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Publications (3)

  • Source
    Keehwan Kwon · Jeremy Hasseman · Saeeda Latham · [...] · Scott N Peterson
    [Show abstract] [Hide abstract] ABSTRACT: 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. 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. 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.
    Full-text Article · May 2011 · BMC Biochemistry
  • Source
    Keehwan Kwon · Jeremy Hasseman · Saeeda Latham · [...] · Scott N Peterson
    [Show abstract] [Hide abstract] ABSTRACT: Table S1 - Data table of protease preparation in five expression vectors and protease activity assay. The selected proteases of Streptococcus pneumoniae, Bacillus anthracis and Yersinia pestis were cloned into five expression vectors, pHis (T02), pMBP (T221), pSP-MBP (T213), pDsbA (T03) and pGST (pEXP7). Data from cloning to protease assay are presented for expression vector. "LR" column represents LR cloning of target ORFs from entry clone. Scores "1" and "0" in "LR" column represent "success" and "fail", respectively. Transformation column represents the number of colonies after transformation into BL21(DE3)pLysS. The scores, "0", "1", "2", and "3" in the columns of 'Expression' and 'Solubility' represent none, low, medium and high expression and solubility, respectively. "[Purified] μg/mL" is the concentration of purified recombinant protein and "Purified (PAGE)" for pGST (pEXP7) represents success ("1") or fail ("0") in purification. "BZAR (S/N)" is the ratio of fluorescence in the presence to the absence of protease after 1 hour incubation. "BZAR (slope)" is the rate of fluorescence change in the presence of a given protease. Combining S/N and slope, protease activity to BZAR was determined and the result is shown in column of "BZAR". The column of "Gelatin" is the result of gelatin zymography, and the numbers represent relative activity-1: weak. 2: medium and 3: strong. "DQ (slope)" is the rate of fluorescence change in the presence of a given protease. "Family" column described peptidase family grouped, and the first letter represents catalytic type: A, aspartic; C, cystein,; M, metallic; S, serine; and U, unknown.
    Full-text Dataset · May 2011
  • [Show abstract] [Hide abstract] ABSTRACT: We have developed and evaluated a highly parallel protein expression and purification system using ORFs derived from the pathogenic bacterium Streptococcus pneumoniae as a representative test case in conjunction with the Gateway cloning technology. Establishing high throughput protein production capability is essential for genome-wide characterization of protein function. In this study, we focused on protein expression and purification outcomes generated from an expression vector which encodes an NH(2)-terminal hexa-histidine tag and a COOH-terminal S-tag. Purified recombinant proteins were validated by SDS-PAGE, followed by in-gel digestion and identification by MALDI-TOF/TOF analysis. Starting with 1360 sequence-validated destination clones we examined correlation analyses of expression and solubility of a wide variety of recombinant proteins. In total, 428 purified proteins (31%) were recovered in soluble form. We describe a semi-quantitative scoring method using an S-tag assay to improve the throughput and efficiency of expression and solubility studies for recombinant proteins. Given a relatively large dataset derived from proteins representing all functional groups in a microbial genome we correlated various protein characteristics as they relate to protein expression outcomes.
    Article · Nov 2007 · Protein Expression and Purification