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Recombinant Expression Systems (recombinant + expression_system)

Distribution by Scientific Domains

Chemistry	75%

Selected Abstracts

Recombinant production, crystallization and preliminary X-ray analysis of PCNA from the psychrophilic archaeon Methanococcoides burtonii DSM 6242

ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 11 2009
Miranda L. Byrne-Steele
Proliferating cell nuclear antigen (PCNA) is a DNA-clamping protein that is responsible for increasing the processivity of the replicative polymerases during DNA replication and repair. The PCNA from the eurypsychrophilic archaeon Methanococcoides burtonii DSM 6242 (MbPCNA) has been targeted for protein structural studies. A recombinant expression system has been created that overproduces MbPCNA with an N-terminal hexahistidine affinity tag in Escherichia coli. As a result, recombinant MbPCNA with a molecular mass of 28.3,kDa has been purified to at least 95% homogeneity and crystallized by vapor-diffusion equilibration. Preliminary X-ray analysis revealed a trigonal hexagonal R3 space group, with unit-cell parameters a = b = 102.5, c = 97.5,Å. A single MbPCNA crystal was subjected to complete diffraction data-set collection using synchrotron radiation and reflections were measured to 2.40,Å resolution. The diffraction data were of suitable quality for indexing and scaling and an unrefined molecular-replacement solution has been obtained. [source]

Stabilization of human papillomavirus virus-like particles by non-ionic surfactants

JOURNAL OF PHARMACEUTICAL SCIENCES, Issue 7 2005
Li Shi
Abstract Human papillomavirus (HPV) virus-like-particles (VLPs) produced by recombinant expression systems are promising vaccine candidates for prevention of cervical cancers as well as genital warts. At high protein concentrations, HPV VLPs, comprised of the viral capsid protein L1 and expressed and purified from yeast, are protected against detectable aggregation during preparation and storage by high concentrations of NaCl. At low protein concentrations, however, high salt concentration alone does not fully protect HPV VLPs from aggregation. Moreover, the analytical analysis of HPV VLPs proved to be a challenge due to surface adsorption of HPV VLPs to storage containers and cuvettes. The introduction of non-ionic surfactants into HPV VLP aqueous solutions provides significantly enhanced stabilization of HPV VLPs against aggregation upon exposure to low salt and protein concentration, as well as protection against surface adsorption and aggregation due to heat stress and physical agitation. The mechanism of non-ionic surfactant stabilization of HPV VLPs was extensively studied using polysorbate 80 (PS80) as a representative non-ionic surfactant. The results suggest that PS80 stabilizes HPV VLPs mainly by competing with the VLPs for various container surfaces and air/water interfaces. No appreciable binding of PS80 to intact HPV VLPs was observed although PS80 does bind to the denatured HPV L1 protein. Even in the presence of stabilizing level of PS80, however, an ionic strength dependence of HPV VLP stabilization against aggregation is observed indicating optimization of both salt and non-ionic surfactant levels is required for effective stabilization of HPV VLPs in solution. © 2005 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 94:1538,1551, 2005 [source]

Cell-free expression and selective isotope labelling in protein NMR

MAGNETIC RESONANCE IN CHEMISTRY, Issue S1 2006
David Staunton
Abstract Isotope labelling is a very powerful tool in NMR studies of proteins and has been employed in various ways for over 40 years. 15N and 13C incorporation, using recombinant expression systems, is now commonplace because heteronuclear experiments assist with the fundamental problems of peak resolution and assignment. The use of selective labelling for peak assignment has been restricted by the scrambling of isotope label through metabolic pathways within the expression host organism. The availability of efficient cell-free expression systems with low levels of metabolic conversion allow the increasing use of selective isotope labelling as a tool in protein NMR. We describe two examples, one where a selective labelling scheme can identify backbone amide peaks from unassigned 1H15N HSQC and HNCO spectra of a 84 residue protein, and another where a specific backbone amide in a 198 residue construct of the ninth and tenth Type III repeats from human fibronectin can be labelled and rapidly identified using a simple HSQC experiment. Copyright © 2006 John Wiley & Sons, Ltd. [source]

A chloroplast transgenic approach to hyper-express and purify Human Serum Albumin, a protein highly susceptible to proteolytic degradation

PLANT BIOTECHNOLOGY JOURNAL, Issue 2 2003
Alicia Fernández-San Millán
Summary Human Serum Albumin (HSA) accounts for 60% of the total protein in blood serum and it is the most widely used intravenous protein in a number of human therapies. HSA, however, is currently extracted only from blood because of a lack of commercially feasible recombinant expression systems. HSA is highly susceptible to proteolytic degradation in recombinant systems and is expensive to purify. Expression of HSA in transgenic chloroplasts using Shine-Dalgarno sequence (SD), which usually facilitates hyper-expression of transgenes, resulted only in 0.02% HSA in total protein (tp). Modification of HSA regulatory sequences using chloroplast untranslated regions (UTRs) resulted in hyper-expression of HSA (up to 11.1% tp), compensating for excessive proteolytic degradation. This is the highest expression of a pharmaceutical protein in transgenic plants and 500-fold greater than previous reports on HSA expression in transgenic leaves. Electron micrographs of immunogold labelled transgenic chloroplasts revealed HSA inclusion bodies, which provided a simple method for purification from other cellular proteins. HSA inclusion bodies could be readily solubilized to obtain a monomeric form using appropriate reagents. The regulatory elements used in this study should serve as a model system for enhancing expression of foreign proteins that are highly susceptible to proteolytic degradation and provide advantages in purification, when inclusion bodies are formed. [source]