S70 Abstracts / Journal of Biotechnology 185S (2014) S37–S125
Characterization of a wheat homologue ofapurinic/apyrimidinic endonuclease
Joldybayeva Botagoz 1,∗, Prorok Paulina 2, AkishevZhiger 1, Ishchenko Alexander 2, SaparbaevMurat 2, Bissenbaev Amangeldy 1
1 Department of Molecular Biology and Genetics,Al-Farabi Kazakh National University, Almaty,Kazakhstan2 Group DNA Repair, Institute Gustave Rossy,Villejuif, France
E-mail address: [email protected] (J. Botagoz).
Until now, molecular characterization of the DNA repair mech-anisms have been mainly focused on Escherichia coli, yeast andmammalian cells, whereas little is known on the mechanismsthat maintain genome stability in plants. In this work a Triticumaestivum cDNA encoding for a putative homologue of ExoIII fam-ily AP endonucleases has been isolated and its protein productpurified and characterized. We report that the putative wheat APendonuclease (TaApe1L) contains AP endonuclease, 3′-repair phos-phodiesterase, 3′-phosphatase and 3′ → 5′ exonuclease activities.Surprisingly, in contrast to bacterial and human AP endonucle-ases, addition of Mg2+ and Ca2+ to the reaction mixture inhibitedTaApe1L whereas the presence of Mn2+, Co2+ and Fe2+ cationsstrongly stimulated all its DNA repair activities. Optimization ofthe reaction conditions revealed that the wheat enzyme requireslow divalent cation concentration (0.1 mM), mildly acidic pH (6–7),low ionic strength (20 mM KCl) and has a temperature opti-mum at around 20 ◦C. The steady-state kinetic parameters ofenzymatic reactions indicate that TaApe1L removes 3′-blockingsugar-phosphate and 3′-phosphate groups with good efficiency(kcat/KM = 630 and 485 �M−1 min−1, respectively) but possesses avery weak AP endonuclease activity as compared to the humanhomologue, APE1.
http://dx.doi.org/10.1016/j.jbiotec.2014.07.236
Rapid E. coli-based cloning and expressionsystem for production of recombinant proteins
Boguslaw Lupa, Krzysztof Stawujak ∗, IgorRozanski, Justyna Stec-Niemczyk
BioCentrum Ltd, Selvita Group, Poland
E-mail address: [email protected] (K. Stawu-jak).
The novel system of ligation independent cloning for generationof genetic constructs designed for the production of proteins ofinterest in Escherichia coli has been applied. The system is basedon the modular approach, in which the indispensable elements ofthe expression vector such as the promoter, selection marker, originof replication, and gene of interest are generated by the PCR. Eachmodule contains the complementary fragments that allow joiningthe modules in the specific PCR, termed the CPEC. The modulesoccur in versions for generation and selection of the genetic con-structs with the highest levels of protein production. Under theexpression inducing conditions the levels of target protein produc-tion are assessed in the multi-well plate format. The clones withthe highest gene expressions are used for scaled-up production ofproteins, which are purified using the affinity chromatographiesbased on common fusion tags. Each genetic construct contains theClean-Cut recognition site, which together with Clean-Cut a novel,proprietary, unique and highly specific recombinant serine pro-tease facilitate the tag removal. The presented approach is ideal
for determination of the optimal conditions for the overexpressionand production of the range of recombinant and heterologouslyproduced proteins surpassing the standard expression tests.
http://dx.doi.org/10.1016/j.jbiotec.2014.07.237
Targeting of a CDNB(1-chloro-2,4-dinitrobenzene) substrate specificglutathione-S-transferase gene fromTetrahymena thermophila to be used a newbiotechnology product
Handan Acelya Kapkac ∗, Muhittin Arslanyolu
Department of Biology, Anadolu University,Eskisehir, Turkey
E-mail address: [email protected] (H.A. Kapkac).
Today, glutathione-S-transferase (GST) enzymes are commonlyused as an affinity tag in the recombinant fusion protein purifi-cation as well as in the kits for enzyme kinetics teaching at highschool and undergraduate education. The goal of this study is totarget a highly responsive CDNB substrate specific glutathione S-transferase gene from Tetrahymena thermophila by analyzing totalGST activity and mRNA expression responds. First, CDNB nontoxic(0.018–0.072 mM) and toxic doses (0.109–0.210 mM) were deter-mined by MTT test (LD50; 0.079; Probit) in the CDNB treated T.thermophila cells. After third division (9th hours), T. thermophilasignificantly overcame only the initial toxicity of 0.018 mM CNDBtreatment (t-test: p ≤ 0.05) by keeping total GST activity constant.Real time PCR analysis showed that cells in nontoxic doses have sig-nificant mRNA increase in the three of the four T. thermophila GSTgenes. The results from MTT, GST activity and real time analysesconclude that the possible target GST genes could be GSTmu19 orGSTmu34 for recombinant protein expression and enzyme kineticsanalyses.
http://dx.doi.org/10.1016/j.jbiotec.2014.07.238
Production of a novel collagenase andapplications
Ana Sofia Duarte 1,∗, Sandra Vieira 2, Odete DaCruz E. Silva 2, António Correia 1, Ana CristinaEsteves 1
1 Department of Biology & CESAM, University ofAveiro, Portugal2 Department of Health Sciences & Centro deBiologia Celular, University of Aveiro, Portugal
E-mail address: [email protected] (A.S. Duarte).
A collagenase gene was identified in a waterborne bacterialpathogen, and its complete nucleotide sequence was determined.The gene is 2782 nucleotides long and the deduced 927-aminoacid protein revealed distinct domain architecture when comparedto Vibrio and Clostridium collagenases. The amino acid sequencecontains a putative signal sequence and a zinc metalloproteaseconsensus sequence, the HEXXH motif. A BLASTP search in theMEROPS database indicated that this enzyme is a new collagen-ase, sharing only 52% identity with the bacterial metalloproteasefrom V. parahaemolyticus, collagenase V. The gene was clonedin Escherichia coli and the recombinant mature collagenase wasexpressed and purified. The enzyme is stable and able to digest heatdenatured collagen, and to physically interact with type-I colla-gen. Moreover, it can act in non-cytotoxic concentrations, reducingthe intracellular production and extracellular deposition of type-I
