title: 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.
authors.affiliation: Pathogen Functional Genomics Resource Center, J, Craig Venter Institute, Rockville, Maryland 20850, USA. scottp@jcvi.org
authors.forename: Keehwan
authors.initials: K
authors.lastname: Kwon
authors.forename: Jeremy
authors.initials: J
authors.lastname: Hasseman
authors.forename: Saeeda
authors.initials: S
authors.lastname: Latham
authors.forename: Carissa
authors.initials: C
authors.lastname: Grose
authors.forename: Yu
authors.initials: Y
authors.lastname: Do
authors.forename: Robert D
authors.initials: RD
authors.lastname: Fleischmann
authors.forename: Rembert
authors.initials: R
authors.lastname: Pieper
authors.forename: Scott N
authors.initials: SN
authors.lastname: Peterson
mesh_terms: D001408:Bacillus anthracis / Q000201:enzymology / Q000235:genetics; D001419:Bacteria / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D019295:Computational Biology; D057075:Enzyme Assays / Q000379:methods*; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D058977:Molecular Sequence Annotation; D010447:Peptide Hydrolases / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods*; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D013296:Streptococcus pneumoniae / Q000201:enzymology / Q000235:genetics; D015010:Yersinia pestis / Q000201:enzymology / Q000235:genetics
chemical_list: D011993:Recombinant Fusion Proteins; D010447:Peptide Hydrolases