, PBST, TS and SB and three washing buffers, PBS, PBST and SB were examined for in situ purification of FolE and LacI. Based upon the estimation of the relative purity levels of FolE and LacI, PBST delivered the best results as a spotting and wash buffer for both Cu2+/IDA/PEG and Ni2+/NTA slides. In Figure 2, relative purities of FolE and LacI, printed with four spotting buffers on both slide surfaces and washed with PBST, were compared. The relative purities of FolE with two spotting buffers, PBS and PBST were similar but the relative purity of Lac I with PBST spotting buffer was nearly 20% higher than that with PBS. Apparently, the nonionic detergent Tween-20 mediates partial denaturation of recombinant proteins and enhances the exposure of His-tags to the Cu2+ or Ni2+ on the slides. This effect may also increase competitive binding of recombinant proteins to histidine-rich contaminant E. coli proteins.

Bottom Line: An optimized wash buffer (PBST) composed of 10 mM phosphate buffer, 2.7 mM KCl, 140 mM NaCl and 0.05% Tween 20, pH 7.4, further improved protein purity levels.The methodology is compatible with higher throughput formats of in vivo protein expression, eliminates the need for resin-based purification and circumvents protein solubility and denaturation problems caused by buffer exchange steps and freeze-thaw cycles, which are associated with resin-based purification, intermittent protein storage and deposition on microarrays.In the context of immunoproteomics, such protein microarrays are complimentary to approaches using non-recombinant methods to discover and characterize bacterial antigens.

View Article: PubMed Central - HTML - PubMed

Affiliation: Pathogen Functional Genomics Resource Center, J. Craig Venter Institute, 9704 Medical Center Drive, Rockville, Maryland 20850, USA. kkwon@jcvi.org

ABSTRACT

Background: In the postgenomic era, high throughput protein expression and protein microarray technologies have progressed markedly permitting screening of therapeutic reagents and discovery of novel protein functions. Hexa-histidine is one of the most commonly used fusion tags for protein expression due to its small size and convenient purification via immobilized metal ion affinity chromatography (IMAC). This purification process has been adapted to the protein microarray format, but the quality of in situ His-tagged protein purification on slides has not been systematically evaluated. We established methods to determine the level of purification of such proteins on metal chelate-modified slide surfaces. Optimized in situ purification of His-tagged recombinant proteins has the potential to become the new gold standard for cost-effective generation of high-quality and high-density protein microarrays.

Results: Two slide surfaces were examined, chelated Cu2+ slides suspended on a polyethylene glycol (PEG) coating and chelated Ni2+ slides immobilized on a support without PEG coating. Using PEG-coated chelated Cu2+ slides, consistently higher purities of recombinant proteins were measured. An optimized wash buffer (PBST) composed of 10 mM phosphate buffer, 2.7 mM KCl, 140 mM NaCl and 0.05% Tween 20, pH 7.4, further improved protein purity levels. Using Escherichia coli cell lysates expressing 90 recombinant Streptococcus pneumoniae proteins, 73 proteins were successfully immobilized, and 66 proteins were in situ purified with greater than 90% purity. We identified several antigens among the in situ-purified proteins via assays with anti-S. pneumoniae rabbit antibodies and a human patient antiserum, as a demonstration project of large scale microarray-based immunoproteomics profiling. The methodology is compatible with higher throughput formats of in vivo protein expression, eliminates the need for resin-based purification and circumvents protein solubility and denaturation problems caused by buffer exchange steps and freeze-thaw cycles, which are associated with resin-based purification, intermittent protein storage and deposition on microarrays.

Conclusion: An optimized platform for in situ protein purification on microarray slides using His-tagged recombinant proteins is a desirable tool for the screening of novel protein functions and protein-protein interactions. In the context of immunoproteomics, such protein microarrays are complimentary to approaches using non-recombinant methods to discover and characterize bacterial antigens.

Show MeSH
Related in: MedlinePlus