Recombinant collagen production optimization in Escherichia coli

Thesis (M. Eng.)--Massachusetts Institute of Technology, Dept. of Electrical Engineering and Computer Science, 2005.This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.Includes bibliographical references (p. 151-158).An Escherichia coli-based collagen-production process was used to investigate several process optimization objectives for use at the industrial scale. The effect of cooling on fermentation growth kinetics was studied, with preliminary results indicating that cooling an early to mid-batch phase fermentation (OD₆₀₀ = 2-4) to 10⁰C for up to 40 hours was not detrimental to the recovery of fermentation growth or the culture's ability to reach high cell density (OD₆₀₀> 50). A cost-effective assay of collagen-like polymer was examined under high-cell density conditions (as opposed to previously studied low density conditions) and an experimental design for tailoring the assay to high cell density fermentation samples is presented. In addition, a dual-plasmid strain of E. coli was designed for use in a novel process for the mass production of collagen-like polymers: one plasmid contains a thermally inducible recombinant collagen gene (CLP3.1-his), and the other contains an arabinose-inducible lytic gene (bacteriophage T4 t-holin) along with the basally expressed T7 lysozyme gene from the pLysS plasmid. A methodology for the optimization of the sequential induction of CLP3.1-his followed by induction of the t-holin is presented; a review of the literature suggests that decreased growth rate is detrimental to lytic efficiency (both the time required for lysis and the degree of lysis). The resulting process will enable lysis to take place in the bioreactor, thus avoiding the extra time and monetary cost of a separate cell homogenization step for E. coli disruption and endogenous protein release..by Brett A. Whittemore.M.Eng

The solution structure of the N-terminal domain of human vitronectin: Proximal sites that regulate fibrinolysis and cell migration

The three-dimensional structure of an N-terminal fragment comprising the first 51 amino acids from human plasma vitronectin, the somatomedin B (SMB) domain, has been determined by two-dimensional NMR approaches. An average structure was calculated, representing the overall fold from a set of 20 minimized structures. The core residues (18-41) overlay with a root mean square deviation of 2.29 ± 0.62 Å. The N- and C-terminal segments exhibit higher root mean square deviations, reflecting more flexibility in solution and/or fewer long-range NOEs for these regions. Residues 26-30 form a unique single-turn α-helix, the locus where plasminogen activator inhibitor type-1 (PAI-1) is bound. This structure of this helix is highly homologous with that of a recombinant SMB domain solved in a co-crystal with PAI-1 (Zhou, A., Huntington, J. A., Pannu, N. S., Carrell, R. W., and Read, R. J. (2003) Nat. Struct. Biol. 10, 541-544), although the remainder of the structure differs. Significantly, the pattern of disulfide cross-links observed in this material isolated from human plasma is altogether different from the disulfides proposed for recombinant forms. The NMR structure reveals the relative orientation of binding sites for cell surface receptors, including an integrin-binding site at residues 45-47, which was disordered and did not diffract in the co-crystal, and a site for the urokinase receptor, which overlaps with the PAI-1-binding site

Obstructive hydrocephalus and intracerebral mass secondary to Epicoccum nigrum

Here we report a case of a 14-week-old girl with a history of intrauterine drug exposure and hypoxic ischemic encephalopathy secondary to cardiac arrest requiring prolonged resuscitation at birth presented with irritability and a bulging anterior fontanelle. After neurosurgical resection, pathologic examination showed fungal hyphae, an

Assignment of the four disulfides in the N-terminal somatomedin B domain of native vitronectin isolated from human plasma

The primary sequence of the N-terminal somatomedin B (SMB) domain of native vitronectin contains 44 amino acids, including a framework of four disulfide bonds formed by 8 closely spaced cysteines in sequence patterns similar to those found in the cystine knot family of proteins. The SMB domain of vitronectin was isolated by digesting the protein with endoproteinase Glu-C and purifying the N-terminal 1-55 peptide by reverse-phase high performance liquid chromatography. Through a combination of techniques, including stepwise reduction and alkylation at acidic pH, peptide mapping with matrix-assisted laser desorption ionization mass spectrometry and NMR, the disulfide bonds contained in the SMB domain have been determined to be Cys5:Cys9, Cys 19:Cys31, Cys21:Cys32, and Cys 25:Cys39. This pattern of disulfides differs from two other connectivities that have been reported previously for recombinant forms of the SMB domain expressed in Escherichia coli. This arrangement of disulfide bonds in the SMB domain from native vitronectin forms a rigid core around the Cys19: Cys31 and Cys21:Cys32 disulfides. A small positively charged loop is created at the N terminus by the Cys5: Cys9 cystine. The most prominent feature of this disulfide-bonding pattern is a loop between Cys25 and Cys 39 similar to cystine-stabilized α-helical structures commonly observed in cystine knots. This α-helix has been confirmed in the solution structure determined for this domain using NMR (Mayasundari, A., Whittemore, N. A., Serpersu, E. H., and Peterson, C. B. (2004) J. Biol. Chem. 279, 29359-29366). It confers function on the SMB domain, comprising the site for binding to plasminogen activator inhibitor type-1 and the urokinase receptor

Preferred strategies for secondary infrainguinal bypass: Lessons learned from 300 consecutive reoperations

AbstractPurpose: To determine the optimal surgical strategies in reoperative infrainguinal bypass, we reviewed our results in 300 consecutive secondary bypasses in 251 patients operated on between Jan. 1, 1975, and Nov. 1, 1993.Methods: There were 168 men (67%) and 83 women (33%), with a mean age of 64.8 years and a typical distribution of risk factors including smoking (76.4%), diabetes (33.7%), and coronary artery disease (47.1%). The indications for surgery were limb-threatening ischemia in 83.5% and severe claudication in 16.5% of patients. The majority of conduits (n = 213) were autogenous vein and were composed of a single segment of greater saphenous vein in 121 bypasses (57%) and various alternative veins including composite, arm, and lesser saphenous vein in 92 bypasses (43%). Prosthetic conduits included 69 polytetrafluoroethylene, 16 umbilical vein, and two Dacron grafts.Results: There was one perioperative death (0.3%) and a 25% total morbidity rate including a 1.7% myocardial infarction rate. There was a 28.6% early (<30 days) graft failure and 10.7% early amputation rate for prosthetic bypass grafts compared with 13.6% early graft failure and 5.6% early amputation rates for vein grafts. Autogenous vein bypasses had higher 5-year secondary patency rates than had prosthetic grafts (51.5% ± 4.6% vs 27.4% ± 6.1%, p < 0.001). Results with autogenous vein bypass improved significantly from the 1975 to 1984 to the 1985 to 1993 interval with 5-year secondary patency rates increasing from 38.3% ± 6.9% to 59.1% ± 5.8% (p = 0.017) and 5-year limb-salvage rates increasing from 40.4% ± 7.6% to 72.4% ± 6.6% (p < 0.001). Vein grafts to the popliteal and tibial outflow levels had equivalent long-term results. Vein grafts completed for claudication demonstrated results superior to those for limb salvage, with a 5-year secondary patency rate of 75.8% ± 8.1% versus 52.3% ± 7.9% (p = 0.048). Secondary autogenous vein bypass grafting performed after early primary graft failure (< 3 months) did particularly poorly, with only a 27.2% ± 7.7% 4-year secondary patency rate. Greater saphenous veins tended to perform better than alternative vein bypasses, with a 5-year secondary patency rate of 68.5% ± 6.0% compared with 48.3% ± 10.5% (p = 0.09) and a 5-year limb-salvage rate of 77.8% ± 7.4% versus 54.2% ± 11.8% (p = 0.046).Conclusions: When patients suffer a recurrence of limb-threatening ischemia at the time of infrainguinal graft failure, aggressive attempts at secondary revascularization with autogenous vein are warranted based on the low surgical morbidity and mortality rates and the improved patency and limb salvage rates that are currently attainable. (J VASC SURG 1995;21:282-95.

Telomerase gene therapy ameliorates the effects of neurodegeneration associated to short telomeres in mice

Neurodegenerative diseases associated with old age such as Alzheimer's disease present major problems for society, and they currently have no cure. The telomere protective caps at the ends of chromosomes shorten with age, and when they become critically short, they can induce a persistent DNA damage response at chromosome ends, triggering secondary cellular responses such as cell death and cellular senescence. Mice and humans with very short telomeres owing to telomerase deficiencies have an earlier onset of pathologies associated with loss of the regenerative capacity of tissues. However, the effects of short telomeres in very low proliferative tissues such as the brain have not been thoroughly investigated. Here, we describe a mouse model of neurodegeneration owing to presence of short telomeres in the brain as the consequence of telomerase deficiency. Interestingly, we find similar signs of neurodegeneration in very old mice as the consequence of physiological mouse aging. Next, we demonstrate that delivery of telomerase gene therapy to the brain of these mice results in amelioration of some of these neurodegeneration phenotypes. These findings suggest that short telomeres contribute to neurodegeneration diseases with aging and that telomerase activation may have a therapeutic value in these diseases