Methods for producing soluble, biologically-active disulfide-bond containing eukaryotic proteins in bacterial cells

Disclosed are methods of producing eukaryotic disulfide bond-containing polypeptides in bacterial hosts, and compositions resulting therefrom. Co-expression of a eukaryotic foldase and a disulfide bond-containing polypeptide in a bacterial host cell is demonstrated. In particular embodiments, the methods have been used to produce mammalian pancreatic trypsin inhibitor and tissue plasminogen activator (tPA) in soluble, biologically-active forms, which are isolatable from the bacterial periplasm. Also disclosed are expression systems, recombinant vectors, and transformed host cells.Board of Regents, University of Texas Syste

Circular Permutation in the Ω-Loop of TEM-1 β-Lactamase Results in Improved Activity and Altered Substrate Specificity

Generating diverse protein libraries that contain improved variants at a sufficiently high frequency is critical for improving the properties of proteins using directed evolution. Many studies have illustrated how random mutagenesis, cassette mutagenesis, DNA shuffling and similar approaches are effective diversity generating methods for directed evolution. Very few studies have explored random circular permutation, the intramolecular relocation of the N- and C-termini of a protein, as a diversity-generating step for directed evolution. We subjected a library of random circular permutations of TEM-1 β-lactamase to selections on increasing concentrations of a variety of β-lactam antibiotics including cefotaxime. We identified two circularly permuted variants that conferred elevated resistance to cefotaxime but decreased resistance to other antibiotics. These variants were circularly permuted in the Ω-loop proximal to the active site. Remarkably, one variant was circularly permuted such that the key catalytic residue Glu166 was located at the N-terminus of the mature protein

Phase diagram of turbulence in superfluid 3He-B

In superfluid 3He-B mutual-friction damping of vortex-line motion decreases roughly exponentially with temperature. We record as a function of temperature and pressure the transition from regular vortex motion at high temperatures to turbulence at low temperatures. The measurements are performed with non-invasive NMR techniques, by injecting vortex loops into a long column in vortex-free rotation. The results display the phase diagram of turbulence at high flow velocities where the transition from regular to turbulent dynamics is velocity independent. At the three measured pressures 10.2, 29.0, and 34 bar, the transition is centered at 0.52--0.59Tc and has a narrow width of 0.06Tc while at zero pressure turbulence is not observed above 0.45Tc.Comment: To be published in J. Low Temp. Phys. (QFS2004 proceedings

Transition to superfluid turbulence governed by an intrinsic parameter

Hydrodynamic flow in both classical and quantum fluids can be either laminar or turbulent. To describe the latter, vortices in turbulent flow are modelled with stable vortex filaments. While this is an idealization in classical fluids, vortices are real topologically stable quantized objects in superfluids. Thus superfluid turbulence is thought to hold the key to new understanding on turbulence in general. The fermion superfluid 3He offers further possibilities owing to a large variation in its hydrodynamic characteristics over the experimentally accessible temperatures. While studying the hydrodynamics of the B phase of superfluid 3He, we discovered a sharp transition at 0.60Tc between two regimes, with regular behaviour at high-temperatures and turbulence at low-temperatures. Unlike in classical fluids, this transition is insensitive to velocity and occurs at a temperature where the dissipative vortex damping drops below a critical limit. This discovery resolves the conflict between existing high- and low-temperature measurements in 3He-B: At high temperatures in rotating flow a vortex loop injected into superflow has been observed to expand monotonically to a single rectilinear vortex line, while at very low temperatures a tangled network of quantized vortex lines can be generated in a quiescent bath with a vibrating wire. The solution of this conflict reveals a new intrinsic criterion for the existence of superfluid turbulence.Comment: Revtex file; 5 pages, 2 figure

Instability of vortex array and transitions to turbulent states in rotating helium II

We consider superfluid helium inside a container which rotates at constant angular velocity and investigate numerically the stability of the array of quantized vortices in the presence of an imposed axial counterflow. This problem was studied experimentally by Swanson {\it et al.}, who reported evidence of instabilities at increasing axial flow but were not able to explain their nature. We find that Kelvin waves on individual vortices become unstable and grow in amplitude, until the amplitude of the waves becomes large enough that vortex reconnections take place and the vortex array is destabilized. The eventual nonlinear saturation of the instability consists of a turbulent tangle of quantized vortices which is strongly polarized. The computed results compare well with the experiments. Finally we suggest a theoretical explanation for the second instability which was observed at higher values of the axial flow

Genetic Selection for Enhanced Folding In Vivo Targets the Cys14-Cys38 Disulfide Bond in Bovine Pancreatic Trypsin Inhibitor

The periplasm provides a strongly oxidizing environment; however, periplasmic expression of proteins with disulfide bonds is often inefficient. Here, we used two different tripartite fusion systems to perform in vivo selections for mutants of the model protein bovine pancreatic trypsin inhibitor (BPTI) with the aim of enhancing its expression in Escherichia coli. This trypsin inhibitor contains three disulfides that contribute to its extreme stability and protease resistance. The mutants we isolated for increased expression appear to act by eliminating or destabilizing the Cys14-Cys38 disulfide in BPTI. In doing so, they are expected to reduce or eliminate kinetic traps that exist within the well characterized in vitro folding pathway of BPTI. These results suggest that elimination or destabilization of a disulfide bond whose formation is problematic in vitro can enhance in vivo protein folding. The use of these in vivo selections may prove a valuable way to identify and eliminate disulfides and other rate-limiting steps in the folding of proteins, including those proteins whose in vitro folding pathways are unknown. Antioxid. Redox Signal. 14, 973-984.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/90494/1/ars-2E2010-2E3712.pd

Can I Be Me With You at Work? Examining Relational Authenticity and Discretionary Behaviors in the Workplace

Management scholars have long been interested in the topic of authenticity in the workplace, evidenced by the history of scholarship on authentic leadership and the many new authenticity constructs that have emerged. In this article, we take a narrower view of authenticity and focus on relational authenticity in the workplace, which we define as being genuine in workplace relationships. Adapting a validated relational authenticity scale to the organizational context, we explore the ways in which feeling authentic in workplace relationships has ramifications for discretionary behaviors. Specifically, we build on belongingness theory to posit that relational authenticity will result in an increase in engagement in both altruistic and sportsmanship behaviors. We also explore the moderating effect of proactive personality on these relationships. Results from our two-sample study (N = 352; 500) indicate that relational authenticity is positively associated with engagement in both altruistic and sportsmanship behaviors

GLUE-IT and PEDEL-AA: new programmes for analyzing protein diversity in randomized libraries

There are many methods for introducing random mutations into nucleic acid sequences. Previously, we described a suite of programmes for estimating the completeness and diversity of randomized DNA libraries generated by a number of these protocols. Our programmes suggested some empirical guidelines for library design; however, no information was provided regarding library diversity at the protein (rather than DNA) level. We have now updated our web server, enabling analysis of translated libraries constructed by site-saturation mutagenesis and error-prone PCR (epPCR). We introduce GLUE-Including Translation (GLUE-IT), which finds the expected amino acid completeness of libraries in which up to six codons have been independently varied (according to any user-specified randomization scheme). We provide two tools for assisting with experimental design: CodonCalculator, for assessing amino acids corresponding to given randomized codons; and AA-Calculator, for finding degenerate codons that encode user-specified sets of amino acids. We also present PEDEL-AA, which calculates amino acid statistics for libraries generated by epPCR. Input includes the parent sequence, overall mutation rate, library size, indel rates and a nucleotide mutation matrix. Output includes amino acid completeness and diversity statistics, and the number and length distribution of sequences truncated by premature termination codons. The web interfaces are available at http://guinevere.otago.ac.nz/stats.html

Rotating Superfluid Turbulence

Almost all studies of vortex states in helium II have been concerned with either ordered vortex lattices or disordered vortex tangles. This work studies numerically what happens in the presence of both rotation (which induces order) and thermal counterflow (which induces disorder). We find a new statistically steady state in which the vortex tangle is polarized along the rotational axis. Our results are used to interpret an instability which was discovered experimentally by Swanson et al. years ago but has been unexplained until now