Vaccinia Virus Expression Vector (Retrovirus/AIDS/Virus Assembly/Reverse Transcriptase) Velissarios KARACOSTAS*, Kunio Nagashimat, MATTHEW A

Total Page:16

File Type:pdf, Size:1020Kb

Vaccinia Virus Expression Vector (Retrovirus/AIDS/Virus Assembly/Reverse Transcriptase) Velissarios KARACOSTAS*, Kunio Nagashimat, MATTHEW A Proc. Nail. Acad. Sci. USA Vol. 86, pp. 8964-8967, November 1989 Medical Sciences Human immunodeficiency virus-like particles produced by a vaccinia virus expression vector (retrovirus/AIDS/virus assembly/reverse transcriptase) VELISSARIos KARACOSTAS*, KUNIo NAGASHIMAt, MATTHEW A. GONDAt, AND BERNARD MOSS*t *Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892; and tLaboratory of Cell and Molecular Structure, Program Resources, Inc., National Cancer Institute, Frederick Cancer Research Facility, Frederick, MD 21701 Contributed by Bernard Moss, August 11, 1989 ABSTRACT Infectious retrovirus particles consist of a MATERIALS AND METHODS core structure containing RNA and gag-pol polypeptides sur- rounded by a lipid membrane studded with env proteins. A Plasmid Construction. The plasmid sp64/HXB.2, contain- recombinant vaccinia virus was designed to express the entire ing an infectious cDNA copy of HIV-1 isolate HXB.2 (21) gag-pol precursor protein of the human immunodeficiency inserted into the Xba I site of plasmid sp64, was provided by virus type 1. Synthesis and processing of gag proteins occurred F. Wong-Staal and R. C. Gallo (National Cancer Institute) in mammalian cells infected with this live recombinant virus, and served as the source of the gag-pol gene. A 2300- and reverse transcriptase was detected largely in the medium. base-pair Sac I-EcoRV fragment of sp64/HXB.2 was in- Electron micrographs revealed immature retrovirus-like par- serted into the Sac I-Sma I sites of the replicative form of ticles budding from the plasma membrane and extracellular bacteriophage M13mpl8. Oligonucleotide-directed mutagen- particles with morphological characteristics of immature and esis was carried out to place a Sal I site followed by a mature human immunodeficiency virus. The latter contained consensus sequence for eukaryotic translation (22) before the functional reverse transcriptase as well as processed p24 and start codon of the gag gene. The gag-pol gene was inserted p17 gag polypeptides. Thus, assembly and maturation of into a modified form of the vector pSC11 (23) giving rise to human immunodeficiency virus-like particles can occur in the pVK3, the final plasmid used to obtain the recombinant virus absence ofeither infectious RNA molecules or env proteins. The (vVK1) expressing the HIV gag-pol genes. Restriction endo- production of noninfectious virus-like particles by expression nuclease analysis and DNA hybridization were used to vectors should be useful for biochemical studies and could characterize the plasmid pVK3. provide a safe source of material for the development of Viruses and Cells. Vaccinia virus (strain WR) was originally vaccines. obtained from the American Type Culture Collection. Re- combinant vaccinia virus vVK1 was isolated using the plas- mid pVK3 and was purified as reported (23). CV-1 monkey Human immunodeficiency virus type 1 (HIV-1), the caus- kidney- cells were propagated as monolayers in minimal ative agent ofacquired immunodeficiency syndrome (AIDS), essential medium (Quality Biologicals, Gaithersburg, MD) contains an RNA genome that encodes gag, pol, and env supplemented with 2.5% (vol/vol) fetal bovine serum. Cells proteins, as well as additional regulatory proteins (1-4). The were infected with 30 plaque-forming units of virus per cell primary gag translation product is a 55-kDa precursor, p55, for 2 hr; the virus was then removed, and the cells were that is proteolytically processed to p24, p17, and p15, the washed and overlayed with fresh medium. The cells were major core proteins. The pol open reading frame encodes the harvested at appropriate times after infection by scraping the protease, reverse transcriptase, and integrase (5-11). monolayers in isotonic phosphate-buffered saline (PBS), Expression of the protease, as well as other products of the washed with PBS, and lysed in 0.5% Nonidet P-40/PBS. pol gene, requires a relatively inefficient ribosomal frame- Immunoblotting. The detergent-disrupted cytoplasmic ex- shifting event within the gag gene (12) that leads to the tracts were treated with SDS and 2-mercaptoethanol prior to formation ofsmall amounts ofthe putative gag-pol precursor. polyacrylamide gel electrophoresis. The electrophoretically A myristic acid residue is present at the N terminus of p17 as separated polypeptides were transferred by electroblotting well as the gag precursor (13, 14) and by analogy with other onto a nitrocellulose membrane, blocked with 5% (wt/vol) retroviruses is likely to be required for transport to the nonfat dry milk/0.3% Tween 20 in PBS, incubated with the plasma membrane (15), into which the glycosylated envelope antibody in 0.3% Tween/PBS for 1 hr, washed three times proteins are inserted. Studies with defective avian and mu- with 0.3% Tween/PBS, incubated with 125I-labeled protein rine retroviruses, however, have shown that neither infec- A, washed three times with 0.3% Tween/PBS, dried, and tious RNA nor env protein is required for particle assembly subjected to autoradiography. (16-19). In this context, Gheysen et al. (20) observed the Reverse Transcriptase Assay. Cells were infected as de- formation of immature particles containing unprocessed p55 scribed above, and reverse transcriptase activity was mea- in insect cells that were infected with a baculovirus contain- sured using 5 ,ul of cell extract or medium, poly(A), oligo(dT), ing only the gag gene of HIV-1. In this communication, we and [a-32P]dTTP in 30 Al as described (24). After 2 hr at 37°C, describe HIV-like particle formation in mammalian cells 10lO was spotted on DE81 ion-exchange chromatography infected with a recombinant vaccinia virus expressing the paper (Whatman), air-dried, and washed four times in 2X entire gag-pol gene under control of a vaccinia virus pro- standard saline citrate (0.3 M NaCI/0.03 M sodium citrate, moter. Production of noninfectious HIV-1 particles using pH 7.0). The paper was dried and the radioactivity present expression vectors could be valuable both for studies of was determined in a Beckman scintillation counter. assembly and for vaccine purposes. Purification of Particles. Medium, collected 15 hr after infection of CV-1 cells with vVK1, was clarified by centrif- The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviation: HIV-1, human immunodeficiency virus type 1. in accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed. 8964 Downloaded by guest on September 23, 2021 Medical Sciences: Karacostas et al. Proc. Natl. Acad. Sci. USA 86 (1989) 8%5 ugation at 2000 X g, and layered on top of a 20% (wt/vol) with time (Fig. 2). These data suggested that reverse tran- sucrose cushion. The pellet, formed by centrifugation at scriptase was activatedjust before or after its release from the 27,000 rpm in an SW 27 rotor for 90 min, was suspended in cell. Control experiments confirmed the absence of detect- PBS, and layered on top of a preformed 20-60% sucrose able reverse transcriptase activity from cells infected with gradient. After centrifugation at 100,000 x g for 18 hr in an wild-type vaccinia virus. It is also important to note that SW 41 rotor, 12 equal fractions were collected starting at the vaccinia virus (strain WR) particles remain almost entirely bottom. intracellular. Electron Microscopy. CV-1 cells infected with recombinant Characterization of Purified Particles. To understand the vaccinia virus expressing HIV-1 gag-pol genes were har- physical nature of the secreted reverse transcriptase, the vested in PBS and centrifuged at 1500 x g. The pellets were medium was collected and placed over a sucrose cushion. fixed in 1.25% (vol/vol) gluteraldehyde and then in 1% After centrifugation, all ofthe activity was found in the pellet osmium tetroxide, dehydrated in graded alcohols, and em- fraction, suggesting that it was in a particulate form (Fig. 2). bedded in epoxy resins. Virus pellets were prepared by The pelleted material was resuspended and subjected to centrifugation at 100,000 x g for 90 min and then processed equilibrium density sucrose gradient sedimentation. Almost for electron microscopy as above. Thin sections were cut and all of the reverse transcriptase activity was found in fractions stained with uranyl acetate and lead citrate. 5, 6, and 7, corresponding to a density of -1.16 g/cm3 (Fig. 3 Upper). Additional samples from each gradient fraction were ana- RESULTS lyzed by immunoblotting after polyacrylamide gel electro- Expression and Processing of HIVgag-pol. When cells were phoresis (Fig. 3 Lower). Although material reactive with infected with the recombinant vaccinia virus, synthesis and AIDS patient antiserum was widely dispersed, the processed processing of the gag-pol proteins occurred. In cell extracts gag polypeptides p24 and p17 were predominantly located in prepared 4 hr after infection, the most prominent polypep- the fractions with reverse transcriptase as expected for tides that reacted with HIV-1-specific antiserum were -55, mature HIV particles. 46, and 41 kDa (Fig. 1). Also present were small amounts of Electron Microscopy of Retroviral Particles. Electron mi- high molecular mass material (>100 kDa), presumably re- crographs ofthin sections ofvVK1-infected cells revealed the sulting from frame-shifting, as also shown by Gowda et al. presence of 100- to 120-nm particles budding from the plasma (25). With time, three small polypeptides of 32, 24, and 17 membrane (Fig. 4A). These retrovirus-like particles were detected at 4 hr and increased greatly in amount from 8 hr on. kDa increased in amount and the higher molecular mass The morphogenesis of the particles, produced by vVK1, was polypeptides diminished. Pulse-chase experiments with The [35S]methionine were consistent with precursor-product re- similar to that of HIV-1 and other retroviruses (26-29).
Recommended publications
  • Gene Therapy Glossary of Terms
    GENE THERAPY GLOSSARY OF TERMS A • Phase 3: A phase of research to describe clinical trials • Allele: one of two or more alternative forms of a gene that that gather more information about a drug’s safety and arise by mutation and are found at the same place on a effectiveness by studying different populations and chromosome. different dosages and by using the drug in combination • Adeno-Associated Virus: A single stranded DNA virus that has with other drugs. These studies typically involve more not been found to cause disease in humans. This type of virus participants.7 is the most frequently used in gene therapy.1 • Phase 4: A phase of research to describe clinical trials • Adenovirus: A member of a family of viruses that can cause occurring after FDA has approved a drug for marketing. infections in the respiratory tract, eye, and gastrointestinal They include post market requirement and commitment tract. studies that are required of or agreed to by the study • Adeno-Associated Virus Vector: Adeno viruses used as sponsor. These trials gather additional information about a vehicles for genes, whose core genetic material has been drug’s safety, efficacy, or optimal use.8 removed and replaced by the FVIII- or FIX-gene • Codon: a sequence of three nucleotides in DNA or RNA • Amino Acids: building block of a protein that gives instructions to add a specific amino acid to an • Antibody: a protein produced by immune cells called B-cells elongating protein in response to a foreign molecule; acts by binding to the • CRISPR: a family of DNA sequences that can be cleaved by molecule and often making it inactive or targeting it for specific enzymes, and therefore serve as a guide to cut out destruction and insert genes.
    [Show full text]
  • Lecture 3 Types, Biology and Salient Features of Vectors in Recombinant
    NPTEL – Bio Technology – Genetic Engineering & Applications MODULE 1- LECTURE 3 TYPES, BIOLOGY AND SALIENT FEATURES OF VECTORS IN RECOMBINANT DNA TECHNOLOGY – PLASMID 1-3.1 Introduction: DNA molecule used for carrying an exogenous DNA into a host organism and facilitates stable integration and replication inside the host system is termed as Vector. Molecular cloning involves series of sequential steps which includes restriction digestion of DNA fragments both target DNA and vector, ligation of the target DNA with the vector and introduction into a host organism for multiplication. Then the fragments resulted after digestion with restriction enzymes are ligated to other DNA molecules that serve as vectors. In general, vectors should have following characteristics: • Capable of replicating inside the host. • Have compatible restriction site for insertion of DNA molecule (insert). • Capable of autonomous replication inside the host (ori site). • Smaller in size and able to incorporate larger insert size. • Have a selectable marker for screening of recombinant organism. 1-3.2 Plasmids: • Plasmids are naturally occurring extra chromosomal double-stranded circular DNA molecules which can autonomously replicate inside bacterial cells. Plasmids range in size from about 1.0 kb to over 250 kb. • Plasmids encode only few proteins required for their own replication (replication proteins) and these proteins encoding genes are located very close to the ori. All the other proteins required for replication, e.g. DNA polymerases, DNA ligase, helicase, etc., are provided by the host cell.Thus, only a small region surrounding Joint initiative of IITs and IISc – Funded by MHRD Page 28 of 84 NPTEL – Bio Technology – Genetic Engineering & Applications the ori site is required for replication.
    [Show full text]
  • Molecular Biology and Applied Genetics
    MOLECULAR BIOLOGY AND APPLIED GENETICS FOR Medical Laboratory Technology Students Upgraded Lecture Note Series Mohammed Awole Adem Jimma University MOLECULAR BIOLOGY AND APPLIED GENETICS For Medical Laboratory Technician Students Lecture Note Series Mohammed Awole Adem Upgraded - 2006 In collaboration with The Carter Center (EPHTI) and The Federal Democratic Republic of Ethiopia Ministry of Education and Ministry of Health Jimma University PREFACE The problem faced today in the learning and teaching of Applied Genetics and Molecular Biology for laboratory technologists in universities, colleges andhealth institutions primarily from the unavailability of textbooks that focus on the needs of Ethiopian students. This lecture note has been prepared with the primary aim of alleviating the problems encountered in the teaching of Medical Applied Genetics and Molecular Biology course and in minimizing discrepancies prevailing among the different teaching and training health institutions. It can also be used in teaching any introductory course on medical Applied Genetics and Molecular Biology and as a reference material. This lecture note is specifically designed for medical laboratory technologists, and includes only those areas of molecular cell biology and Applied Genetics relevant to degree-level understanding of modern laboratory technology. Since genetics is prerequisite course to molecular biology, the lecture note starts with Genetics i followed by Molecular Biology. It provides students with molecular background to enable them to understand and critically analyze recent advances in laboratory sciences. Finally, it contains a glossary, which summarizes important terminologies used in the text. Each chapter begins by specific learning objectives and at the end of each chapter review questions are also included.
    [Show full text]
  • Regulation of Transgene Expression in Genetic Immunization
    Brazilian Journal of Medical and Biological Research (1999) 32: 155-162 Regulation of transgene expression 155 ISSN 0100-879X Regulation of transgene expression in genetic immunization J.S. Harms1, 1Department of Animal Health and Biomedical Sciences, University of Wisconsin, S.C. Oliveira2 Madison, WI, USA and G.A. Splitter1 2Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brasil Abstract Correspondence The use of mammalian gene expression vectors has become increas- Key words J.S. Harms ingly important for genetic immunization and gene therapy as well as · DNA vaccine AHABS basic research. Essential for the success of these vectors in genetic · Gene therapy 1655 Linden Drive · immunization is the proper choice of a promoter linked to the antigen Regulation Madison, WI 53706 · Transcription of interest. Many genetic immunization vectors use promoter elements USA · Transgene Fax: +1-608-262-7420 from pathogenic viruses including SV40 and CMV. Lymphokines E-mail: [email protected] produced by the immune response to proteins expressed by these vectors could inhibit further transcription initiation by viral promot- Research supported by the Robert ers. Our objective was to determine the effect of IFN-g on transgene Draper Technology Innovation Fund expression driven by viral SV40 or CMV promoter/enhancer and the (No. 135-0546) of the University mammalian promoter/enhancer for the major histocompatibility com- of Wisconsin, Madison. plex class I (MHC I) gene. We transfected the luciferase gene driven Presented at the International by these three promoters into 14 cell lines of many tissues and several Symposium “The Third Revolution species.
    [Show full text]
  • RNA Viruses As Tools in Gene Therapy and Vaccine Development
    G C A T T A C G G C A T genes Review RNA Viruses as Tools in Gene Therapy and Vaccine Development Kenneth Lundstrom PanTherapeutics, Rte de Lavaux 49, CH1095 Lutry, Switzerland; [email protected]; Tel.: +41-79-776-6351 Received: 31 January 2019; Accepted: 21 February 2019; Published: 1 March 2019 Abstract: RNA viruses have been subjected to substantial engineering efforts to support gene therapy applications and vaccine development. Typically, retroviruses, lentiviruses, alphaviruses, flaviviruses rhabdoviruses, measles viruses, Newcastle disease viruses, and picornaviruses have been employed as expression vectors for treatment of various diseases including different types of cancers, hemophilia, and infectious diseases. Moreover, vaccination with viral vectors has evaluated immunogenicity against infectious agents and protection against challenges with pathogenic organisms. Several preclinical studies in animal models have confirmed both immune responses and protection against lethal challenges. Similarly, administration of RNA viral vectors in animals implanted with tumor xenografts resulted in tumor regression and prolonged survival, and in some cases complete tumor clearance. Based on preclinical results, clinical trials have been conducted to establish the safety of RNA virus delivery. Moreover, stem cell-based lentiviral therapy provided life-long production of factor VIII potentially generating a cure for hemophilia A. Several clinical trials on cancer patients have generated anti-tumor activity, prolonged survival, and
    [Show full text]
  • Clone the Unclonable – Vectors and Cells to Capture and Express Problematic Dnas
    Clone the Unclonable – Vectors and Cells to Capture and Express Problematic DNAs Ron Godiska, Ph.D. May, 2016 Agenda Improve Cloning and Expression of Problematic DNA • What makes DNA difficult to clone or express? • Circular vectors for difficult DNAs • Linear vectors for “impossible” DNAs • Enzyme-free cloning and compatible protein expression vectors • E. coli strains for toxic recombinant proteins or unstable DNA What is Unclonable DNA? Characteristics of Difficult DNA • Toxic coding sequences • Promoters • A-T Rich DNA • Large fragments (>10 kb) • Trace amounts Which types of difficult DNA have you worked with in the lab? Common Vector Traits Cause Cloning Problems Vector driven transcription into the insert – Deleterious expression – Destabilize 2o structure Insert driven transcription out into the vector – False negatives (blue or lethal) – Transcriptional interference pUC19 – Replication interference High copy number – Increased effect of the above issues – Active origin – Many copies/cell Supercoiling – Deletion of secondary structures CloneSmart® Technology “Silencing” DNA Inserts to Improve Cloning • No Transcriptional Interference – Stably maintain “unclonable” DNA • No blue/white screening – No false positives/negatives • Minimal vector size • Variable copy number • Variety of drug selection markers • Very low empty-vector background U.S. Patent 6,709,861 pSMART Vectors Clone Toxic Inserts and Promoters Test DNA Fragments: • RNase coding sequence (350 bp; no promoter) • Phage Lambda PR Promoter (400 bp) pSMART Vectors cDNA Clones are More Stable Deletion Rates: <1% in pSMART HC Kan vs. 32% in pUC % Deleted Clones Insert Size (kb) • Teleost fish cDNA library • Clones were grown in liquid medium, diluted, and regrown • Each final culture was diluted, plated, and grown up into individual colonies • Plasmid DNA was isolated from colonies for each clone and analyzed - Oleksiak M.F., Crawford D.L., 2001.
    [Show full text]
  • Important Vector Factors for Gene Expression Technical Reference Guide
    BioResearch Important Vector Factors for Gene Expression Technical Reference Guide vectors expressing the same luciferase gene in different backbones and expression cassettes and obtained highly discriminative expres- sion levels. In this guideline, we show the most important aspects to be considered when choosing an expression vector and their impact on your experimental results. 1. Promoter Strength Introduction Is your promoter appropriate for the cell type that you are working with? Our Scientific Support Team is commonly asked: “Why don’t I get the Table 1 describes the promoter strengths as a relative percent of the same expression level of my gene if I use various vector backbones?” strength of the CMV promoter for various cells for which Lonza has There are many components to a vector that can have an effect on the Optimized Protocols. The CMV promoter activity is set to 100% based level of gene expression. Below you will find the 10 most important on their CAT assay values from the referenced publications. Although factors to consider when looking at vectors. CMV is a strong promoter in many mammalian cells, another promoter The selection of an appropriate expression vector is crucial for efficient may give stronger expression in your cells (e.g., promoter SV40 in gene expression. Just take a look at Figure 1. We tried 10 different BHK-21 cells). n 1600 1400 1200 1000 800 [% of pGL3-CMV at 24 h] 24 at pGL3-CMV [% of 600 400 Relative luciferase-expressio Relative 200 0 Control Program Only pGL3-Control pGL3-CMV pmax Cloning™ pSG5 pcDNA3.1 pFP pIRES-MCS B pcDNA4 HisMax pCMVbeta pCMV-TNT 4 hours 24 hours 48 hours n 2000 1500 1000 500 0 Control Program Only pGL3-Control pGL3-CMV pmax Cloning™ pSG5 pcDNA3.1 pFP pIRES-MCS B pcDNA4 HisMax pCMVbeta pCMV-TNT [% of pGL3-CMV at 24 h] 24 at pGL3-CMV [% of 4 hours 24 hours 48 hours Relative luciferase-expressio Relative Figure 1: Luciferase expression levels depend on vector backbones.
    [Show full text]
  • Cloning Technologies for Protein Expression and Purification
    Cloning technologies for protein expression and purification James L Hartley Detailed knowledge of the biochemistry and structure of expedite the manipulations that are often required to individual proteins is fundamental to biomedical research. To obtain pure, active recombinant proteins. further our understanding, however, proteins need to be purified in sufficient quantities, usually from recombinant sources. Cloning for protein expression and Although the sequences of genomes are now produced in purification automated factories purified proteins are not, because their The required amount, purity, homogeneity, and activity behavior is much more variable. The construction of plasmids of the protein of interest usually determine the choice of and viruses to overexpress proteins for their purification is often expression context, vector and host. Usually the first tedious. Alternatives to traditional methods that are faster, easier consideration is what host cell to use. Expression in and more flexible are needed and are becoming available. Escherichia coli is fast, inexpensive and scaleable, and minimal post-translational modifications make proteins Addresses Protein Expression Laboratory, Research Technology Program, purified from E. coli relatively homogeneous and highly SAIC-Frederick, Inc., NCI-Frederick, Frederick, Maryland 21702, USA desirable for structural studies. However, in our experi- ence most eukaryotic proteins larger than 30 kDa are Corresponding author: Hartley, James L ([email protected]) unlikely to be properly folded when expressed in E. coli (JL Hartley et al., unpublished). Expression in mamma- Current Opinion in Biotechnology 2006, 17:359–366 lian cells is best for activity and native structure (includ- ing post-translational modifications), but yields are much This review comes from a themed issue on lower and costs are high.
    [Show full text]
  • Complete Control Retroviral Inducible Mammalian Expression System
    Complete Control Retroviral Inducible Mammalian Expression System Instruction Manual Catalog #217564 (pFB-ERV), #240028 (pCFB-EGSH) Revision D.0 For Research Use Only. Not for use in diagnostic procedures. 217564-12 LIMITED PRODUCT WARRANTY This warranty limits our liability to replacement of this product. No other warranties of any kind, express or implied, including without limitation, implied warranties of merchantability or fitness for a particular purpose, are provided by Agilent. Agilent shall have no liability for any direct, indirect, consequential, or incidental damages arising out of the use, the results of use, or the inability to use this product. ORDERING INFORMATION AND TECHNICAL SERVICES Email [email protected] World Wide Web www.genomics.agilent.com Telephone Location Telephone United States and Canada 800 227 9770 Austria 01 25125 6800 Benelux 02 404 92 22 Denmark 45 70 13 00 30 Finland 010 802 220 France 0810 446 446 Germany 0800 603 1000 Italy 800 012575 Netherlands 020 547 2600 Spain 901 11 68 90 Sweden 08 506 4 8960 Switzerland 0848 8035 60 UK/Ireland 0845 712 5292 All Other Countries Please visit www.agilent.com/genomics/contactus Complete Control Retroviral Inducible Mammalian Expression System Materials Provided .............................................................................................................................. 1 Storage Conditions .............................................................................................................................. 1 Additional Materials
    [Show full text]
  • An Episomal Expression Vector for Screening Mutant Gene Libraries in Pichia Pastoris
    Plasmid 54 (2005) 80–85 www.elsevier.com/locate/yplas Short communication An episomal expression vector for screening mutant gene libraries in Pichia pastoris Charles C. Lee*, Tina G. Williams, Dominic W.S. Wong, George H. Robertson USDA-ARS-WRRC, 800 Buchanan St., Albany, CA 94710, USA Received 7 October 2004, revised 6 December 2004 Available online 20 January 2005 Communicated by Manuel Espinosa Abstract Screening mutant gene libraries for isolating improved enzyme variants is a powerful technique that benefits from effective and reliable biological expression systems. Pichia pastoris is a very useful organism to express proteins that are inactive in other hosts such as Escherichia coli and Saccharomyces cerevisiae. However, most P. pastoris expression plasmids are designed to integrate into the host chromosome and hence are not as amenable to high-throughput screen- ing projects. We have designed a P. pastoris expression vector, pBGP1, incorporating an autonomous replication sequence that allows the plasmid to exist as an episomal element. This vector contains the a-factor signal sequence to direct secretion of the mutant enzymes. Expression of the genes is driven by the constitutive GAP promoter, thus eliminating the need for timed or cell density-specific inductions. The pBGP1 plasmid was used to screen a xylanase gene library to isolate higher activity mutants. Published by Elsevier Inc. Keywords: Pichia pastoris; Episomal expression vector 1. Introduction formed with libraries of randomly mutated genes encoding the enzyme of interest (Cohen et al., There are many efforts directed at improving 2001). Clones that demonstrate an increased activ- enzymes involved in industrial processes in order ity relative to wild type can be isolated and further to increase cost and energy efficiency.
    [Show full text]
  • Retroviral Gene Transfer and Expression User Manual Table of Contents I
    Takara Bio USA, Inc. Retroviral Gene Transfer and Expression User Manual Cat. Nos. Many (081419) Takara Bio USA, Inc. 1290 Terra Bella Avenue, Mountain View, CA 94043, USA U.S. Technical Support: [email protected] United States/Canada Asia Pacific Europe Japan Page 1 of 31 800.662.2566 +1.650.919.7300 +33.(0)1.3904.6880 +81.(0)77.565.6999 Retroviral Gene Transfer and Expression User Manual Table of Contents I. Introduction ..................................................................................................................................................................... 3 II. List of Components ....................................................................................................................................................... 10 III. Additional Materials Required .................................................................................................................................. 13 IV. Safety Guidelines for Working with Retroviruses .................................................................................................... 16 V. Plasmid Vector Manipulations ...................................................................................................................................... 17 VI. Working with Retroviral Packaging Cell Lines ........................................................................................................ 18 A. General Cell Culture and Retrovirus Information ....................................................................................................
    [Show full text]
  • Advances in the Development of Genetically Modified Animals
    Advances in the Development of Genetically Modified Animals Louis-Marie Houdebinea, Martín Alfredo Lemab, Moisés Burachikc* aBiologie du Développement et Reproduction, INRA, UMR1198, 78350 Jouy en Josas, France bNational University of Quilmes. Roque Sáenz Peña 352, Bernal B1876BXD, Buenos Aires Province, Argentina cINDEAR (Agrobiotechnology Institute Rosario), Ocampo 210 Bis (Predio CCT), (2000) Rosario, Argentina *E-mail: [email protected] (corresponding author) Abstract A wide range of genetically modified (GM) animal/trait combinations are being developed with a great variety of purposes: to enhance food production or nutritional quality; to improve human or animal health; as sources of products (pharmaceuticals) or tissues (transplantation) for human therapeutic use; to control vectors of human infectious diseases; and to improve animal farming operations, amongst others. This review will focus on developments in three main animal classes: mammals, fish and insects. Several developments of GM mammals and their derived products shall be reviewed as examples of the potential impacts of this technology on farming practices through to human health. Fish, on the other hand, pose a particular problem because of the possible effects of their entry into natural ecosystems as a result of their unintended escape from fish farming operations. Two main objectives drive the development of GM insects, namely pest management and control of vector-borne infectious diseases. These will require the deliberate release of GM insects directly
    [Show full text]