Improved yields of full-length functional human FGF1 can be achieved using the methylotrophic yeast Pichia pastoris

We have produced human fibroblast growth factor 1 (hFGF1) in the methylotrophic yeast Pichia pastoris in order to obtain the large amounts of active protein required for subsequent functional and structural characterization. Four constructs were made to examine both intracellular and secreted expression, with variations in the location of the His6 tag at either end of the peptide. hFGF1 could be produced from all four constructs in shake flasks, but production was optimized by growing only the highest-yielding of these strains, which produced hFGF1 intracellularly, under tightly controlled conditions in a 3 L fermentor. One hundred and eight milligrams of pure protein was achieved per liter culture (corresponding to 0.68 mg of protein per gram of wet cells), the function of which was verified using NIH 3T3 cell cultures. This is a 30-fold improvement over previously reported yields of full-length hFGF1. © 2006 Elsevier Inc. All rights reserved.

Production, purification and characterization of recombinant, full-length human claudin-1

The transmembrane domain proteins of the claudin superfamily are the major structural components of cellular tight junctions. One family member, claudin-1, also associates with tetraspanin CD81 as part of a receptor complex that is essential for hepatitis C virus (HCV) infection of the liver. To understand the molecular basis of claudin-1/CD81 association we previously produced and purified milligram quantities of functional, full-length CD81, which binds a soluble form of HCV E2 glycoprotein (sE2). Here we report the production, purification and characterization of claudin-1. Both yeast membrane-bound and detergent-extracted, purified claudin-1 were antigenic and recognized by specific antibodies. Analytical ultracentrifugation demonstrated that extraction with n-octyl-ß-d-glucopyranoside yielded monodispersed, dimeric pools of claudin-1 while extraction with profoldin-8 or n-decylphosphocholine yielded a dynamic mixture of claudin-1 oligomers. Neither form bound sE2 in line with literature expectations, while further functional analysis was hampered by the finding that incorporation of claudin-1 into proteoliposomes rendered them intractable to study. Dynamic light scattering demonstrated that claudin-1 oligomers associate with CD81 in vitro in a defined molar ratio of 1:2 and that complex formation was enhanced by the presence of cholesteryl hemisuccinate. Attempts to assay the complex biologically were limited by our finding that claudin-1 affects the properties of proteoliposomes. We conclude that recombinant, correctly-folded, full-length claudin-1 can be produced in yeast membranes, that it can be extracted in different oligomeric forms that do not bind sE2 and that a dynamic preparation can form a specific complex with CD81 in vitro in the absence of any other cellular components. These findings pave the way for the structural characterization of claudin-1 alone and in complex with CD81.

Structural characterization of recombinant human CD81 produced in Pichia pastoris

Human CD81 (hCD81) protein has been recombinantly produced in the methylotrophic yeast Pichia pastoris. The purified protein, produced at a yield of 1.75 mg/L of culture, was shown to interact with Hepatitis C virus E2 glycoprotein. Immunofluorescent and flow cytometric staining of P. pastoris protoplasts with monoclonal antibodies specific for the second extracellular loop (EC2) of hCD81 confirmed the antigenicity of the recombinant molecule. Full-length hCD81 was solubilized with an array of detergents and subsequently characterized using circular dichroism (CD) and analytical ultracentrifugation. These biophysical techniques confirmed that the protein solution comprises a homogenous species possessing a highly-defined alpha-helical secondary structure. The predicted alpha-helical content of the protein from CD analysis (77.1%) fits remarkably well with what would be expected (75.2%) from knowledge of the protein sequence together with the data from the crystal structure of the second extracellular loop. This study represents the first biophysical characterization of a full-length recombinant tetraspanin, and opens the way for structure-activity analyses of this ubiquitous family of transmembrane proteins.

Fibronectin-tissue transglutaminase matrix rescues RGD-impaired celladhesion through syndecan-4 and β integrin co-signaling

Heterotropic association of tissue transglutaminase (TG2) with extracellular matrix-associated fibronectin (FN) can restore the adhesion of fibroblasts when the integrin-mediated direct binding to FN is impaired using RGD-containing peptide. We demonstrate that the compensatory effect of the TG-FN complex in the presence of RGD-containing peptides is mediated by TG2 binding to the heparan sulfate chains of the syndecan-4 cell surface receptor. This binding mediates activation of protein kinase Ca (PKCa) and its subsequent interaction with ß1 integrin since disruption of PKCa binding to ß1 integrins with a cell-permeant competitive peptide inhibits cell adhesion and the associated actin stress fiber formation. Cell signaling by this process leads to the activation of focal adhesion kinase and ERK1/2 mitogen-activated protein kinases. Fibroblasts deficient in Raf-1 do not respond fully to the TG-FN complex unless either the full-length kinase competent Raf-1 or the kinase-inactive domain of Raf-1 is reintroduced, indicating the involvement of the Raf-1 protein in the signaling mechanism. We propose a model for a novel RGD-independent cell adhesion process that could be important during tissue injury and/or remodeling whereby TG-FN binding to syndecan-4 activates PKCa leading to its association with ß1 integrin, reinforcement of actin-stress fiber organization, and MAPK pathway activation.

The effects of the environment on the stability and expression of genetically engineered plasmids

The lac promoter is widely used in plasmid expression systems, even though it is prone to catabolite repression. As a consequence glycerol is often used as an alternative carbon source. Three plasmids containing various sizes of the staphylococcal protein A (SPA) gene, which are under the control of the lac promoter were investigated in continuous culture, to evaluate the effects of nutrient limitations on their stability and expression. The fears of catabolite repression were dispelled as a low expression plasmid (pPA16) produced a greater amount of truncated SPA under glucose limiting conditions (11 ug mg-1 cell protein) when compared to that using glycerol (8 ug mg-1 cell protein). Segregational instability was also observed under glycerol limiting conditions at all the dilution rates investigated. Whereas pPA16 was relatively stable under glucose limiting conditions, with SPA production being continuous. Experiments using excess glycerol with limited ammonium increased the stability of pPA16, (when compared to limited glycerol) with expression of SPA being continuous but reduced (6 ug mg-1 cell protein). With excess glucose and limited ammonium the copy numbers remained high but expression of SPA paralled that produced under glucose limiting conditions. This might indicate that the higher levels of glucose are reducing expression (catabolite repression) or that the low level of ammonium is affecting protein production. A high expression plasmid (pPA31) produced nearly 100 ug full length SPA mg-1 cell protein, while another high expression plasmid (pPA34) producing truncated SPA proved to be very unstable. An ELISA was developed to detect the SPA produced by these experiments, which could be adapted for western blotting or immunogold probing using electron microscopy. SPA was localised in electron lucent areas present in the periplasmic space of the E. coli host harbouring pPA16. While in the same host containing pPA31, SPA was localised not only in electron lucent areas but also around the whole of the outer-membrane.

Structural characterization of CD81-Claudin-1 hepatitis C virus receptor complexes

Tetraspanins are thought to exert their biological function(s) by co-ordinating the lateral movement and trafficking of associated molecules into tetraspanin-enriched microdomains. A second four-TM (transmembrane) domain protein family, the Claudin superfamily, is the major structural component of cellular TJs (tight junctions). Although the Claudin family displays low sequence homology and appears to be evolutionarily distinct from the tetraspanins, CD81 and Claudin-1 are critical molecules defining HCV (hepatitis C virus) entry; we recently demonstrated that CD81-Claudin-1 complexes have an essential role in this process. To understand the molecular basis of CD81-Claudin-1 complex formation, we produced and purified milligram quantities of full-length CD81 and Claudin-1, alone and in complex, in both detergent and lipid contexts. Structural characterization of these purified proteins will allow us to define the mechanism(s) underlying virus-cell interactions and aid the design of therapeutic agents targeting early steps in the viral life cycle.

Autocrine activity of soluble Flt-1 controls endothelial cell function and angiogenesis

Background - The negative feedback system is an important physiological regulatory mechanism controlling angiogenesis. Soluble vascular endothelial growth factor (VEGF) receptor-1 (sFlt-1), acts as a potent endogenous soluble inhibitor of VEGF- and placenta growth factor (PlGF)-mediated biological function and can also form dominant-negative complexes with competent full-length VEGF receptors. Methods and results - Systemic overexpression of VEGF-A in mice resulted in significantly elevated circulating sFlt-1. In addition, stimulation of human umbilical vein endothelial cells (HUVEC) with VEGF-A, induced a five-fold increase in sFlt-1 mRNA, a time-dependent significant increase in the release of sFlt-1 into the culture medium and activation of the flt-1 gene promoter. This response was dependent on VEGF receptor-2 (VEGFR-2) and phosphoinositide-3'-kinase signalling. siRNA-mediated knockdown of sFlt-1 in HUVEC stimulated the activation of endothelial nitric oxide synthase, increased basal and VEGF-induced cell migration and enhanced endothelial tube formation on growth factor reduced Matrigel. In contrast, adenoviral overexpression of sFlt-1 suppressed phosphorylation of VEGFR-2 at tyrosine 951 and ERK-1/-2 MAPK and reduced HUVEC proliferation. Preeclampsia is associated with elevated placental and systemic sFlt-1. Phosphorylation of VEGFR-2 tyrosine 951 was greatly reduced in placenta from preeclamptic patients compared to gestationally-matched normal placenta. Conclusion - These results show that endothelial sFlt-1 expression is regulated by VEGF and acts as an autocrine regulator of endothelial cell function.

Editorial

The American Academy of Optometry (AAO) had their annual meeting in San Diego in December 2005 and the BCLA and CLAE were well represented there. The BCLA does have a reasonable number of non-UK based members and hopefully in the future will attract more. This will certainly be beneficial to the society as a whole and may draw more delegates to the BCLA annual conference. To increase awareness of the BCLA at the AAO a special evening seminar was arranged where BCLA president Dr. James Wolffsohn gave his presidential address. Dr. Wolffsohn has given the presidential address in the UK, Ireland, Hong Kong and Japan – making it the most travelled presidential address for the BCLA to date. Aside from the BCLA activity at the AAO there were numerous lectures of interest to all, truly a “something for everyone” meeting. All the sessions were multi-track (often up to 10 things occurring at the same time) and the biggest dilemma was often deciding what to attend and more importantly what you will miss! Nearly 200 new AAO Fellows were inducted at the Gala Dinner from many countries including 3 new fellows from the UK (this year they all just happened to be from Aston University!). It is certainly one of the highlights of the AAO to see fellows from different schools of training from around the world fulfilling the same criteria and being duly rewarded for their commitment to the profession. BCLA members will be aware that 2006 sees the introduction of the new fellowship scheme of the BCLA and by the time you read this the first set of fellowship examinations will have taken place. For more details of the FBCLA scheme see the BCLA web site http://www.bcla.org.uk. Since many of CLAE's editorial panel were at the AAO an informal meeting and dinner was arranged for them where ideas were exchanged about the future of the journal. It is envisaged that the panel will meet twice a year – the next meeting will be at the BCLA conference. The biggest excitement by far was the fact that CLAE is now Medline/PubMed indexed. You may ask why is this significant to CLAE? PubMed is the free web-based service from the US National Library of Medicine. It holds over 15 million biomedical citations and abstracts from the Medline database. Medline is the largest component of PubMed and covers over 4800 journals published in more than 70 countries. The impact of this is that CLAE is starting to attract more submissions as researchers and authors are not worried that their work will not be hidden from other colleagues in the field but rather the work is available to view on the World Wide Web. CLAE is one of a very small number of contact lens journals that is indexed this way. Amongst the other CL journals listed you will note that the International Contact Lens Clinic has now merged with CLAE and the journal CLAO has been renamed Eye and Contact Lenses – making the list of indexed CL journals even smaller than it appears. The on-line submission and reviewing system introduced in 2005 has also made it easier for authors to submit their work and easier for reviewers to check the content. This ease of use has lead to quicker times from submission to publication. Looking back at the articles published in CLAE in 2005 reveals some interesting facts. The majority of the material still tends to be from UK groups related to the field of Optometry, although we hope that in the future we will attract more work from non-UK groups and also from non-Optometric areas such as refractive surgery or anterior eye pathology. Interestingly in 2005 the most downloaded article from CLAE was “Wavefront technology: Past, present and future” by Professor W. Neil Charman, who was also the recipient of the Charles F. Prentice award at the AAO – one of the highest awards honours that the AAO can bestow. Professor Charman was also the keynote speaker at the BCLA's first Pioneer's Day meeting in 2004. In 2006, readers of CLAE will notice more changes, firstly we are moving to 5 issues per year. It is hoped that in the future, depending on increased submissions, a move to 6 issues may be feasible. Secondly, CLAE will aim to have one article per issue that carries CL CET points. You will see in this issue there is an article from Professor Mark Wilcox (who was a keynote speaker at the BCLA conference in 2005). In future articles that carry CET points will be either reviews from BCLA conference keynote speakers, members of the editorial panel or material from other invited persons that will be of interest to the readership of CLAE. Finally, in 2006, you will notice a change to the Editorial Panel, some of the distinguished panel felt that it was good time to step down and new members have been invited to join the remaining panel. The panel represent some of the most eminent names in the fields of contact lenses and/or anterior eye and have varying backgrounds and interests from many of the prominent institutions around the world. One of the tasks that the Editorial Panel undertake is to seek out possible submissions to the journal, either from conferences they attend (posters and papers that they will see and hear) and from their own research teams. However, on behalf of CLAE I would like to extend that invitation to seek original articles to all readers – if you hear a talk and think it could make a suitable publication to CLAE please ask the presenters to submit the work via the on-line submission system. If you found the work interesting then the chances are so will others. CLAE invites submissions that are original research, full length articles, short case reports, full review articles, technical reports and letters to the editor. The on-line submission web page is http://www.ees.elsevier.com/clae/.