DOCSLIB.ORG

Structure and Application of Antifreeze Proteins from Antarctic Bacteria Patricio A

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Structure and Application of Antifreeze Proteins from Antarctic Bacteria Patricio A Muñoz et al. Microb Cell Fact (2017) 16:138 DOI 10.1186/s12934-017-0737-2 Microbial Cell Factories RESEARCH Open Access Structure and application of antifreeze proteins from Antarctic bacteria Patricio A. Muñoz1*, Sebastián L. Márquez1,2, Fernando D. González‑Nilo3, Valeria Márquez‑Miranda3 and Jenny M. Blamey1,2* Abstract Background: Antifreeze proteins (AFPs) production is a survival strategy of psychrophiles in ice. These proteins have potential in frozen food industry avoiding the damage in the structure of animal or vegetal foods. Moreover, there is not much information regarding the interaction of Antarctic bacterial AFPs with ice, and new determinations are needed to understand the behaviour of these proteins at the water/ice interface. Results: Diferent Antarctic places were screened for antifreeze activity and microorganisms were selected for the presence of thermal hysteresis in their crude extracts. Isolates GU1.7.1, GU3.1.1, and AFP5.1 showed higher thermal hysteresis and were characterized using a polyphasic approach. Studies using cucumber and zucchini samples showed cellular protection when samples were treated with partially purifed AFPs or a commercial AFP as was determined using toluidine blue O and neutral red staining. Additionally, genome analysis of these isolates revealed the presence of genes that encode for putative AFPs. Deduced amino acids sequences from GU3.1.1 (gu3A and gu3B) and AFP5.1 (afp5A) showed high similarity to reported AFPs which crystal structures are solved, allowing then generating homology models. Modelled proteins showed a triangular prism form similar to β-helix AFPs with a linear distribution of threonine residues at one side of the prism that could correspond to the putative ice binding side. The statistically best models were used to build a protein-water system. Molecular dynamics simulations were then performed to compare the antifreezing behaviour of these AFPs at the ice/water interface. Docking and molecular dynamics simulations revealed that gu3B could have the most efcient antifreezing behavior, but gu3A could have a higher afnity for ice. Conclusions: AFPs from Antarctic microorganisms GU1.7.1, GU3.1.1 and AFP5.1 protect cellular structures of frozen food showing a potential for frozen food industry. Modeled proteins possess a β-helix structure, and molecular dock‑ ing analysis revealed the AFP gu3B could be the most efcient AFPs in order to avoid the formation of ice crystals, even when gu3A has a higher afnity for ice. By determining the interaction of AFPs at the ice/water interface, it will be possible to understand the process of adaptation of psychrophilic bacteria to Antarctic ice. Keywords: Antifreeze proteins, Antarctica, Psychrophiles, Frozen food, Ice binding proteins Background ecosystem. Although diferent microorganisms have been Antarctica is an extreme continent with the coldest isolated from several places, biological records are avail- temperatures, low precipitations, dryness, and almost able for only a miniscule fraction of them from land and completely covered by ice. In this continent, microor- surrounding waters [1]. ganisms dominate the genetic pool and biomass play- Psychrophilic microorganisms dominate the major- ing an important role maintaining the operation of the ity of Antarctic habitats [2]. For their survival, they have developed several growing strategies on ice including the *Correspondence: [email protected]; [email protected] production of compatible solutes, exopolysaccharides, 1 Fundación Científca y Cultural Biociencia, José Domingo Cañas, 2280, and antifreeze proteins [3]. Ñuñoa, Santiago, Chile Full list of author information is available at the end of the article © The Author(s) 2017. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Muñoz et al. Microb Cell Fact (2017) 16:138 Page 2 of 13 Initially, antifreeze proteins (AFPs) were described screw and 50 mL plastic tubes. Temperature and pH of in marine fshes [4, 5] and defned as cryoprotectants the selected area for sampling were measured and regis- [6]. Currently, AFPs have been obtained from diferent tered. Samples were kept at 0–4 °C in a cooler and imme- sources including snow molds fungi [7], sea ice diatoms diately transported to operation stations for processing. [8], snow alga [9] and bacteria [10–13]. AFPs are a diverse group of ice-binding proteins that Enrichment of environmental samples and bacterial prevent ice growing through the depression of the freez- isolation ing point of a solution to below the melting point [14]. Solid environmental samples were directly inoculated Tis diference between freezing and melting point is into 20 mL of TGY medium, containing (g/L) 5.0 tryp- referred as thermal hysteresis (TH) and results from the tone, 3.0 yeast extract, and 1.0 glucose. Te pH was adsorption of the AFP on the crystal surface of ice. Tis adjusted to 7.0. Meanwhile, ice samples were thawed in interaction causes the ice growth to take place in a con- seawater at 4 °C in order to avoid hypotonic shocking of vex surface between adjacent AFPs, thus decreasing the bacteria, centrifuged at 9000×g during 10 min at 4 °C, freezing point [15]. Additionally, these proteins act at low and inoculated on TGY medium. All cultures were incu- concentration and they are 200–300 times more efective bated aerobically at 4 °C for 4 weeks or until microbial than ideal solutes [16], being AFPs from insects 10–100 growth were observed. more than those from fsh [17]. Enriched cultures were isolated using serial dilutions Te full biotechnological potential of AFPs has not on liquid TGY and streaking on plates of TGY supple- been fully exploited [18]. Tey possess potential applica- mented with 1.5% (p/p) of agar. Colonies were transferred tions in cryopreservation [19], as additive in CO2 hydrate to a liquid TGY medium. Incubations were performed at slurry production [20] and frozen food preparation [21]. 4 °C. Tese procedures were repeated until a single and On this last case, cucumbers and zucchinis, which have homogeneous morphology was observed. high water content, loose their crispness when they are freeze–thaw cycles, resulting in poor product qual- Freeze–thaw resistant bacteria selection ity. Antarctica possesses a wide unexplored diversity of Isolates were exposed to repetitive freeze–thaw cycles microorganisms to search for AFPs in order to produce between −20 and 4 °C in order to select which micro- these proteins in large commercial scales. organisms have cryoprotective abilities, including AFPs Tis study was focused on searching bacterial AFPs production, according to the protocol described by Wil- from diferent places in Antarctica, including continental son et al. [22]. and insular sites. Several microorganisms were isolated and subjected to continued cycles of freezing and thaw- Microbial identifcation and characterization ing in order to select those who produce AFPs. Crude Genomic DNA from the isolates was extracted by the extracts were prepared from several isolates and TH was chloroform:isoamyl alcohol method [23]. 16S rRNA determined using a Nanoliter Osmometer. AFP-produc- gene was amplifed by PCR using bacteria specifc prim- ing isolates (AFP5.1, GU1.7.1 and GU3.1.1) were char- ers 27F and 1492R [24]. Te reaction mix contained acterized using a polyphasic approach and AFPs were 2.5 U Taq DNA polymerase, 200 μM of each deoxynu- partially purifed from these microorganisms in order cleotide (dATP, dCTP, dGTP and dTTP), 1× reaction to study their protective efect on cucumber and zuc- bufer, 0.75 mM MgCl2 and 0.5 mM of each primer. PCR chini samples when they were frozen and thawed in the consisted of 45 cycles: 95 °C for 45 s, 55 °C for 45 s and presence or absence of these proteins. AFP-coding genes 72 °C for 45 s. A fnal elongation step of 72 °C for 10 min were obtained from AFP5.1 and GU3.1.1 allowing deduc- was included. Amplifcation reactions were carried out ing amino acids sequences in order to obtain homology using a Palm Gradient Cycler (Corbett). PCR product models for these proteins and analyze in silico their inter- was observed on 1.0% agarose gel prepared in 1× TAE action with ice crystal. bufer (40 mM Tris–acetate, 10 mM EDTA) and visual- ized under UV light using a 1× GelRed (Biotium) in TAE Methods bufer. PCR products were sequenced using the primers Sampling described above and manually edited using ChromasPro Sample collection was performed at diferent places software (Technelysium Pty Ltd.) for fnal sequences of in insular and continental Antarctica during Chilean 1200 bp. Partial sequences were compared to GenBank Antarctic Scientifc Expeditions ECA 50–52. Samples using Blastn software. consisting of sediment or small stones were collected A phenotypic characterization was performed accord- aseptically using metallic spoons, and Whirl–Pak bags ing to the procedure described by Correa-Llantén et al. (8 × 8 inch). Ice samples were taken using a metallic [25]. Muñoz et al. Microb Cell Fact (2017) 16:138 Page 3 of 13 TH measurement O (TBO) or neutral red (NR) according to procedures TH was measured using a Nanolitre Osmometer (Otago described by González et al. [28] to determine the efect Osmometers) using the procedure described by Bra- of AFPs over plant cell integrity and cell viability (CV), slavsky and Drori [26]. respectively. Integrity of cell walls was visualized using TBO stain- AFP induction and purifcation ing. Samples were mounted to microscope slides with Te isolates GU1.7.1, GU3.1.1 and AFP5.1 were grown in a small drop of deionized water and a drop of 0.025% 10 L of TGY medium without shaking.
Load more

Recommended publications
  • Distribution and Functional Characterisation of Antifreeze Proteins in Polar Diatoms
    Living inside Sea Ice - Distribution and Functional Characterisation of Antifreeze Proteins in Polar Diatoms Dissertation zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften - Dr. rer. Nat. - am Fachbereich 2 (Biologie/Chemie) der Universit¨atBremen vorgelegt von Christiane Uhlig Bremen Oktober, 2011 1. Pr¨ufer: Prof. Kai Bischof 2. Pr¨ufer: Prof. Ulrich Bathmann F¨urmeine Mutter Monika Was Menschen in die Polargebiete trieb, war die Macht des Unbekannten ¨uber den men- schlichen Geist. Sie treibt uns zu den verborgenen Kr¨aftenund Geheimnissen der Natur, hinab in die unermesslich kleine mikroskopische Welt und desgleichen hinaus in die uner- forschten Weiten des Universums. Sie l¨asstuns keine Ruhe, bis wir den Planeten, auf dem wir leben, von der tiefsten Tiefen des Ozeans bis zu den h¨ochsten Schichten der Atmosph¨arekennen. Fridtjof Nansen Danksagung Ich danke Prof. Ulrich Bathmann und Prof. Kai Bischof f¨urdie Begutachtung dieser Arbeit. Prof. Rudolf Amann danke ich, dass er sich kurzfristig bereit erkl¨arthat die Aufgabe des 3. Pr¨uferzu ¨ubernehmen. Bei Prof. Kai Bischof und Andreas Krell bedanke ich mich f¨urdie Hilfe zur Einwerbung des Stipendiums, welches diese Arbeit ¨uberhaupt erst erm¨oglicht hat. Der Studienstiftung des deutschen Volkes danke ich f¨ur die finanzielle Unterst¨utzung. Ich bedanke mich ganz besonders bei der Meereis-Gruppe, die mehr ist als nur eine Ar- beitsgruppe. Besonders danke ich Gerhard Dieckmann, dass ich in Deiner Arbeitsgruppe meine Arbeit durchf¨uhrendurfte und f¨ur Deine Unterst¨utzungin allen m¨oglichen organ- isatorischen und pers¨onlichen Dingen. Klaus Valentin danke ich f¨urdie Unterst¨utzung, kreativen Titelvorschl¨ageund den Einsatz daf¨ur,dass ich noch eine Weile am AWI bleiben kann.
    [Show full text]
  • Effects of Antifreeze Protein III on Sperm Cryopreservation of Pacific Abalone, Haliotis Discus Hannai
    International Journal of Molecular Sciences Article Effects of Antifreeze Protein III on Sperm Cryopreservation of Pacific Abalone, Haliotis discus hannai Shaharior Hossen 1 , Md. Rajib Sharker 1,2, Yusin Cho 1, Zahid Parvez Sukhan 1 and Kang Hee Kho 1,* 1 Department of Fisheries Science, College of Fisheries and Ocean Sciences, Chonnam National University, 50 Daehak-ro, Yeosu 59626, Jeonnam, Korea; [email protected] (S.H.); [email protected] (M.R.S.); [email protected] (Y.C.); [email protected] (Z.P.S.) 2 Department of Fisheries Biology and Genetics, Faculty of Fisheries, Patuakhali Science and Technology University, Patuakhali 8602, Bangladesh * Correspondence: [email protected]; Tel.: +82-616-597-168; Fax: +82-616-597-169 Abstract: Pacific abalone (Haliotis discus hannai) is a highly commercial seafood in Southeast Asia. The aim of the present study was to improve the sperm cryopreservation technique for this valuable species using an antifreeze protein III (AFPIII). Post-thaw sperm quality parameters including motility, acrosome integrity (AI), plasma membrane integrity (PMI), mitochondrial membrane potential (MMP), DNA integrity, fertility, hatchability, and mRNA abundance level of heat shock protein 90 (HSP90) were determined to ensure improvement of the cryopreservation technique. Post-thaw motility of sperm cryopreserved with AFPIII at 10 µg/mL combined with 8% dimethyl sulfoxide (DMSO) (61.3 ± 2.7%), 8% ethylene glycol (EG) (54.3 ± 3.3%), 6% propylene glycol (PG) (36.6 ± 2.6%), or 2% glycerol (GLY) (51.7 ± 3.0%) was significantly improved than that of sperm cryopreserved without AFPIII. Post-thaw motility of sperm cryopreserved with 2% MeOH and 1 µg/mL of AFPIII was also improved than that of sperm cryopreserved without AFPIII.
    [Show full text]
  • Replacement of the Antifreeze-Like Domain of Human N-Acetylneuraminic Acid Phosphate Synthase with the Mouse Antifreeze-Like
    Replacement of the antifreeze-like domain of human N-acetylneuraminic acid phosphate synthase with the mouse antifreeze-like domain impacts both N-acetylneuraminic acid 9-phosphate synthase and 2-keto-3-deoxy-D-glycero-D- galacto-nonulosonic acid 9-phosphate synthase activities Marshall Louis Reaves1,2, Linda Carolyn Lopez1 & Sasha Milcheva Daskalova1,* 1The Biodesign Institute, Arizona State University, Tempe Arizona, 85287, 2Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544, USA Human NeuNAc-9-P synthase is a two-domain protein with In prokaryotes, the final stage of formation of N-ace- ability to synthesize both NeuNAc-9-P and KDN-9-P. Its tyl-D-neuraminic acid (NeuNAc) the most common sialic mouse counterpart differs by only 20 out of 359 amino acids acid in nature involves condensation of N-Acetyl-D-mannos- but does not produce KDN-9-P. By replacing the AFL domain amine (ManNAc) with phosphoenol pyruvate (PEP). The proc- of the human NeuNAc-9-P synthase which accommodates 12 ess, when irreversible, is catalyzed by the enzyme NeuNAc of these differences, with the mouse AFL domain we examined synthase (E.C. 4.1.3.19). The first bacterial gene, neuB gene, its importance for the secondary KDN-9-P synthetic activity. coding for the E. coli enzyme was identified in 1995 (2) and The chimeric protein retained almost half of the ability of the this later allowed overexpression and purification of sufficient human enzyme for KDN-9-P synthesis while the NeuNAc-9-P amount of protein for detailed characterization (3). Using the production was reduced to less than 10%.
    [Show full text]
  • Enhancement of Insect Antifreeze Protein Activity by Solutes of Low Molecular Mass
    The Journal of Experimental Biology 201, 2243–2251 (1998) 2243 Printed in Great Britain © The Company of Biologists Limited 1998 JEB1557 ENHANCEMENT OF INSECT ANTIFREEZE PROTEIN ACTIVITY BY SOLUTES OF LOW MOLECULAR MASS NING LI, CATHY A. ANDORFER AND JOHN G. DUMAN* Department of Biological Sciences, University of Notre Dame, Notre Dame, IN 46556, USA *Author for correspondence (e-mail: [email protected]) Accepted 20 May; published on WWW 14 July 1998 Summary Antifreeze proteins (AFPs) lower the non-equilibrium Glycerol is the only one of these enhancing solutes that is freezing point of water (in the presence of ice) below the known to be present at these concentrations in melting point, thereby producing a difference between the overwintering D. canadensis, and therefore the freezing and melting points that has been termed thermal physiological significance of most of these enhancers is hysteresis. In general, the magnitude of the thermal unknown. The mechanism(s) of this enhancement is also hysteresis depends upon the specific activity and unknown. concentration of the AFP. This study describes several low- The AFP used in this study (DAFP-4) is nearly identical molecular-mass solutes that enhance the thermal hysteresis to previously described D. canadensis AFPs. The mature activity of an AFP from overwintering larvae of the beetle protein consists of 71 amino acid residues arranged in six 12- Dendroides canadensis. The most active of these is citrate, or 13-mer repeats with a consensus sequence consisting of which increases the thermal hysteresis nearly sixfold from Cys-Thr-X3-Ser-X5-X6-Cys-X8-X9-Ala-X11-Thr-X13, where 1.2 °C in its absence to 6.8 °C.
    [Show full text]
  • Antifreeze Protein Improves the Cryopreservation Efficiency of Hosta Capitata by Regulating the Genes Involved in the Low-Temper
    horticulturae Study Protocol Antifreeze Protein Improves the Cryopreservation Efficiency of Hosta capitata by Regulating the Genes Involved in the Low-Temperature Tolerance Mechanism Phyo Phyo Win Pe 1,2,†, Aung Htay Naing 3,† , Chang Kil Kim 3,* and Kyeung Il Park 1,* 1 Department of Horticulture and Life Science, Yeungnam University, Gyeongsan 38541, Korea; [email protected] 2 Department of Horticulture, Yezin Agricultural University, Nay Pyi Taw 15013, Myanmar 3 Department of Horticulture, Kyungpook National University, Daegu 41566, Korea; [email protected] * Correspondence: [email protected] (C.K.K.); [email protected] (K.I.P.) † Phyo Phyo Win Pe and Aung Htay Naing equally contributed to this work. Abstract: In this study, whether the addition of antifreeze protein (AFP) to a cryopreservative solution (plant vitrification solution 2 (PVS2)) is more effective in reducing freezing injuries in Hosta capitata than PVS2 alone at different cold exposure times (6, 24, and 48 h) is investigated. The upregulation of C-repeat binding factor 1 (CBF1) and dehydrin 1 (DHN1) in response to low temperature was observed in shoots. Shoots treated with distilled water (dH2O) strongly triggered gene expression 6 h after cold exposure, which was higher than those expressed in PVS2 and PVS2+AFP. However, 24 h after cold exposure, gene expressions detected in dH2O and PVS2 treatments were similar and Citation: Pe, P.P.W.; Naing, A.H.; higher than PVS2 + AFP. The expression was highest in PVS2+AFP when the exposure time was Kim, C.K.; Park, K.I. Antifreeze extended to 48 h. Similarly, nitric reductase activities 1 and 2 (Nia1 and Nia2) genes, which are Protein Improves the responsible for nitric oxide production, were also upregulated in low-temperature-treated shoots, Cryopreservation Efficiency of Hosta as observed for CBF1 and DHN1 expression patterns during cold exposure periods.
    [Show full text]
  • Supporting Information
    Supporting Information Deng et al. 10.1073/pnas.1007883107 Fig. S1. Nucleotide sequence alignment of the C-terminal domain coding exon (exon6) of Ld sialic acid synthase (SAS)-A and LdSAS-B and exon2 of three typical antifreeze protein (AFP)III genes, LdψAFPIII, LdAFPIII-1, and LdAFPIII-2, from the Lycodichthys dearborni AFPIII locus. Among AFPIII genes, LdψAFPIII shares the greatest sequence similarity with the LdSAS genes, including having six additional nucleotides (shown in red) not present in the other two AFPIII genes, suggesting that LdψAFPIII is the earliest member of the AFPIII gene family. Asterisks indicate identical nucleotides between the six sequences, and the asterisks in red indicate identical nucleotides shared between LdψAFPIII and the LdSAS genes. The stop codon (TGA for SAS and TAG for AFPIII) is underlined. Fig. S1 (PDF) Fig. S2. Sequence alignments of LdSAS-A, LdSAS-B, and LdψAFPIII, the putative primitive AFPIII gene members from L. dearborni.(A) Nucleotide sequence alignment and deduced amino acid sequence of LdSAS-A and LdSAS-B. The proximal 5′ flanking sequences (∼600 nt) of the two genes share no sequence similarity and thus, are not homologous. The locations of the six exons are labeled, and exon sequences and translated amino acids are bold. Amino acidsand codons of LdSAS-B that differ from LdSAS-A are shown in blue. (B) Nucleotide sequence alignment of the homologous regions between LdSAS-B and LdψAFPIII. The proximal 5′ flanking regions of the two genes are homologous. The color highlighted LdSAS-B sequence regions (in the ancestral LdSAS-B) gave rise to the AFPIII gene.
    [Show full text]
  • Biodiversity Research and Innovation in Antarctica and the Southern Ocean
    bioRxiv preprint doi: https://doi.org/10.1101/2020.05.03.074849; this version posted May 3, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 1 Biodiversity Research and Innovation in Antarctica and the 2 Southern Ocean 3 2020 4 Paul Oldham and Jasmine Kindness1 5 Abstract 6 This article examines biodiversity research and innovation in Antarctica and the Southern 7 Ocean based on a review of 150,401 scientific articles and 29,690 patent families for 8 Antarctic species. The paper exploits the growing availability of open access databases, 9 such as the Lens and Microsoft Academic Graph, along with taxonomic data from the Global 10 Biodiversity Information Facility (GBIF) to explore the scientific and patent literature for 11 the Antarctic at scale. The paper identifies the main contours of scientific research in 12 Antarctica before exploring commercially oriented biodiversity research and development 13 in the scientific literature and patent publications. The paper argues that biodiversity is not 14 a free good and must be paid for. Ways forward in debates on commercial research and 15 development in Antarctica can be found through increasing attention to the valuation of 16 ecosystem services, new approaches to natural capital accounting and payment for 17 ecosystem services that would bring the Antarctic, and the Antarctic Treaty System, into 18 the wider fold of work on the economics of biodiversity.
    [Show full text]
  • Antifreeze Proteins and Their Practical Utilization in Industry, Medicine, and Agriculture
    biomolecules Review Antifreeze Proteins and Their Practical Utilization in Industry, Medicine, and Agriculture Azadeh Eskandari 1,2, Thean Chor Leow 1,3,4 , Mohd Basyaruddin Abdul Rahman 5 and Siti Nurbaya Oslan 1,2,4,* 1 Enzyme and Microbial Technology Research Centre, Universiti Putra Malaysia, UPM, Serdang 43400, Selangor, Malaysia; [email protected] (A.E.); [email protected] (T.C.L.) 2 Department of Biochemistry, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, UPM, Serdang 43400, Selangor, Malaysia 3 Department of Cell and Molecular Biology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, UPM, Serdang 43400, Selangor, Malaysia 4 Enzyme Technology Laboratory, Institute of Bioscience, Universiti Putra Malaysia, UPM, Serdang 43400, Selangor, Malaysia 5 Department of Chemistry, Faculty of Science, Universiti Putra Malaysia, UPM, Serdang 43400, Selangor, Malaysia; [email protected] * Correspondence: [email protected]; Tel.: +60-39769-6710; Fax: +60-39769-7590 Received: 8 October 2020; Accepted: 30 November 2020; Published: 9 December 2020 Abstract: Antifreeze proteins (AFPs) are specific proteins, glycopeptides, and peptides made by different organisms to allow cells to survive in sub-zero conditions. AFPs function by reducing the water’s freezing point and avoiding ice crystals’ growth in the frozen stage. Their capability in modifying ice growth leads to the stabilization of ice crystals within a given temperature range and the inhibition of ice recrystallization that decreases the drip loss during thawing. This review presents the potential applications of AFPs from different sources and types. AFPs can be found in diverse sources such as fish, yeast, plants, bacteria, and insects.
    [Show full text]
  • ENGINEERING ANTIFREEZE PROTEIN MULTIMERS THROUGH GENETIC FUSION to SELF-ASSEMBLING PROTEIN OLIGOMERS Sean William Phippen
    ENGINEERING ANTIFREEZE PROTEIN MULTIMERS THROUGH GENETIC FUSION TO SELF-ASSEMBLING PROTEIN OLIGOMERS by Sean William Phippen A thesis submitted to the Department of Biomedical and Molecular Sciences In conformity with the requirements for the degree of Master of Science Queen’s University Kingston, Ontario, Canada (August, 2017) Copyright © Sean William Phippen, 2017 Abstract Ice-binding proteins (IBPs) serve many purposes for the organisms producing them, enabling them to thrive in ice-laden environments. Antifreeze proteins (AFPs), a subset of this group, act as biological antifreezes and inhibit ice recrystallization. These ice-binding activities have many applications in the field of biotechnology, and to this end AFPs have been engineered in several ways to increase their activity. One of these strategies is to connect multiple AFPs together using a scaffold, which has been accomplished previously through bioconjugation of AFPs to the reactive termini of branched polymers. The inefficiency of such linkage reactions, however, resulted in heterogeneous mixtures of particles with incomplete occupancy of the available termini. To address this shortcoming, AFPs were genetically fused to the C termini of subunits forming self-assembling protein oligomers, in such a way that the ice-binding site of the AFP was optimally exposed for contact with ice. First, two different type of AFPs were separately fused to 12 of the 24 subunits of a self-assembling protein cage. The subunits assembled into the designed structures, and the AFP multimers showed greatly enhanced freezing point depression and ice recrystallization inhibition over the corresponding monomeric AFPs. A moderately active fish AFP was also fused to a subunit designed to assemble into two- dimensional arrays, and the resulting fusion protein showed increased freezing point depression over the monomer.
    [Show full text]
  • The Study of Evolutionary Origin of the Antifreeze Gene in ​Rhagium
    Roskilde Universitet / Roskilde University Den Naturvidenskabelige Bacheloruddannelse / The Natural Science Bachelor Programme _____________________________________________ The study of evolutionary origin of the antifreeze gene in Rhagium mordax. __________________________​ ____________​ _​ ___________________ Group members: Saudat Alishayeva Nicolaj Stelzner Grønvall Alexander Varnich Hansen Philip Kruse Nina Štrancar Project supervisor: Peter Kamp Busk Semester: 5th Semester Date: 18/12/2018 Abstract This project report details the attempt to isolate and sequence candidate sequences for the evolution of the antifreeze protein gene present in Rhagium mordax. Larvae of ​ ​ the R.mordax species were collected, their DNA extracted and underwent PCR ​ ​ amplification with the use of specially designed primers which targeted sequences similar to ones coding for antifreeze genes. In addition, to validate the species we have applied DNA barcoding method. PCR amplifications were analysed with gel electrophoresis. Several PCR products were observed and sent for direct Sanger sequencing. Due to a problem with sequencing, we have been unable to identify the sequences and draw the conclusions of the origin of the antifreeze gene. Although the sequencing failed, results from the previous steps indicate that the methods utilized in this project were feasible for the study of R. mordax genetics. ​ ​ 1 Table of contents Abstract 1 Table of contents 2 Introduction 4 1.1 Hypothesis 5 2. Theory 6 2.1 Historical Overview of the Discovery of the Antifreeze Proteins 6 2.2 Convergent Evolution and R. mordax 6 2.3 Mutations - Gene Duplication 8 2.4 Antifreeze Proteins and Their Function 10 2.5 Degenerate primer design 10 3. Experimental methods 15 3.1 Сollection of R.
    [Show full text]
  • Antifreeze Protein Prolongs the Life-Time of Insulinoma Cells During Hypothermic Preservation
    Antifreeze Protein Prolongs the Life-Time of Insulinoma Cells during Hypothermic Preservation Tatsuro Kamijima1, Mami Sakashita1, Ai Miura1, Yoshiyuki Nishimiya1, Sakae Tsuda1,2* 1 Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Sapporo, Hokkaido, Japan, 2 Graduate School of Life Science, Hokkaido University, Sapporo, Hokkaido, Japan Abstract It is sometimes desirable to preserve mammalian cells by hypothermia rather than freezing during short term transplantation. Here we found an ability of hypothermic (+4uC) preservation of fish antifreeze protein (AFP) against rat insulinoma cells denoted as RIN-5F. The preservation ability was compared between type I–III AFPs and antifreeze glycoprotein (AFGP), which could be recently mass-prepared by a developed technique utilizing the muscle homogenates, but not the blood serum, of cold-adapted fishes. For AFGP, whose molecular weight is distributed in the range from 2.6 to 34 kDa, only the proteins less than 10 kDa were examined. The viability rate was evaluated by counting of the preserved RIN-5F cells unstained with trypan blue. Significantly, either AFPI or AFPIII dissolved into Euro-Collins (EC) solution at a concentration of 10 mg/ml could preserve approximately 60% of the cells for 5 days at +4uC. The 5-day preserved RIN-5F cells retained the ability to secrete insulin. Only 2% of the cells were, however, preserved for 5 days without AFP. Confocal photomicroscopy experiments further showed the significant binding ability of AFP to the cell surface. These results suggest that fish AFP enables 5-day quality storage of the insulinoma cells collected from a donor without freezing.
    [Show full text]
  • The Use of Antifreeze Proteins for Frost Protection in Sensitive Crop Plants
    Environmental and Experimental Botany 106 (2014) 60–69 Contents lists available at ScienceDirect Environmental and Experimental Botany journal homepage: www.elsevier.com/locate/envexpbot The use of antifreeze proteins for frost protection in sensitive crop plants John G. Duman a,∗, Michael J. Wisniewski b a Department of Biological Sciences, University of Notre Dame, Notre Dame, IN 46556, USA b United States Department of Agriculture, Agricultural Research Service, Kearneysville, WV 25430, USA article info abstract Article history: Antifreeze proteins (AFPs), also known as ice binding proteins (IBPs), have evolved as an important adap- Received 14 November 2013 tation in numerous organisms exposed to subzero temperatures. Plant AFPs have only been identified Received in revised form in freeze tolerant species (those able to survive extracellular freezing). Consequently, plant AFPs have 17 December 2013 very low specific activities as they have not evolved to completely prevent ice formation in the plant. Accepted 3 January 2014 In contrast, fish and most insect AFPs function to prevent freezing in species that have evolved freeze Available online 13 January 2014 avoidance mechanisms. Therefore, the activity of these AFPs, especially those of insects (as they are gen- erally exposed to considerably lower temperatures than fish), is much greater. The ability of AFPs to Keywords: Antifreeze proteins non-colligatively lower the freezing point of water (thermal hysteresis) has led to the idea that frost- Plant cold-tolerance sensitive crop plants could avoid damage resulting from common minor frost events in late spring and Transgenic plants early autumn by expressing high activity AFPs that permit them to remain unfrozen to temperatures of ◦ Plant frost injury approximately −5 C.
    [Show full text]