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

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.

Figure 1

Gateway compatible expression vectors with different fusion tags.

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).

Figure 3

Comparison of success frequencies of expression vectors at each stage of purification. Selected entry clones of 187 ORFs in Bacillus anthracis Ames, Streptococcus pneumonia TIGR4 and Yersinia pestis KIM, were used to prepare expression clones using 5 different Gateway comparable vectors, pHis, pMBP, pSP-MBP, pDsbA and pGST. Target proteins were expressed in 96-well blocks by IPTG induction at OD_{600 nm} = 0.7-0.9 for 20 hours at 20°C. The harvested cell pellets were resuspended in lysis buffer [50 mM Tris-HCl, 100 mM NaCl, 1 mM DTT at pH 7.8 at 4°C] and lysed using PopCulture and lysonase. The recombinant proteins were purified using Ni-NTA agarose column as described in 'Methods and Materials'.

Figure 4

Dependence of purification success rates on fusion tags and sub-cellular localization of proteins. Analysis of purification success rate by expression vectors and (A) subcellular localization of proteins or (B) presence of signal peptide.

Figure 5

Detection of protease activity using gelatin zymogram. A putative collagenase in Bacillus anthracis Ames was cloned in pMBP and expressed in BL21(DE3)pLysS. Expression (E) and solubility (S) of the putative collagenase (BA0555) were confirmed by His-vision staining on Nu-PAGE. The purified protein was run on Nu-PAGE gel and stained with Coomassie blue to confirm the purity (P). Gelatin zymogram was performed to examine protease activity using 2 ng of the purified putative collagenase (C). Purification background using E. coli supernatant (B) and 2.5 ng of trypsin (T) were used as a negative and a positive control, respectively.

Figure 6

Protease activity screen using a fluorescence substrate, BZAR. A. The BZAR (Rhodamine 110, bis-(N-CBZ-L-arginine amide), dihydrochloride) is a general substrate of serine proteases. Upon protease cleavage, the nonfluorescent bisamide derivative of rhodamine 110 is first converted to the fluorescent monoamide and then to rhodamine 110, with a further increase in fluorescence. B. Time course measurements of protease cleavage of BZAR. The protease cleavage reactions were montiored by time fluorescence measurements (λ_ex: 485 nm, λ_em: 535 nm) using a GENeo Pro (Tecan). The increase of fluorescence represents protease cleavage reactions. Δ: y3353, putative kinase, ○: trypsin, ◊: y1992: putative carboxypeptidase, and □: SP1780, oligoendopeptidase.