Ген pal, кодирующий L-фенилаланин-аммоний-лиазу Rhodosporidium toruloides (GenBank: X12702.1), последовательность которого была оптимизирована, клонирован в состав экспрессирующего вектора pET28a. В результате оптимизации экспрессии, проводившейся по трем параметрам (тип индуктора, время индукции и температура индукции) был получен штамм-продуцент рекомбинантного белка L-фенилаланин-аммоний-лиазы с максимальной продуктивностью, составляющей 35±1% от общего клеточного белка при использовании в качестве индуктора 0,2% лактозы (по Штудиеру), времени индукции 18 ч и температуры культивирования 37°С. В результате определения растворимости L-фенилаланин-аммоний-лиазы было показано, что рекомбинантный белок на 99% находится в нерастворимой фракции. Использование в качестве индуктора не 0,2% лактозы, а 1 мМ ИПТГ не изменило растворимость белка, также не изменилась растворимость белка при культивировании бактерий при различных температурах: 25°С и 37°С.
L-фенилаланин-аммоний-лиаза, клонирование, экспрессия, рекомбинантный белок, индукция, L-фенилаланин, фенилкетонурия.
L-Phenylalanine ammonia-lyase (PAL, EC 220.127.116.11) catalyzes the reaction of reversible deamination of L-phenylalanine to trans-cinnamic acid and ammonia . PAL is the key enzyme of phenylpropanoids metabolism in plants and fungi, where it is involved in biosynthesis of secondary metabolites (flavonoids, furanocoumarins, and components of cell wall) and exists in multiple isoforms . First three-dimensional structure of PAL from the yeast species Rhodosporidium toruloides has been determined at 2.1 Å resolution. Molecular weight of PAL is 76880 Da. Molecule of the enzyme contains 716 amino acid residues. Typically, optimal pH values for PAL are in the range of 8.2 to 9.0. Optimal temperature lies within the range of 35 to 55°C in function of the enzyme source .
Enzyme isolation is of interest for application as a therapeutic agent in treatment of phenylketonuria; it may be used directly as a drug in phenylketonuria therapy or in production of phenylalanine-free food [1, 2]. In addition to medicinal applications, PAL may be used in biotechnology to produce L-phenylalanine from trans-cinnamic acid .
Application of Escherichia coli strain producing recombinant L-phenylalanine ammonia-lyase as a source of the enzyme in industry seems promising [4, 5].
Escherichia coli is one of the most efficient and simple ways of large-scale production of recombinant proteins in view of the well-studied genetics of the microorganism, availability of convenient expression vector systems and host strains, simple use, low price, and high levels of target gene expression reaching 40-45% to the total cell protein [5, 6].
The aims of the present work were cloning of L-phenylalanine ammonia-lyase gene and its expression in E. coli cells, as well as characterization of the expression product.
MATERIALS AND METHODS
Reagents. Acrylamide, N',N'-methylene-bisacrylamide, sodium dodecylsulfate (SDS), bromophenol blue, glycogen, glycerol, 2-mercaptoethanol, ammonium persulfate, Tween 20, Triton X-100, Tris(hydroxymethyl)aminomethane (Tris), N,N,N',N'-tetramethylethylenediamine (TEMED), ethylenediaminetetraacetic acid (EDTA), and glucose from Serva (Germany); agarose, ethidium bromide, bovine serum albumin (BSA), deoxyribonucleoside 5'-triphosphates, mineral oil, protease K, isopropyl β-D-1-thiogalactopyranoside (IPTG), and lysozyme from Sigma (United States); yeast extract, bacto-tryptone, and agar from Dafco (United Kingdom). Phenol, lysozyme, chloroform, ethanol, acids, alkalis, and salts (analytically and chemically pure grades) from Reakhim (Russia); LB medium from Gibco BRL (United States); kanamycin sulfate from Sintez (Kurgan, Russia); restriction endonucleases NcoI and HindIII, T3 DNA ligase, Pfu-pol, and Taq-pol from Sibenzim (Russia).
Bacterial strains. Cells of E. coli strain BL21[DE3]Star (Invitrogen, United States) of F- ompT hsdSB (rB-mB-) gal dcm rne131 (DE3) phenotype containing λDe3 lyzogene and rne131 mutation in the genome were used for the target protein expression. Mutant gene rne (rne131) codes for a shortened RNAase E form, which decreases intracellular degradation of mRNA leading to increase in its enzymatic stability.
Plasmid DNA. Vector pET28a, containing promoter for T7 phage polymerase, lac-operon, ribosomal complex binding site (RBS), starting codon for translation of the cloned fragments, and a polyhistidine-tag fragment within the reading frame, was used for expression in E. coli cells. Any nucleotide sequence cloned in the vector is expressed as a protein fused with polyhistidine for convenience of further purification by immobilized metal affinity chromatography.
Gene synthesis was performed in such a way that it would contain restriction sites NcoI and HindIII for amplification and further insertion into the gene fragment of polylinker pET28a.
Amplification of the pal gene was performed by the method of polymerase chain reaction (PCR). Oligonucleotide primers were designed using the OLIGO (version 3.3) software taking into account the data on primary structure of the pal gene. To amplify the coding region of the pal gene from R. toruloides, sequence from GenBank database (X12702.1) was used as a template. At their 5'-ends, primers contained additional sequences incorporating restriction sites NcoI in case of the forward primer and HindIII, in case of the reverse one, in order to amplify the gene structural region and insert it into a polylinker of the pET28a expressing vector at relevant sites. Reverse primer was constructed so that the amplicon would not contain a stop codon and joining of the reading frames of the gene and His6 sequences would be ensured.
Polymerase chain reaction was performed in 20-50 μL solution prepared on the basis of ten-fold buffer for Taq polymerase containing deoxynucleoside triphosphate, 200 μM each, 0.5 μM of each primer, 2 mM MgSO4, 10 ng template, 2 units Taq DNA polymerase, and 0.1 units Pfu DNA polymerase. Temperature of annealing of oligonucleotides was calculated according to an empiric formula Tm = 67.5 + 34[% GC] - 395/n, where %GC = (G + C)/n, n is the number of nucleotides. Analysis of PCR products was performed by electrophoresis in 1% agarose gel.
Sanger sequencing was performed on an ABI3730xl (Applied Biosystems, United States) equipment using BigDye® Terminator v3.1 Cycle Sequencing Kit according to the manufacturers' protocols.
DNA hydrolysis by restriction endonucleases NcoI and HindIII was performed in buffer solutions under optimal conditions of incubation medium recommended for each of the enzymes by the manufacturers. Completion of hydrolysis was controlled by electrophoresis in agarose gel. Reaction mixture was purified from the reaction products using the QuickClean kit.
Isolation of DNA fragments from agarose gel. Samples of DNA were separated by electrophoresis in Tris-acetate buffer in a 0.7-0.8% agarose gel (Bio-Rad, United States) containing 0.3 μg/mL ethidium bromide and analyzed by fluorescence under ultraviolet light at 254 nm. Gel pieces containing fragments of interest were cut out and transferred into microcentrifuge tubes, then DNA fragments were eluted from the gel using the "Isolation of DNA from agarose gels" kit (Boeringer Mannheim, Germany). Sodium perchlorate was added to the tubes in the amount of 400 μL per 100 mg weight of the cut out gel. The mixture was heated to 65°C, then agarose was dissolved in salt buffer. Glass milk microbeads were introduced into the suspension at the amount of 20 µL per 100 mg of gel weight. In the salt solution, DNA contained in the gel adsorbed on the surface of the microbeads. They were washed (consecutive precipitation-resuspension) with the same salt solution once and with 70% ethanol, two times. DNA was desorbed from the beads by resuspension in TE buffer (10 mM Tris-HCl buffer, pH 7.4, 1 mM EDTA) in the amount of 50 μL per 100 mg gel weight.
Ligation. Products of hydrolysis of the vector DNA obtained as described above and the pal gene amplicon were ligated by phage T4 DNA ligase. Concentrations of the vector and gene in the reaction mixture were 5 ng/mL each. Concentration of phage T4 DNA ligase was 5 units/μL. Reaction was performed at 15°C during 24 h.
Preparation of competent E. coli cells for transformation. To prepare competent cells for the following transformation by electroporation, individual colony was grown on LB agar and placed into 5 mL LB medium. Cells were grown during night at 37°C and constant stirring (250 rpm). Two milliliter of the night culture were placed into 200 mL LB medium. Cells were grown at 37°C at constant stirring (250 rpm) till the optical density at 600 nm reached 0.6, then they were sedimented by centrifugation during 10 min at 4000 g at 4°C. Cells were washed in deionized water in the initial volume followed by centrifugation. The procedure of washing was preformed three times. After washing, cell sediment was resuspended in small volume of deionized water and centrifuged during 30 s at 5000 rpm in a microcentrifuge. Three volumes (of the cell sediment volume) of 15% glycerol solution were added to the sediment, it was resuspended and quickly frozen in liquid nitrogen. Cells ready for transformation were stored at -70°C.
Transformation of E. coli cells. Transformation of competent cells was performed by electroporation. Plasmid DNA (2 μL) at concentration of 0.3-1.0 ng/μL was added to 12 μL of competent cells, and mixed; electroporation was performed in a GVI-1 generator of high-voltage impulses in sterile cells under electrical impulse strength of 10 kV/cm and duration of 4 ms. After transformation, cells were put in 1 mL SOC medium (2% bacto-tryptone, 0.5% yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2, 10 mM MgSO4, and 20 mM glucose) and incubated for 40 min at 37°C. After incubation 10-250 μL cell suspension were inoculated into a selective LB medium containing kanamycin (25 μg/mL) to select the recombinant clones.
Induction of gene expression with IPTG. Induction of expression of genes coding for the recombinant enzyme in producer strain was performed using IPTG at final concentration of 1 mM. For this purpose, a single producer strain colony was inoculated onto standard liquid medium LB containing kanamycin at concentration of 25 μg/mL and 1% glucose and fermented at 37°C in a rotor-type temperature-controlled shaker overnight at 250 rpm. Then, after optical density at 600 nm was measured, the culture was diluted with the LB liquid medium containing kanamycin at concentration of 25 μg/mL to the optical density of 0.1 OU and fermented during 2-3 h at 37°C to the optical density of 0.6-0.8 OU. Then, the culture was divided into two equal parts: IPTG was added to one of the parts to the final concentration of 1 mM and it was fermented during 5 h at temperature of 25°C or 37°C, cell aliquots collected for analysis at certain time intervals. The aliquots were stored at -20°C.
Autoinduction of expression with 0.2% lactose. To induce autoinduction of expression according to Studier , modified PYP-5052 medium, containing 1% peptone, 0.5% yeast extract, 50 mM Na2HPO4, 50 mM K2HPO4, 25 mM (NH4)2SO4, 2mM MgSO4, 0.5% glycerol, 0.05% glucose, and 0.2% lactose, was used.
A single colony of producer strain was inoculated into PYP-5052 medium containing 25 μg/mL kanamycin. After that, the colony was fermented at 25°C or 37°C in a temperature-controlled rotor-type shaker at 250 rpm during 32, or 18 h, or till no significant change in optical density at 600 nm occurred per 1 h. Then, an aliquot of cells was collected for analysis. Aliquots were stored at -20°C.
Polyacrylamide gel electrophoresis (PAGE). Electrophoresis of cell lysates and proteins was performed according to disk-electrophoresis procedure in 10% PAGE under denaturing conditions according to Laemmli.
Destruction of bacterial cells under native conditions. Bacterial cells were destroyed under native conditions using ultrasonic treatment. Wet cell sediment obtained from 300 μL culture medium were resuspended in 30 mL buffer (50 mM Tris-HCl, pH 8.0, 20 mM EDTA, pH 8.0) and sonicated for 10 min at the amplitude of 60%, sonication duration 30 s, pause of 30 s, and a working temperature of 4°C. Destruction was controlled by inoculation of cells after sonication on a standard agarized LB medium containing kanamycin at concentration of 25 μg/mL. After sonication, cell lysate was centrifuged during 20 min at 15000 g, and the precipitate and sediment were used for analysis of p17 protein localization.
Computer methods of data analysis. Analyses of nucleotide and amino acid sequences were performed using a Lasergene v.7.1.0 (DNAStar, United States) and BioEdit v.5.0.9 software packages.
Search for homologous sequences was performed using the BLAST2 (http://www.wbi.ac.uk/blastall/) software. Comparison of amino acid sequences was performed using a ClustalW1.8 (http://www.ebi.ac.uk/clustalw/index.html) software.
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