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Recombinant expression and functional analysis of proteases from Streptococcus pneumoniae, Bacillus anthracis, and Yersinia pestis.

Kwon K, Hasseman J, Latham S, Grose C, Do Y, Fleischmann RD, Pieper R, Peterson SN - BMC Biochem. (2011)

Bottom Line: Overall, 86.1% of selected protease genes including hypothetical proteins were expressed and purified using a combination of five different expression vectors.To detect novel proteolytic activities, zymography and fluorescence-based assays were performed and the protease activities of more than 46% of purified proteases and 40% of hypothetical proteins that were predicted to be proteases were confirmed.The combinatorial functional analysis of the purified proteases using fluorescence assays and zymography confirmed their function.

View Article: PubMed Central - HTML - PubMed

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

ABSTRACT

Background: Uncharacterized proteases naturally expressed by bacterial pathogens represents important topic in infectious disease research, because these enzymes may have critical roles in pathogenicity and cell physiology. It has been observed that cloning, expression and purification of proteases often fail due to their catalytic functions which, in turn, cause toxicity in the E. coli heterologous host.

Results: In order to address this problem systematically, a modified pipeline of our high-throughput protein expression and purification platform was developed. This included the use of a specific E. coli strain, BL21(DE3) pLysS to tightly control the expression of recombinant proteins and various expression vectors encoding fusion proteins to enhance recombinant protein solubility. Proteases fused to large fusion protein domains, maltosebinding protein (MBP), SP-MBP which contains signal peptide at the N-terminus of MBP, disulfide oxidoreductase (DsbA) and Glutathione S-transferase (GST) improved expression and solubility of proteases. Overall, 86.1% of selected protease genes including hypothetical proteins were expressed and purified using a combination of five different expression vectors. To detect novel proteolytic activities, zymography and fluorescence-based assays were performed and the protease activities of more than 46% of purified proteases and 40% of hypothetical proteins that were predicted to be proteases were confirmed.

Conclusions: Multiple expression vectors, employing distinct fusion tags in a high throughput pipeline increased overall success rates in expression, solubility and purification of proteases. The combinatorial functional analysis of the purified proteases using fluorescence assays and zymography confirmed their function.

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Coomassie blue stained Nu-PAGE gel of purified proteases. The proteins were expressed using pMBP vector and purified using Ni-NTA agarose resin as described in Materials and Methods. The purity of proteases was confirmed on the Nu-PAGE gel and the concentrations were determined by Bradford assay with BSA standard curve. (M: marker, 1: SP_1467, conserved hypothetical protein, 2: SP1477, hypothetical protein, 3: y0125, sigma cross-reacting protein 27A, 4: y0137, protease DO, 5: y0720, putative PhnP protein, 6: y0746, cytosol aminopeptidase, 7: y1280, pyrrolidone-carboxylate peptidase, 8: y2013, putative pepetidase, 9: y2057, peptidase U7 family SohB protein, 10: y2527, prolyl oligopeptidase family protein, 11: y2694, conserved hypothetical protein, 12: y3230,4-methyl-5(b-hydroxyethyl)-thiazole monophosphate biosynthesis protein, 13: y3297, proline-specific aminopeptidase, 14: y3855, oligopeptidase A, 15: y3857, putative alkaline metalloproteinase).
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Figure 2: Coomassie blue stained Nu-PAGE gel of purified proteases. The proteins were expressed using pMBP vector and purified using Ni-NTA agarose resin as described in Materials and Methods. The purity of proteases was confirmed on the Nu-PAGE gel and the concentrations were determined by Bradford assay with BSA standard curve. (M: marker, 1: SP_1467, conserved hypothetical protein, 2: SP1477, hypothetical protein, 3: y0125, sigma cross-reacting protein 27A, 4: y0137, protease DO, 5: y0720, putative PhnP protein, 6: y0746, cytosol aminopeptidase, 7: y1280, pyrrolidone-carboxylate peptidase, 8: y2013, putative pepetidase, 9: y2057, peptidase U7 family SohB protein, 10: y2527, prolyl oligopeptidase family protein, 11: y2694, conserved hypothetical protein, 12: y3230,4-methyl-5(b-hydroxyethyl)-thiazole monophosphate biosynthesis protein, 13: y3297, proline-specific aminopeptidase, 14: y3855, oligopeptidase A, 15: y3857, putative alkaline metalloproteinase).

Mentions: The high throughput protein production pipeline is based on a 96-well format procedure. The pipeline consists of cloning, DNA sequence validation, expression, purification, confirmation of protein identity by mass spectroscopy and storage. One of the essential features of our pipeline is the cloning of ORFs into multiple expression vectors. Gateway cloning technology was introduced in the pipeline in order to maintain efficient cloning in this regard (Figure 1). When a single expression vector encoding the His-tag is used to overexpress randomly selected recombinant proteins in E. coli, generally, yielded purified, soluble proteins in less than 40% of target genes [29,47]. If the selected genes are toxic to the host cells, the final yields are decreased further. However, when multiple expression vectors are used to prepare protein, the overall success rates of expression, solubility and purification of target proteins can be significantly increased. The Gateway cloning system represents an ideal cloning method to produce multiple expression vectors for use in a high throughput protein production pipeline. Once entry clones are prepared and their sequences are validated, the entry clones may be used to generate expression clones using destination vectors encoding a variety of fusion tags by simply shuttling ORFs into multiple Gateway compatible expression vectors using the recombinase, LR clonase. Multiple expression vectors shown in Figure 1 were constructed based on T7 expression pET system and Gateway cloning system in order to improve expression and solubility of recombinant proteins. Unlike methods using restriction enzymes, Gateway cloning is very efficient and sequence validated entry clones can be used for any Gateway destination vector without further DNA sequence validation. Although the pET system is very powerful, it lacks tight control of expression. The expression control of proteins is critical for successful expression of potentially toxic proteins. Proteases represent a strong example for which expression control is important due to cytotoxic proteolytic activities. The early undesired expression of target proteins using pET expression system was suppressed by addition of glucose in the media and using BL21(DE3)pLysS. A primary carbon source for E. coli host, glucose binds to the lac repressor and shut off transcription of the T7 RNA polymerase gene under the control of the lac UV5 promoter. Low level expression of T7 lysozyme from pLysS binds to and inhibits T7 RNA polymerase. Approximately, 86.1% of cloned ORFs were expressed and purified with at least one of expression vectors. By batch purification using 2 mL 96-well filter block, between 2 - 200 μg of recombinant proteins were obtained. Purity of the recombinant proteins was dependent upon the amount of soluble expressed recombinant protein and ranged between 80-95%. The purity of the recombinant proteins were confirmed by Nu-PAGE gel analysis (Figure 2) and the identity of the purified proteins were confirmed by in-gel trypsin digestion followed by MALDI-TOF/TOF analysis.


Recombinant expression and functional analysis of proteases from Streptococcus pneumoniae, Bacillus anthracis, and Yersinia pestis.

Kwon K, Hasseman J, Latham S, Grose C, Do Y, Fleischmann RD, Pieper R, Peterson SN - BMC Biochem. (2011)

Coomassie blue stained Nu-PAGE gel of purified proteases. The proteins were expressed using pMBP vector and purified using Ni-NTA agarose resin as described in Materials and Methods. The purity of proteases was confirmed on the Nu-PAGE gel and the concentrations were determined by Bradford assay with BSA standard curve. (M: marker, 1: SP_1467, conserved hypothetical protein, 2: SP1477, hypothetical protein, 3: y0125, sigma cross-reacting protein 27A, 4: y0137, protease DO, 5: y0720, putative PhnP protein, 6: y0746, cytosol aminopeptidase, 7: y1280, pyrrolidone-carboxylate peptidase, 8: y2013, putative pepetidase, 9: y2057, peptidase U7 family SohB protein, 10: y2527, prolyl oligopeptidase family protein, 11: y2694, conserved hypothetical protein, 12: y3230,4-methyl-5(b-hydroxyethyl)-thiazole monophosphate biosynthesis protein, 13: y3297, proline-specific aminopeptidase, 14: y3855, oligopeptidase A, 15: y3857, putative alkaline metalloproteinase).
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Related In: Results  -  Collection

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Figure 2: Coomassie blue stained Nu-PAGE gel of purified proteases. The proteins were expressed using pMBP vector and purified using Ni-NTA agarose resin as described in Materials and Methods. The purity of proteases was confirmed on the Nu-PAGE gel and the concentrations were determined by Bradford assay with BSA standard curve. (M: marker, 1: SP_1467, conserved hypothetical protein, 2: SP1477, hypothetical protein, 3: y0125, sigma cross-reacting protein 27A, 4: y0137, protease DO, 5: y0720, putative PhnP protein, 6: y0746, cytosol aminopeptidase, 7: y1280, pyrrolidone-carboxylate peptidase, 8: y2013, putative pepetidase, 9: y2057, peptidase U7 family SohB protein, 10: y2527, prolyl oligopeptidase family protein, 11: y2694, conserved hypothetical protein, 12: y3230,4-methyl-5(b-hydroxyethyl)-thiazole monophosphate biosynthesis protein, 13: y3297, proline-specific aminopeptidase, 14: y3855, oligopeptidase A, 15: y3857, putative alkaline metalloproteinase).
Mentions: The high throughput protein production pipeline is based on a 96-well format procedure. The pipeline consists of cloning, DNA sequence validation, expression, purification, confirmation of protein identity by mass spectroscopy and storage. One of the essential features of our pipeline is the cloning of ORFs into multiple expression vectors. Gateway cloning technology was introduced in the pipeline in order to maintain efficient cloning in this regard (Figure 1). When a single expression vector encoding the His-tag is used to overexpress randomly selected recombinant proteins in E. coli, generally, yielded purified, soluble proteins in less than 40% of target genes [29,47]. If the selected genes are toxic to the host cells, the final yields are decreased further. However, when multiple expression vectors are used to prepare protein, the overall success rates of expression, solubility and purification of target proteins can be significantly increased. The Gateway cloning system represents an ideal cloning method to produce multiple expression vectors for use in a high throughput protein production pipeline. Once entry clones are prepared and their sequences are validated, the entry clones may be used to generate expression clones using destination vectors encoding a variety of fusion tags by simply shuttling ORFs into multiple Gateway compatible expression vectors using the recombinase, LR clonase. Multiple expression vectors shown in Figure 1 were constructed based on T7 expression pET system and Gateway cloning system in order to improve expression and solubility of recombinant proteins. Unlike methods using restriction enzymes, Gateway cloning is very efficient and sequence validated entry clones can be used for any Gateway destination vector without further DNA sequence validation. Although the pET system is very powerful, it lacks tight control of expression. The expression control of proteins is critical for successful expression of potentially toxic proteins. Proteases represent a strong example for which expression control is important due to cytotoxic proteolytic activities. The early undesired expression of target proteins using pET expression system was suppressed by addition of glucose in the media and using BL21(DE3)pLysS. A primary carbon source for E. coli host, glucose binds to the lac repressor and shut off transcription of the T7 RNA polymerase gene under the control of the lac UV5 promoter. Low level expression of T7 lysozyme from pLysS binds to and inhibits T7 RNA polymerase. Approximately, 86.1% of cloned ORFs were expressed and purified with at least one of expression vectors. By batch purification using 2 mL 96-well filter block, between 2 - 200 μg of recombinant proteins were obtained. Purity of the recombinant proteins was dependent upon the amount of soluble expressed recombinant protein and ranged between 80-95%. The purity of the recombinant proteins were confirmed by Nu-PAGE gel analysis (Figure 2) and the identity of the purified proteins were confirmed by in-gel trypsin digestion followed by MALDI-TOF/TOF analysis.

Bottom Line: Overall, 86.1% of selected protease genes including hypothetical proteins were expressed and purified using a combination of five different expression vectors.To detect novel proteolytic activities, zymography and fluorescence-based assays were performed and the protease activities of more than 46% of purified proteases and 40% of hypothetical proteins that were predicted to be proteases were confirmed.The combinatorial functional analysis of the purified proteases using fluorescence assays and zymography confirmed their function.

View Article: PubMed Central - HTML - PubMed

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

ABSTRACT

Background: Uncharacterized proteases naturally expressed by bacterial pathogens represents important topic in infectious disease research, because these enzymes may have critical roles in pathogenicity and cell physiology. It has been observed that cloning, expression and purification of proteases often fail due to their catalytic functions which, in turn, cause toxicity in the E. coli heterologous host.

Results: In order to address this problem systematically, a modified pipeline of our high-throughput protein expression and purification platform was developed. This included the use of a specific E. coli strain, BL21(DE3) pLysS to tightly control the expression of recombinant proteins and various expression vectors encoding fusion proteins to enhance recombinant protein solubility. Proteases fused to large fusion protein domains, maltosebinding protein (MBP), SP-MBP which contains signal peptide at the N-terminus of MBP, disulfide oxidoreductase (DsbA) and Glutathione S-transferase (GST) improved expression and solubility of proteases. Overall, 86.1% of selected protease genes including hypothetical proteins were expressed and purified using a combination of five different expression vectors. To detect novel proteolytic activities, zymography and fluorescence-based assays were performed and the protease activities of more than 46% of purified proteases and 40% of hypothetical proteins that were predicted to be proteases were confirmed.

Conclusions: Multiple expression vectors, employing distinct fusion tags in a high throughput pipeline increased overall success rates in expression, solubility and purification of proteases. The combinatorial functional analysis of the purified proteases using fluorescence assays and zymography confirmed their function.

Show MeSH
Related in: MedlinePlus