Heterologous Expression of L-Amino Acid Oxidase from Calloselasma rhodostoma and
Induction of Cell Death
Dissertation
zur Erlangung des akademischen Grades des Doktors der Naturwissenschaften
(Dr. rer. nat.)
an der Universität Konstanz (Fachbereich Biologie)
Vorgelegt von
Phaneeswara Rao Kommoju Konstanz; Germany
2006
Tag der mündlichen Prüfung: 30. Juni 2006 1. Referent: Prof. Dr. Sandro Ghisla 2. Referent: Prof. Dr. Peter Macheroux
Dedicated to my parents and teachers…
i. Acknowledgements
First of all, thanks to Prof. Sandro Ghisla, for providing me the opportunity to work in his lab. I am lucky indeed to have had such a friendly and lively person as my PhD supervisor.
Prof. Peter Macheroux for his constructive criticisms and suggestions during this Ph.D. work and for critical reading of the manuscripts.
Prof. Elisa May for her kindly advices and coordination in completing the publication work on LAAO induced cell death.
Prof. Martin Scheffner for the lively discussions, suggestions and for providing the mammalian expression vectors and for the access to the cell culture facility.
Herr Dr. Thomas Kapitza for patiently teaching me the techniques in baculovirus mediated expression system.
Special thanks to Rajesh Kumar Singh and Michael Desilva for their help in cell culture experiments.
Frau Susanne Feindler-Boeckh for her day-to-day assistance in the lab, without whose help this work would have been difficult to finish.
Herr Karsten Schäfer.for his help in chromatographic techniques.
Mac-Support, Uni-Konstanz for rectifying my entire computer related problems.
Family members for their support and love.
ii. Abbreviations
α- anti- (used for antibodies)
°C degree celcius
aa(s) amino acid(s)
Ab, mAb antibody, monoclonal antibody
AIP apoptosis inducing protein
AOX alcohol oxidase
bp base pairs
cDNA complementary DNA
CIP calf intestinal phosphatase
CV column volume
Da, kDa dalton, kilodalton (molecular weight) DAAO D-amino acid oxidase
DAPI 4´-6-Diamidino-2-phenylidole DMEM Dulbecco ́s modified eagle medium
DMSO Dimethylsulfoxide DNA deoxy ribonucleic acid
e.g. for example
EDTA ethylenediamine-tetra acetic acid ELISA enzyme-linked immunosorbent assay
ER Endoplasmic Reticulum
EtBr ethidium bromide
FACS fluoresence activated cell sorting FAD flavin adenine dinucleotide
FBS fetal bovine serum
FCS fetal calf serum
g, mg, µg, ng gram, milligram, microgram, nanogram H Hour
IgG immunoglobulin IPTG Isopropyl-β-D-thiogalactopyranoside
Kbp kilo basepairs
LAAO L-amino acid oxidase L, mL, µL liter, milliliter, microliter Lpt-medium, LAAO pretreated-medium m, cm, µm, meter, centimeter, micrometer M, mM, µM molar, millimolar, micromolar
mA milli ampere
MeOH methanol
Min minute (s)
Muts, Mut+ methanol utilization slow MWCO molecular weight cut off NAN N-acetyl neuraminic acid
NP-40 nonidet P-40
O.D. optical density
PBS phosphate buffered saline
PCR polymerase chain reaction POD Peroxidase
RNA ribonucleic acid
RPM/rpm rounds per minute
RT room temperature
S, sec Second
SDS sodium dodocyl sulfate
SDS-PAGE SDS-polyacrylamide gel electrophoresis Siglecs sialic acid binding Ig superfamily lectins Sp.activity specific activity
TBE tris borate EDTA buffer TBS tris buffered saline
TCA trichloroacetic acid
UV-VIS ultraviolet-visible V volume V volt W weight
x-gal 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside YNB yeast nitrogen base
Amino acids: Unless specified, all the amino acids are generally L-isomers. D- amino acids are indicated with ´d-´prior to the amino acid (e.g. dM = d-Methionine)
Alanine Ala A Arginine Arg R Asparagine Asn N Aspartic acid Asp D Cysteine Cys C Glutamine Gln G Glutamic acid Glu E Glycine Gly G Histidine His H Isoleucine Ile I
Leucine Leu L
Lysine Lys K
Methionine Met M Phenylalanine Phe F Proline Pro P
Serine Ser S
Threonine Thr T Tryptophan Trp W Tyrosine Tyr Y
Valine Val V
iv. Table of contents
1 GENERAL INTRODUCTION...1
1.1 L-AMINO ACID OXIDASE,CHEMICAL AND PHYSICAL PROPERTIES...1
1.2 GLYCOSYLATION OF CRLAAO ...2
1.3 PHYSIOLOGICAL ROLE OF LAAO...4
1.4 LAAO GLYCOSYLATION AND CELL DEATH...5
2. CHAPTER: I......7
MOLECULAR CLONING, EXPRESSION AND SEQUENCE ANALYSIS OF CRLAAO... ...7
2.1 ABSTRACT...7
2.2 INTRODUCTION...8
2.3 MATERIALS AND METHODS...9
2.3.1 Tools ...9
2.3.2 Chemicals ...10
2.3.3 Buffers, stock solutions and media...10
2.3.4 Primers ...13
2.3.5 Bacterial strains and cell lines. ...15
2.3.6 Kits...16
2.3.7 SDS-PAGE ...16
2.3.8 Chromatography...17
2.3.9 PCR and agarose gel electrophoresis...18
2.3.10 TOPO cloning of PCR products...20
2.3.11 Colony PCR (E.coli) ...20
2.3.12 Preparation of plasmid DNA ...20
2.3.13 DNA digestion with endonucleases...21
2.3.14 DNA purification from agarose gel ...21
2.3.15 Cloning into various vectors...21
2.3.16 E.coli competent cell preparation...22
2.3.17 Transformation of competent E.coli cells ...22
2.3.18 DNA sequencing ...22
2.3.19 Construction of CRLAAO expression vectors. ...23
2.3.20 Expression of CRLAAO ...24
2.3.21 General protein related methods ...28
2.4 RESULTS...30
2.4.1 PCR amplification...30
2.4.2 TOPO® cloning ...31
2.4.3 Expression of CRLAAO in E.coli ...32
2.4.4 Expression of CRLAAO in insect cells ...40
2.4.5 Expression of CRLAAO in mammalian system ...43
2.4.6 Analysis of the C-terminal end of CRLAAO...48
2.5 DISCUSSION...51
3 CHAPTER: II...53
MOLECULAR CLONING AND EXPRESSION OF L-AMINO ACID OXIDASE FROM THE MALAYAN PIT VIPER CALLOSELASMA RHODOSTOMA IN PICHIA PASTORIS...53
3.1 ABSTRACT...53
3.2 INTRODUCTION...54
3.3 MATERIALS AND METHODS...55
3.3.1 Enzymes and chemicals...55
3.3.2 LAAO activity assay...55
3.3.3 Plasmids and strains ...56
3.3.4 Growth media ...56
3.3.5 Construction of the yeast expression plasmid... 57
3.3.6 Pichia transformation... 57
3.3.7 Pichia colony PCR... 57
3.3.8 Determining Mut phenotype ... 58
3.3.9 Expression in Muts strain of Pichia pastoris ... 59
3.3.10 Antibodies and Western blotting... 59
3.3.11 Time course of recombinant CRLAAO expression in Pichia pastoris ... 59
3.3.12 Expression of recombinant protein... 60
3.3.13 Chromatographic techniques... 60
3.4 RESULTS... 61
3.4.1 Preparation of recombinant Pichia pastoris ... 61
3.4.2 Optimization of CRLAAO expression ... 62
3.4.3 Purification and characterization of recombinant CRLAAO... 63
3.5 DISCUSSION... 65
4 CHAPTER: III ...69
MECHANISMS OF CELL DEATH INDUCTION BY L-AMINO ACID OXIDASE, A MAJOR COMPONENT OF OPHIDIAN VENOM ...69
4.1 ABSTRACT... 69
4.2 INTRODUCTION... 70
4.3 MATERIALS AND METHODS... 72
4.3.1 General chemicals, reagents and enzymes ... 72
4.3.2 Cell culture and incubation conditions... 73
4.3.4 Measurement of H2O2 production... 73
4.3.5 Viability assays and membrane alterations ... 73
4.3.6 LAAO and DAAO activity assay ... 74
4.3.7 Amino acid analysis... 74
4.3.8 Preparation of medium depleted of L-amino acids (Lpt-medium)... 75
4.3.9 Measurement of caspase activity ... 75
4.3.10 Desialylation of LAAO... 75
4.3.11 Generation of antibodies specific for LAAO... 76
4.3.12 Western blotting... 77
4.4 RESULTS... 77
4.4.1 L-amino acid oxidase (LAAO) induces different modes of cell death in Jurkat cells . 77 4.4.2 Apoptosis is induced by LAAO but not by the related amino acid oxidase DAAO ... 80
4.4.3 Desialylation counteracts the proapoptotic activity of LAAO ... 85
4.4.4 Interaction of LAAO and desialylated LAAO with Jurkat cells ... 87
4.5 DISCUSSION... 88
4.6 CONCLUSION... 92
5 SUMMARY...93
6 ZUSAMMENFASSUNG ...94
7 LITERATURE CITED ...95
8. EIGENABGRENZUNG...107
9 LIST OF PUBLICATIONS ...108
1 GENERAL INTRODUCTION
1.1 L-amino acid oxidase, Chemical and Physical Properties
L-Amino acid oxidase (LAAO) occurs in many different organisms such as bacteria (Corynebacterium [1], Proteus [2], cyanobacteria (Synococcus [3]), fungi (Neurospora crassa [4]), green algae (Chlamydomonas rheinhardtii [5]), fish [6], sea hares [7] [8], snails [9] and most prominently venomous snakes such as crotalids elapids and virapids [10]. LAAO is the only FAD-dependent oxidase found in snake venom and is thought to contribute to its toxicity, possibly through generation of hydrogen peroxide. The reaction mechanism of L-amino acid oxidase is shown in scheme. 1.
R
COO- H
NH3+
O2 H2O2 R
COO-
NH2
H2O R
COO-
O
LAAO
+ NH4+ + H+
Scheme 1: LAAO-catalyzed dehydrogenation of L-amino acids. The products of this reaction, the α-imino acids, hydrolyze spontaneously to the α-keto acid and ammonia. The reducing equivalents derived from dehydrogenation are transferred to the FAD cofactor, which is readily oxidized by dioxygen to yield hydrogen peroxide and reform (oxidized) LAAO.
LAAOs from different sources are also distinct with regard to molecular mass, substrate specificity, posttranslational modifications (glycosylation) and their regulation. This diversity suggests that LAAOs have undergone large evolutionary changes since their separation from a putative ancestral protein. As LAAO from snake venom, in particular that of Crotalus adamanteus can be easily purified [11], it
has become an attractive subject of enzymological, kinetic, and mechanistic investigations (as reviewed in [12]). The peculiar and fully reversible freeze- and pH- induced inactivation of the enzyme was the subject of interest in the 1960s and 70s [12] [13]. It can be summarized as follows: (a) inactivation occurs upon freezing of the enzyme to subzero temperatures (with a maximal effect at −20 °C) with the rate of inactivation depending on pH and buffer composition; (b) pH-induced inactivation takes place upon increasing the pH to above neutrality in the absence of monovalent ions which prevent this type of inactivation. The most favourable reactivation conditions for both inactivated forms involve heat treatment at low pH (e.g. 37 °C and pH 5 [14], [15]). The activation/inactivation process is associated with shifts of absorbance maxima of the FAD cofactor, which can be utilized to monitor the interconversions. Because the UV/visible absorbance and circular dichroism spectra appear to be different for the freeze- and pH-inactivated forms, it was suggested that these two inactive forms are structurally distinct [15]. However these observations were contradicted by the studies carried out on CRLAAO from the Malayan pit viper (Calloselasma rhodostoma) by Macheroux et.al. [16] which suggests that the two inactive forms provide similar environments for the FAD cofactor. How ever this phenomenon of interconversion of active and inactive forms of LAAO and its physiological role could not be understood and are still open to debate.
1.2 Glycosylation of CRLAAO
Macheroux et al. have reported that CRLAAO shows 83% identity to LAAOs from the Eastern and Western diamondback rattle snakes (Crotalus adamanteus and Crotalus atrox respectively). The most interesting feature among the ophidian LAAOs is the number of glycosylation sites: all three ophidian enzymes share a N- glycosylation consensus sequence in the C-terminal part (Asn361 in the CRLAAO sequence). An additional N-glycosylation site was found in CRLAAO in the N- terminal part of the protein at Asn172. The glycosylation pattern is remarkable in that it is highly homogeneous, in contrast to the case of other glycoproteins from the same origin. Studies by Macheroux and co-workers [17] have shown that the enzyme
contains up to 3.7 kDa of glycosylation per protomer. The dimeric CRLAAO contains 62 kDa protomers with three distinctive domains, (Helical, substrate binding and FAD binding domains) as shown in the Fig. 1.
Fig. 1. Crystal structure of Calloselasma rhodostoma LAAO (CRLAAO). The FAD binding domain is shown in red, the substrate binding domain in green and the helical domain in blue.
A. Structural model of the homodimeric glyco protein LAAO (Source: Geyer et.al. 2001 [17]). B. Monomeric form of CRLAAO (Source: Pawelek et. al 2000 [18]). FAD visible in the active center and the sugar moieties at Asn 172 and Asn 362 on the surface are represented as ball and stick model.
Following the elucidation of the three-dimensional structure CRLAAO [18]
the chemical entity of the glycan constituents was deduced from NMR studies [17].
The glycosylation pattern is remarkable in that it is highly homogeneous, the major component is a bis-sialylated, biantennary, corefucosylated dodecasaccharide (Fig. 2) in contrast to the case of other glycoproteins from the same origin [17]
Fig. 2. Chemical structure of the dodecasaccharide moiety as deduced by NMR-spectroscopy and mass analysis. The sugar composition is as follows: a, b, e, and i are N- acetylglucosamine residues; c, d, and h are mannose residues; f and j are galactose residues; g and k are N-acetylneuraminic acid residues and l is a fucose residue. R à a,b-hydroxyl group in the released free oligosaccharide and b-Asn172 and b-Asn361 in the glycoprotein (Source:
Geyer et.al. 2001 [17]).
An interesting possibility with regard to the homogeneity of the glycan moiety is that it is a functional requirement connected with the biological activities ascribed to LAAO. As shown in (Fig. 1), the CRLAAO carries two bisialylated oligosaccharides at its surface, one of which (Asn361) is located in direct vicinity to the channel leading to the active site of the enzyme (Fig. 3).
Putative binding of LAAO to Siglecs via its sialylated glycan moiety may then result in production of local high concentrations of H2O2 in or near the binding interface. This in turn, could lead to the oxidative damage of the siglec or other adjacent cell structural elements. This scenario is an attractive hypothesis to rationalize the biological effects observed with ophidian LAAO and would support the proposals by Suhr et al.[19] who initially suggested that LAAO might bind to the cellular surfaces of some cell lines. The antibacterial activity may proceed via an analogous mechanism.
Fig. 3. Active site channel of CRLAAO showing the orientation and substrate entry leading to the active site. (Source: Pawelek et. al 2000 [18])
1.3 Physiological role of LAAO
Although the LAAOs from various snake origins are well characterized, the physiological role of LAAOs from other species is rather obscure. Expression of LAAO from Chlamydomonas reinhardtii is induced during the deprivation of primary nitrogen source to produce ammonia. AIP (apoptosis-inducing protein) is a protein purified from a sea fish Chub Mackerel [6], reported to have LAAO activity.
It was reported that AIP was produced for natural defence when the fish (Chub mackeral) is infected with the larval nematode, Anisakis simplex. AIP was later shown to induce apoptosis in various mammalian cells including human tumour cell lines. AIP has structural and functional homology to other known L-amino acid oxidases [6]. Achacin another l-amino acid oxidase from giant African snail was shown have antibacterial activity [20]. Recently it was reported that the LAAO (apoxin 1) from Crotalus atrox induces apoptosis in human umbilical vein endothelial, human promyelocytic leukemia HL-60, human ovarian carcinoma A2780, and mouse endothelial KN-3 cells [21]. The glycosylation of apoxin I appears to be involved in the maturation and secretion of active enzyme and possibly also in the apoptosis inducing effect. The role of the enzyme in mammals has been questioned, what was once an L-amino acid oxidase of rat kidney is now an L- hydroxy acid oxidase, apparently located both in mitochondria and in peroxisomes [22]. IL-4 induced gene product, FIG1 might be having a role in host defence mechanisms also it shares a significant homology with the known LAAOs. Its expression is strikingly limited to B cells, but not T cells or mast cells [23]. Later it was shown to be the unique mammalian LAAO targeted to lysosomes, an important subcellular compartment involved in Ag processing. [24]. Thus based on these reports LAAOs in general are involved in (a), the first line of defence against the pathogens as in case of AIP of fish, Chub Mackeral, Achacin of giant African snail, and APIT in of sea Hares; and (b), in immunity as in the case of FIG1.
1.4 LAAO glycosylation and cell death
In the past few years some 25 publications have addressed the induction of apoptosis processes by LAAOs from various snake species [19, 21, 25-35]. LAAO activity appears to be, at least in part, responsible for the cytotoxic action of the venom. Following the first report by Araki et al. in 1993 that snake venom induces apoptosis [35], Suhr and Kim described in 1996 the identification of LAAO as the causative agent and the enzymatic H2O2 generation is one of the apoptosis-inducing factors [19]. However, it was also reported that cell survival was only partially recovered by simultaneous addition of catalase [19]. This has led to the concepts that
further mechanisms exist which induce or contribute to the cell death and may be mediated by posttranslational modifications, and specifically by the glycosylation of the enzyme. Suhr and Kim reported that the apoptotic cell death induced by LAAO is not due solely to the hydrogen peroxide produced by the enzymatic reaction [36].
Secondly, induction of apoptosis and hemorrhagic activity was also reported by Souza et al for a purified LAAO from a new world snake [25]. Interestingly LAAO from Eristocophis macmahoni has been reported by Voelters group [28] did not exhibit haemorrhagic effects. This again suggested that H2O2 is not the sole causative agent.
In 1997 Torii et al. reported on the isolation of another snake venom LAAO, apoxin I which induces apoptosis [21]. In the preliminary studies using FITC labelled apoxin I they reported that the enzyme accumulated in the plasma membranes of the treated cells. Later they have expressed apoxin I in 293T cells and showed that N- glycosylation is important for the secretion and its apoptosis inducing activity [27].
However contradictory to the previous report, the immunohistochemical studies in this report did not show any interaction of the LAAO to the plasma membranes.
It became important to assess the role(s) of the glycan moiety of LAAO in more detail by heterologous expression of the ophidian enzyme in a suitable host as well as by enzymatic de-sialylation and deglycosylation of the native enzyme. This thesis mainly focuses on the attempts made to obtain recombinant CRLAAO in various heterologous expression systems. In brief, the recombinant LAAO was insoluble when expressed in E.coli and very low yields were obtained when the gylcosylated active soluble form was expressed in mammalian cells. The C-terminal end of CRLAAO was analyzed to some extent inorder to infer any correlation with the diminished levels of protein secretion and localization of expressed LAAO in membrane organelles. Finally the role of sialic acids in the induction of apoptosis by CRLAAO was described in detail apart from the underlying molecular mechanisms.
Binding of CRLAAO to the cell surface by the aid of terminal sialic acid residues of the glycan moieties was analysed in detail.
2. CHAPTER: I
Molecular cloning, expression and sequence analysis of CRLAAO
• Contents of this chapter are to be published.
• All the experiments of this chapter were performed by myself.
2.1 Abstract
cDNA of LAAO gene from Calloselasma rhodostoma was cloned into various expression systems. Aim of this part of the project was to standardize heterologous expression and purification of either unglycosylated or glycosylated active CRLAAO. Attempts were made to over express either CRLAAO with N-terminal His10 tag for facilitating purification or thioredoxin tag for increasing solubility. In both cases, high yields of recombinant CRLAAO up to 60 mg/l of E.coli culture were obtained but only in insoluble inclusion bodies. The recombinant LAAO cross reacted with the antibodies developed against the native snake venom LAAO.
Attempts made to solubilize and incorporate the FAD cofactor were failed. For the expression of glycosylated soluble form of CRLAAO, eukaryotic expression systems were chosen. Poor secretion yields were observed in baculovirus expression system and mammalian cells. Western blot analysis and immunohistochemistry studies on transiently transfected H1299 and HEK cells showed that the expressed LAAO localized at the perinuclear region. Sequence of the C-terminal end was analyzed to understand the reasons for intracellular localization. Most of the snake venom LAAOs in general have a C-terminal consensus sequence DNEL which resemble ER retention sequence KDEL.
2.2 Introduction
LAAOs used for most of the biochemical, biophysical studies so far were mainly purified from their natural sources like snake venoms [16, 19, 21, 25, 26, 31, 37-41]), mucus (Snails [42]) or crude cell lysates (Gram positive bacteria, Rhodococcus opacus [43]). However the heterologous expression of various LAAOs was carried out mainly for understanding phylogenetic relations by sequence comparisons, sub cellular localization and to study the mechanisms of induction of cell death. Studies on expression of CRLAAO (Calloselasma rhodostomai CRLAAO) in E.coli, insect cell, and mammalian cell systems are described in this chapter.
Heterologous expression of some LAAOs from prokaryotes and lower eukaryotes has been reported. Expression of Rhodococcus opacus LAAO (RoLAAO in E. coli resulted in the accumulation of insoluble protein, but S. lividans was reported to be a suitable host. I should be noted that RoLAAO is of prokaryotic origin and has no glycosylation [43]. The recombinant LAAO of Lechevalieria aerocolonigenes origin could be obtained when co-over expressed with the molecular chaperonins GroEL and ES in E.coli [44]. Based on this report, attempts were made to co over express CRLAAO along with the chaperonins. Most of the recombinant LAAO was found in the inclusion bodies. Although low yields of soluble form of the expressed LAAO could be obtained from E.coli, no activity could be detected. Due to the fact that CRLAAO is a eukaryotic protein containing two intra molecular disulphide bridges and also the two consensus sequences for N- glycosylation, which might be important for the stability of the protein, these results were not surprising. Moreover attempts to obtain the unglycosylated CRLAAO by PNGase-F treatment resulted in protein aggregation. This indicates that CRLAAO cannot exist in the unglycosylated active form. Inorder to express the active glycosylated form of CRLAAO, baculovirus expression system was chosen.
Although soluble recombinant CRLAAO could be secreted into the culture medium, the poor yields discouraged further expression standardizations. Moreover the insect cultures were quite sensitive to small changes in temperature, shear forces, and infections and hence quite difficult in handling and time consuming.
As far as the research in cell biology is concerned, a detailed study of how LAAO is transported from the area of biosynthesis all the way to the final localization in the sub cellular compartments like endoplasmic reticulum (AIP) [45], Golgi (Apoxin) [21] and lysozomes (FigI) [46] has been carried out previously.
Current work focuses on understanding the role of native signal sequence and the localization of expressed CRLAAO in mammalian cells. H1299 and HEK cells were transiently transfected with mammalian expression constructs encoding CRLAAO with its native signal sequence. Expressed CRLAAO is found to be localizing at the perinuclear region as like apoxin I [21].
2.3 Materials and methods
2.3.1 Tools
All the UV-VIS absorption spectra were measured with a Kontron UVIKON 810, 930 and 933 Spectrophotometer (with thermostatically controlled cell holders).
Quarz (Helma/Müllheim) and plastic cuvettes (Greiner/Nürtingen) were used for UV and visible domains respectively. The centrifugations were performed with Sorvall RC-SB (Du Pont de Nemours/Germany), Heraus Cryofuge 8500, Eppendorf 8504R and Eppendorf Mini Spin centrifuge (Hamburg). The cultures were grown in flasks with or without baffles on the shakers. The transformed cells were disrupted by sonication (with a Sonifier Cell Disruptor B-30). Proteins were separated by an FPLC system (Pharmacia / Freiburg) along with a fractions collector Frac-100, a UV-detector, a UV-1-monitor and a recorder. Protein electrophoresis in SDS-PAGE gels was performed with a device from Biometra (Göttingen). Proteins were concentrated with a Centriprep Centrifugal Filter Unit YM-30, YM-50 or Amicon Stirred Ultra filtrations (Millipore GmbH-Eschborn, Deutschland). Protease inhibitors (Roche, Cat. 11257600). 5 kDa Filter devices (Millipore, Cat.
UFV5BC00).
2.3.2 Chemicals
All chemicals, reagents and solutions were of analytical grade and/or of highest commercially available purity. Wherever not stated the chemicals were purchased from the standard chemical companies like Roth, SIGMA, or Merck.
Microfuge tubes, centrifuge tubes, pipette tips were from Eppendorf (Hamburg), Biozym (Hess. Oldendorf) and TPP (Trassadingen, Switzerland). 96 well plates were from TPP. BioMax Light X-Ray films were from Kodak.
Pfu-Turbo DNA polymerase was obtained from Stratagene. All the restriction enzymes and the appropriate buffers were obtained from either NEB or SIGMA. For ligation, T4-DNA quick ligase kit from NEB was used. Sialidase and PNGase-F were purchased from SIGMA and NEB respectively.
2.3.3 Buffers, stock solutions and media
2.3.3.1 Buffers; Molecular biology
10x TBE: 890 mM Tris base, 890 mM boric acid, and 200 mM EDTA pH 8.0
EtBr: 10 mg/mL in H2O, storage in the dark, 4ºC TE: 10 mM Tris-HCl, 1 mM EDTA, pH 8.0
2.3.3.2 Buffers; Microbiology
All the solutions unless mentioned, were autoclaved for 20 min 121 °C. The thermolabile substances marked with * were filter sterilized. The combination of different solvents was done after cooling to room temperature or to 50 °C
Antibiotics (Stock solutions): 50 mg/mL Kanamycin*, 100 mg/mL Ampicillin*, 35 mg/mL, 100 mg/mL Zeocin*
RM-medium: 2% casamino acids, 0.2% glucose, 1mM MgCl2, 6% Na2HPO4 3%
KH2PO4, 0.5% NaCl, 1% NH4Cl, pH 7.4.
SOB-medium: 2% Peptone, 0.5% yeast extract, 10 mM NaCl, 2.5 mM KCl in water was autoclaved and added sterile 10 mM MgSO4 and 10 mM MgCl2 after cooling to room temperature.
SOC-medium *: 1 mL of 1M glucose added to 50 mL of SOB medium and filter sterilized.
CCMB 80 buffer: Dissolved 11.8 g of CaCl2y2H2O, 4 g of MnCl2y4H2O, 2 g of MgCl2y6H2O, in 10 mL of 1M Potassium acetate (pH 6.5) and 100 mL of glycerol, made up to 1 liter with water and autoclaved.
LB-medium: 170 mM NaCl, 0.5% (w/v) yeast extract, 1% (w/v) peptone, pH 7.5 (1.5
% agar for making plates). Antibiotics were added for selection of recombinants (100 µg/mL ampicillin, 50 µg/mL; kanamycin, 35 µg/mL; chloramphenicol; 100-1000 µg/mL zeocin).
YPD: 10 g/L Tryptone, 5 g/L yeast extract, 5g/L NaCl, (15 g/L Agar for making plates). For the selection of positive Pichia transformants 100 µg/mL Zeocin was added to the agar (15 g/L) containing medium.
2x YT-medium: 16 g Bacto-Tryptone, 10 g yeast extract and 5g NaCl per liter.
10x yeast nitrogen base (YNB): 134 g/L YNB with ammonium sulphate and without amino acids (stored at 4 °C)
500x biotin *: 200 mg/mL biotin 10x D: 200 g/L D-glucose
1M potassium phosphate buffer: 132 mL of 1M K2HPO4, 868 mL of 1M KH2PO4
and the pH was adjusted to 6.0 using either phosphoric acid or KOH.
MD: 1.34% YNB and 1x biotin and 2% dextrose.
Breaking buffer: 6g Na2HPO4, 372 mg EDTA, 50 mL of glycerol were dissolved in 900 mL water and pH was adjusted to 7.4 with NaOH and filled upto 1000 mL.
PBS: 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4.
PBST: PBS, 0.05% Tween 20 (5% skim milk powder added when required).
2.3.3.3 Buffers; SDS-Polyacrylamide gel electrophoresis
5x Resolving Gel buffer: 500 mL of 1.5 M Tris-Cl pH 8.8 solution filtered through 0.4 µm cellulose nitrate filter.
Stacking Gel buffer: 500 mL of 1 M Tris-Cl pH 6.8 solution filtered though 0.4 µm cellulose nitrate filter.
Acrylamide solution (AA:BAA): RotiphoreseTM Gel 40 (37.5 : 1)
Resolving and stacking gel solutions: These solutions were prepared according to the composition indicated in the table 1 shown below.
Table 1. Reagent composition for SDS PAGE.
Resolving gel (mL) Stacking gel (mL) Reagent
10 mL 50 mL 5 mL 25 mL
Acrylamide solution Water
Gel Buffer APS (10%) SDS (10%) TEMED
4.17 1.85 3.75 0.1 0.1 0.01
20.85 9.25 18.75
0.5 0.5 0.05
0.72 3.8 0.66 0.033 0.033 0.005
3.6 19 3.3 0.165 0.165 0.025 5 mL of resolving gel and 2.5 mL stacking gel were sufficient for one 8.5 x 9.5 x 1 mm gel. TEMED was normally added just before poring into the gel cassette.
10x SDS electrophoresis buffer: 30.28 g Tris, 144.3 g Glycine and 10 g SDS in 1 l water. pH should be 8.3.
6x sample buffer: 7 mL 4x TrisyCl/SDS, pH 6.8 (0.28M), 30% (v/v) glycerol, 1%
(w/v) SDS, 0.5 M DTT, 1.2 mg bromophenol blue in dd water. 1 mL aliquots were stored at -70 °C
Coomassie Staining solution: 10% acetic acid, 45% Methanol and 0.25% (w/v) Brillient Blue R in water
Destaining solution: 10% acetic acid and 45% Methanol in water 1x Western blot transfer buffer: 2.1% CAPS, 10% Methanol, pH 11.0
Posceau S: 0.1 g Ponceau S dissolved in 5 mL acetic acid and made up to 100 mL with water
100% TCA:100 g TCA was dissolved in 45.4 mL water and stored in a dark bottle at 4 °C.
2.3.4 Primers
All the PCR and sequencing primers used were obtained from Invitrogen, Karlsruhe. The primers used for amplification of specific regions from the CRLAAO cDNA are listed together with their restriction sites (under lined). The amino acid sequence of the CRLAAO coding sequence and the complementary sequences are also indicated. For better understanding, the primer sequences are provided in bold characters from 5´-3´direction.
CRLAAOS+NdeI.for: cDNA 5´amplification primer
Met Asp Val Phe Phe Met Phe Ser aa8 5´-TT CAT ATG AAT GTC TTC TTT ATG TTC TCG C -3´
3´-AA GTA TAC TTA CAG AAG AAA TAG AAG AGC G -5´
CRLAAOBamHI.rev: cDNA 3´ amplification primer 5´-TT GGA TCC TTA AAG TTC ATT GTC ATT GCT C-3´
3´-AA CCT AGG AAT TTC AAG TAA CAG TAA CGA G-5´
Leu Glu Asn Asp Asn Ser aa6
eCRLAAOS-NdeI.for: Without ER leader signal (-18 N-terminal aas) 5´amplification primer (E.coli pET expression system)
Met Ala Asp Asp Arg Asn Pro Leu Ala aa9 5´-TTC CAT ATG GCA GAT GAC AGA AAC CCT CTA GCG-3´
3´-AAG GTA TAC CGT CTA GTC TCT TTG GGA GAT CGC-5´
eCRLAAOS-Trx.for (For pBAD E.coli expression system) Met Ala Asp Asp Arg Asn Pro Leu Ala aa9 5´-CACC ATG GCA GAT GAC AGA AAC CCT CTA GCG-3´
3´-GTGG GAC CGT CTA GTC TCT TTG GGA GAT CGC-5´
bCRLAAOS-BamHI.for (Baculovirus system)
Asp Pro Ala Asp Asp Arg Asn Pro Leu Ala aa9 5´-TT CAG GAT CCG GCA GAT GAC AGA AAC CCT CTA GCG-3´
3´-AA GCT CTA GGC CGT CTA CTG TCT TTG GGA GAT CGC-5´
bCRLAAOE.coRI.rev (Baculovirus system)
5´-TTC AAG CTT AAG TTA AAG TTC ATT GTC ATT GCT C -3´
3´-AAG TTC GAA TTC AAT TTC AAG TAA CAG TAA CGA-5´
Leu Glu Asn Asp Asn Ser aa6
mCRLAAOS-BamHI.for: (Mammalian expression system)
Ala Gly Ser Ala Asp Asp Arg Asn Pro Leu Ala Gln Cys Phe aa15 5´-GCG GGA TCC GCA GAT GAC AGA AAC CCT CTA GCG GAA TGC TTC C-3´
3´-CGC CCT AGG CGT CTA CTG TCT TTG GGA GAT CGC CTT ACG AAG G-5´
mCRLAAOS+BamHI.for: Native ER signal sequence (Mammalian expression system) Ala Gly Ser Met Asp Val Phe Phe Met Phe Ser Leu Asp aa14
5´-GCG GGA TCC ATG AAT GTC TTC TTT ATG TTC TCG CTG CTG-3´
3´-CGC CCT AGG TAC TTA CAG AAG AAA TAG AAG AGC GAC GAC-5
CRLAAOS-XbaI.rev: (Mammalian and Pichia expression systems) 5´-T CTA GAT TCT AGA TTA AAG TTC ATT GTC ATT GCT C-3´
5´-A GAT CTA AGA TCT AAT TTC AAG TAA CAG TAA CGA G-5´
Leu Glu Asn Asp Asn Ser aa6
yCRLAAOS-Kex2XhoI.for: (Pichia expression system)
Leu Glu Lys Arg Glu Ala Asp Asp Arg Asn Pro Leu Ala aa9 5´-AGG CTC GAG AAA AGA GAG GCA GAT GAC AGA AAC CCT CTA GCG-3´
3´-TCC GAG CTC TTT TCT CTG CGT CTA CTG TCT TTG GGA GAT CGC-5´
CRLAAO372.rev: Control primer (Pichia colony PCR) Gly Val Ile Ile Ala Phe Gly Ile Gly Asp Asp aa11
5´-GGG GTT ATT ATA GCC TTT GGC ATT GGT GAT GAT G-3´
3´-CCC CAA TAA TAT CGG AAA CCG TAA CCA CTA CTA C-5´
The following sequencing primers were directly supplied in the Expression kits from Invitrogen for carrying out the sequencing of the expression constructs generated.
M13.for: (5´-GTA AAA CGA CGG CCA GTG-3´) M13.rev: (5´-GGA AAC AGC TAT GAC CAT G-3´) T7.for : (5´-TAA TAC GAC TCA CTA TAG GG-3´) POH.for: (5´-AAA TGA TAA CCA TCT CGC-3´)
5´AOXI.for: (5´-GAC TGG TTC CAA TTG ACA AGC-3´) 3´AOX1.rev: (5´-GCA AAT GGC ATT CTG ACA TCC-3´) α-Factor.for: (5´TAC TAT TGC CAG CAA TTG CTG C-3´)
2.3.5 Bacterial strains and cell lines.
2.3.5.1 Top10 E.coli
Genotype: F- mcrA ∆(mrr-hsdRMS-mcrBC) Φ80lacZ∆M15 ∆lacX74 recA1 araD139 ∆(araleu)7697 galU galK rpsL (Str®) endA1 nupG
One Shot® Top10 competent cells were purchased from Invitrogen, Karlsruhe. These cells were used for general cloning and plasmid amplification purposes. Top10 cells do not express the lac repressor, so IPTG does not affect the induction of protein production.
2.3.5.2 LMG194 E. coli
Genotype: F´∆ lacX74 galE thi rpsL ∆ phoA (Pvu II) ∆ara714 leu::Tn10.
This strain is resistant for tetracycline and streptomycin and can grow in glucose minimal medium. It was used for expression of CRLAAO with N-terminal thioredoxin fusion.
2.3.5.3 BL21 DE3 E.coli
Genotype: F- ompT hsdSB(rB-mB-) gal dcm (DE3)
BL21 DE3 competent cells were purchased from Invitrogen, Karlsruhe. BL21 DE3 was used as an expression system for the different LAAO variations.
2.3.5.4 Insect cell lines
Sf9 (Spodoptera frugiparda) and Hi-5 (Trichoplusia ni) cell lines for used for virus amplification and expression studies respectively.
2.3.5.5 Mammalian cell lines
The following cell lines were used for the transient transfection experiments.
Immunostaining experiments were performed to localize the expressed CRLAAO in the intracellular compartments.
H-1299 cell line: Human Lung Carcinoma cells with p53 null mutation, which pertains resistance to apoptosis. These cells were kindly provided by Prof. Martin Scheffner, University of Konstanz, Germany.
HEK cell line: Originally derived from Human embryonic kidney cells. These cells were obtained from Prof Hoffer´s lab, University of Konstanz, Germany.
2.3.6 Kits
ECL+: Pierce
QIAquick Gel extraction Kit (QIAGEN, Hilden) QIAquick PCR Purification Kit (QIAGEN, Hilden) QIAGEN Plasmid Maxi Kit (QIAGEN, Hilden) QIAGEN Plasmid Midi Kit (QIAGEN, Hilden)
QIAprep Spin Miniprep Kit, Mini Prep Kit (QIAGEN, Hilden) CIP enzyme, 10x buffer NE3 (NEB)
T4-Ligase, 10x buffer (NEB), sterile water (SIGMA)
Pfu-turbo polymerase, 10x universal buffer and DpnI (Stratagene) SuperFect Transfection Kit (Invitrogen, Karlsruhe)
pBAD Directional TOPO expression kit (Cat.No K4102-01, Invitrogen, Karlsruhe) Pichia expression kit (Invitrogen, Cat No. 1740-1)
Baculovirus mediated expression kit (Inivtrogen, Cat No. V1950-20).
2.3.7 SDS-PAGE
SDS-polyacrylamide-gel electrophoresis was performed according to Laemmli´s method. 12% polyacrylamide gels were used throughout the expression studies. Normally 50 µL samples were mixed with 10 µL of 6x SDS sample buffer, and denatured at 95 °C for 5 min then cooled to room temperature and loaded onto gels. The gels were run at constant voltage of 100 V in 1x SDS-PAGE running buffer. For visualizing the protein bands, gels were stained in Coomassie blue staining solution and destained in destaining solution.
2.3.8 Chromatography
After adjusting the pH at 25 °C, all the running buffers were filtered through 0.45 µm cellulose nitrate filters and degassed prior to application onto the columns.
Unless mentioned, the columns were washed with dd water (2-5 CV) and equilibrated with the starting buffer (Buffer A, 5 CV) before applying the protein samples. All the protein separations were carried at 4 °C unless the buffers have 8M urea. After completing the chromatography, the columns were washed with 0.2 N NaOH (2 CV), dd water (2 CV) and stored after passing 20% ethanol (2 CV)
Protein G chromatography (Ab purification)
Buffer A: 20 mM sodium phosphate pH 7.0 (Binding buffer) Buffer B: 0.1 M Glycine-HCl, pH 2.7 (Elution buffer) Buffer C: 1M Tris pH 9.0 (Neutralization buffer) Ni-NTA chromatography: Buffers were diluted to 1x before using.
8x Binding buffer: 40 mM imidazole, 4M NaCl, 160 mM Tris-Cl pH 7 .9 8x Wash buffer: 480 mM imidazole, 4M NaCl, 160 mM Tris-Cl pH7.9 4x Elution buffer: 4M imidazole, 2M NaCl, 80 mM Tris-Cl pH7.9 4x Stripping buffer: 400 mM EDTA, 2M NaCl, 80 mM Tris-Cl pH 7.9 8x Charge buffer: 400 mM NiSO4
Ion exchange chromatography:
Buffer A: 20 mM Tris-Cl pH 8.0
Buffer B: 20 mM Tris-Cl pH 8.0 + 500 mM NaCl Con-A sepharose affinity chromatography
Buffer A: PBS
Buffer B: 500 mM α-D manno pyranoside in PBS Purification of glycoproteins
Culture supernatants were collected 72 h after infection and concentrated 10 fold and incubated with Con-A sepharose beads (100 µL per 1 mL concentrated
supernatant) in an eppendorf tube for 4 hours to pool down all the glycoproteins.
Beads were washed with PBS (Buffer A) for three times with 10 min incubation periods and centrifugation (1000 rpm for 1 min). The bound glycoproteins were eluted with 200 µL of buffer B (1M α-D-mannopyranoside in PBS). Purification steps were analyzed by western blotting.
Gel filtration chromatography: 100 mM KCl, 100 mM Potassium phosphate buffer pH 6.0
2.3.9 PCR and agarose gel electrophoresis
2.3.9.1 Polymerase chain reaction
For general PCR, Taq DNA polymerase and for cloning pfu DNA polymerase was used. A standard 50 µL PCR mix consisted of 5 µL of 10X PCR buffer, 1 µL of 1 to 10 ng template DNA, 5 µL of 10 µM primers each, 2 µL of 10 µM dNTP mix (2.5 µM each of dATP, dTTP, dGTP and dCTP), 3 µL of 1.5 mM MgCl2. The standard PCR cycling conditions are represented as below.
Scheme. 2. PCR cycle parameters. The extension time was adjusted according to the length of the DNA fragment to be amplified, which is 1min/kbp in case of Taq-polymerase.
The amplification of the DNA fragments with the expected length was verified on 0.8% agarose gels. In case of only one dominating band, the PCR reactions were directly used for the TOPO® cloning experiments.
2.3.9.2 Agarose-gel electrophoresis to separate DNA fragments Agarose gel electrophoresis to separate DNA fragments according to size was carried out using gel chambers filled with 1x TBE buffer and a power supply (Biometra). To prepare the gel, agarose was suspended in 1x TBE buffer, and heated
in a microwave oven until the agarose was completely dissolved. After cooling down to approximately 40 °C the gel solution was filled into a gel cast tray equipped with a comb to create slots for filling in the DNA samples, and the agarose was allowed to solidify. After loading the gel with DNA samples in the DNA sample buffer, electrophoresis is carried out in 1x TBE as running buffer at a voltage of 120 mV, typically resulting in 90 mA of current. Due to its negative charge, DNA migrates to the anode. To visualize the DNA, the agarose gel was subsequently incubated with the DNA-intercalating fluorescent dye EtBr in 1xTBE, with gentle shaking. This was followed by a further incubation in EtBr-free 1xTBE for destaining and UV transillumination and photographic documentation of the fluorescent DNA bands.
The size of the unknown DNA fragments was verified by the use of DNA standard markers (NEB) as listed in the Table 2
Table 2. Standard DNA markers Fragments of λ DNA
E.coRI + HindIII digest (bp)
Fragments of 100 bp DNA ladder (bp)
Fragments of 2-Log DNA ladder (bp) 21,226
5,148 4,973 4,268 3,530 2,027 1,904 1,584 1,375 947 831 564
1,517 1,200 1,000 900 800 700 600 500/517
400 300 200 100
10,000 8,000 6,000 5,000 4,000 3,000 2,000 1,500 1,200 1,000 900 800 700 600 500 400 300 200 100 Note: DNA fragments shown in bold characters have increased intensity, as reference.
2.3.10 TOPO cloning of PCR products
Due to unsuccessful attempts of directly cloning the generated PCR products into the expression vectors after performing a PCR fragments were directly cloned into pCR2.1-TOPO cloning vector according to manufacturers recommendations.
Taq polymerase, which was used in PCR, adds a single deoxyadenosine to the 3’ end of PCR products. In the TA kit the linearized pCR2.1 vector provided with corresponding 3’ deoxythymidine overhangs can efficiently anneal with the PCR product. 1-4 µL of a fresh PCR product was mixed with 1 µL of the template DNA and incubated at room temperature (25 °C) for 5 min. The ligated products were transformed into T0P10F´ competent cells and screened by blue white selection on LB agar selection plates containing X-gal. 5-10 white colonies were picked for colony PCR.
2.3.11 Colony PCR (E.coli)
This method was used after all transformations into E.coli for a rapid determination of positive transformants with the CRLAAO insert integration. In all cases a vector specific forward primer and the insert specific reverse primer were used resulting in 1500 to 1600 bp fragments. The PCR reactions were slightly different from the paragraph 2.2.1.1 in that the cell material picked directly using a toothpick was used as PCR template prior to the denaturation step.
2.3.12 Preparation of plasmid DNA
Small-scale isolation of plasmid DNA was performed by using the QIAprep Spin Miniprep Kit (Qiagen) according to the manufacturer’s instructions. Basically, bacterial were lysed and the plasmids were purified by affinity chromatography.
Large-scale isolation of plasmid DNA was performed by using the QIAprep endotoxin free Maxiprep Kit according to the manufacturer’s instructions. The procedure is based on alkaline lysis of bacteria, followed by adsorption of DNA to silica gel at precisely set pH. The columns were washed with a salt solution inorder toremove RNA, proteins and low molecular weight contaminants. The plasmid DNA was eluted with a high-salt buffer and subsequently precipitated with isopropanol.
2.3.13 DNA digestion with endonucleases
The reaction conditions such as temperature, buffer, incubation time and concentration of DNA and enzyme were in accordance with the manufacturer’s instructions. To avoid inhibition of the enzyme reaction by glycerol, the volume of enzyme solution added to the reaction maintained not more than 1/10 of the total volume.
2.3.14 DNA purification from agarose gel
Purification of plasmid and DNA inserts was carried out on 0.8-2% low melting point agarose gels. EtBr stained gels were visualized on a UV trans- illuminator (265 nm). A clean scalpel was used to excise the insert bands of interest and DNA was extracted using the QIA quick gel extraction kit. Briefly, 3 volumes of buffer QG was added to 1 volume of gel and incubated at 50 °C for 10 min followed by 1 gel volume of isopropanol. The mix was applied to QIA quick column to bind DNA and washed with buffer PE. The DNA was eluted with 10 mM Tris-Cl, pH 8.5.
2.3.15 Cloning into various vectors
2.3.15.1 Directional TOPO cloning (pBAD202)
For cloning in pBAD202 expression vector, no restriction enzymes are involved. PCR product amplified using a forward primer with a 5´sequence CACC followed by ATG of the CRLAAO could be directionally cloned into the vector.
Briefly 1-2 µL of a fresh PCR product mixed with 1 µL of the vector template and transformed into TOP10 E.coli cells after 30 min incubation period at room temperature.
2.3.15.2 T4 DNA ligase mediated cloning
Preparation of DNA: The expression vectors pET16b, pPicZαCRLAAO, pMelBac- B, pcDNA3 HA were double digested using appropriate restriction enzymes. The linearized vectors were treated with calf intestinal phosphatase (CIP). This dephosphorylation of the linearized vectors prevents self-ligation. The DNA fragments of expected size were excised from an agarose gel and eluted using QIAGEN gel extraction kit. For preparation of the insert DNA plasmid preparations
of TOPO-clones with correct inserts were carried out using Qiagen plasmid (Miniprep) kit followed by digestion with appropriate restriction enzymes. The mixture was run through an agarose gel, the insert DNA was excised and eluted using Qiagen gel extraction kit.
Ligation:For ligation reaction 1U of T4 DNA ligase was added in 1:3 molar ratio of digested vector to insert DNA (0.5-1 µg total DNA) in 20 µL 1X ligase buffer mix.
The reaction was incubated at RT for 3-5 h. 5-10 µL of this reaction mix was used to transform 100 µL competent cells.
2.3.16 E.coli competent cell preparation
A single colony of E.coli of 2-3 mm diameter was dissolved in 1 mL of SOB medium by vortexing and mixed with 50 mL of SOB medium. Culture was grown at 30 °C until OD550nm reached to 0.3. Culture was centrifuged at 1000x g for 10 min at 4 °C and the cell pellet was resuspended in 1/3 volume of CCMB80 buffer. Cell suspension was incubated on ice for 20 min and then centrifuged at 11000 g for 10 min at 4°C. Now the cell pellet was resuspended in 1/12 volume of CCMB80. These competent cells were aliquoted (100 µL volumes) in 1.5 mL eppendorf tubes, frozen quickly in liquid nitrogen and stored finally at –70°C.
2.3.17 Transformation of competent E.coli cells
Competent cells were thawed on ice and mixed with 0.1-2 µL of purified plasmid DNA or with ligation mix and incubated on ice for 30 min. Subsequently the cells were heat shocked at 42 °C for exactly 45 s and incubated on ice for 2 min. The cells were added to 250-300 µL of SOC medium and incubated for 45 min at 37 °C with shaking. 10-200 µL of the transformation mix was plated onto LB agar plates with appropriate antibiotics and incubated overnight at 37 °C.
2.3.18 DNA sequencing
Sequencing of all the plasmid constructs was done by GATC-Biotech (Konstanz) with one of the sequencing primers mentioned in the Primers section (Section 2.3.5) and an insert specific reverse primer for complete coverage of the
CRLAAO sequence. Normally 30 µL (50-100 ng/µL) plasmid DNA and 30 µL (10 pmol/µL) primer in 1.5 mL eppendorf tubes were sent for sequencing.
2.3.19 Construction of CRLAAO expression vectors.
Directions for preparation of vector and designing of primers were strictly followed from the instruction manuals provided along with the expression kits.
pTOPO®CRLAAO plasmid DNA was used as template for all the PCR amplifications of CRLAAO coding sequence. Various sets of forward and reverse primers used were named according to the restriction sites attached their sequences.
After amplification the PCR product and the expression vectors were digested with the appropriate restriction enzymes and then gel purified. Vector was CIP treated to prevent self-ligation. The insert was ligated with the corresponding vector and transformed into TOP10 E.coli cells. The transformants were selected based on the antibiotic resistance conferred by the expression vector.
2.3.19.1 pET16bCRLAAO
To express unglycosylated CRLAAO with N-terminal His10-tag, a PCR product amplified using a set of forward primer (eCRLAAOS-NdeI.for) and the reverse primer (eCRLAAOBamHI.rev) was cloned into pET16b expression vector.
The transformants were selected on LB agar medium containing ampicillin. The resultant plasmid clone pET16bCRLAAO was isolated from a single transformant.
2.3.19.2 pBADCRLAAOTrx
This plasmid was constructed to express CRLAAO with N-terminal thioredoxin fusion in E.coli. A PCR product amplified using forward primer eCRLAAOS-Trx.for and reverse primer eCRLAAOBamHI.rev was cloned into pBAD TOPO expression vector. Here since ligation is independent of the restriction sites there is no significance of the restriction site BamHI in the reverse primer.
Transformants were selected on LB agar plates containing kanamycin. Plasmid DNA (pBADCRLAAOTrx) was prepared from a positive clone (LMGCRLAAO) and used for the expression studies.
2.3.19.3 pMelBacCRLAAO
To express CRLAAO in its native form with the post translational modifications such as glycosylation and disulphide bonds, Baculovrus expression system was chosen. A forward primer bCRLAAOS-BamHI.for and reverse primer bCRLAAOS-E.coRI.rev were used for PCR amplification of the coding region of CRLAAO and cloned after the Honey Bee Mellitin (HBM) secretary signal sequence in the pMelBac-B vector. Two additional amino acids (Asn and Pro) were added to the N-terminal end of the mature CRLAAO for creating an optimal HBM signal cleavage site. The transformants were selected on LB agar plates containing ampicillin and the plasmid DNA (pMelBacCRLAAO) was isolated from a single transformant.
2.3.19.4 pcDNACRLAAOS- (or S+)
For studying the effect of native secretory leader sequence and immuno staining experiments, CRLAAO was cloned in a mammalian expression vector pcCDNA-HA. Inserts were prepared by PCR amplification using the forward primer either mCRLAAOS-BamHI.for or mCRLAAOS+BamHI.for and the reverse primer mCRLAAOS-XbaI.rev. The transformants were selected on LB agar plates containing ampicillin. The resultant plasmid clones pcDNACRLAAOS- or pcDNACRLAAOS+ were purified from single transformants.
2.3.20 Expression of CRLAAO
2.3.20.1 Expression in E.coli
For cloning and screening studies of transformants, LB medium was used.
RM medium was used for the expression of CRLAAO trx in LMG194 strain. SOB medium was used for growing the cultures during transformation. 20% arabinose stock solution was used for induction of the growing culture. Culture growth was followed by observing the absorption at 600nm.
E.coli Fermentation
The E. coli cells transformed with the expression plasmids and selected on appropriate medium with antibiotics. Overnight culture was developed from a single colony in 5 mL LB containing antibiotic(s). This preculture was used for inoculating 400 mL medium taken in 2 liter Elnermeyer flasks (with 400 mL LB medium; 1 µg antibiotic/mL medium) and grown until OD600nm reached 0.8 - 1. This culture was transferred to fermentor vessel (B. Braun Biotech International fed-batch fermentor) containing 8 liters of 2x YT-medium containing 100 µg of ampicillin/mL.
Polypropylene glycol 2000 (1-2 mL/8 L) has been used as antifoaming agent.
Expression of recombinant protein was induced at OD600 1.0 with appropriate inducer, and the E.coli cells were harvested after an induction time of about 5 - 6 h with an OD600nm of 8-10. Fed-bach process has been used to obtain high cell-density Ammonium hydroxide 25% is frequently used as a nitrogen source and as a base; 1 M HCl was used for controlling pH of the medium and 95% glycerol as carbon source.
Co-over expression of CRLAAO and Chaparonins
Plasmids pET16bCRLAAO or pBadCRLAAO-Trx were transformed into competent E.coli cells harboring pGroESL plasmid and the transformants were selected on LB agar plates containing appropriate antibiotics. (kanamycin + chloramphenicol for pBAD system and ampicillin + chloramphenicol for pET system). A single transformant was used to overexpress both chaperonins, (GroES / EL) and CRLAAO.
2.3.20.2 Expression in baculovirus system Transfection ofiInsect cell lines
To prepare each transfection mixture, 1.5 mL micro centrifuge tubes containing 10 µL (0.5 µg) of the Bac-N-Blue DNA, 4 µg of pMelBac-LAAO plasmid DNA, 1 mL of grace’s Insect media (without supplements or FBS), and 20 µL of Cellfectin® liposomes mixture were added and mixed briefly by vortexing.
This transfection mixture was incubated at room temperature for 15 min. Meanwhile medium was removed from the insect cells and washed carefully by adding 2 mL of
fresh medium without supplements and FBS. Again the medium was carefully removed and the entire transfection mixture was added drop wise to 60 mm dish plates. These were incubated for 4 h with intermittent mixing to ensure uniform spreading. 1 mL of complete TNM-FH medium was added to each dish. Plates were then incubated with X-gal (150 µg/mL) at 27°C. 4 days after transfection, signs of virus production were observed. Transfected cells gained 25-30% size and nuclei appeared to fill the cells. The recombination process between the pMel-Bac transfer vector and the baculoviral DNA was confirmed by the activity of β-galactosidase that resulted in visible blue plaques. Once the transfection was successful, recombinant virus was purified by plaque assay.
Plaque assay
10 fold virus dilutions (10-1, 10-2 up to 10-8) of 2 mL each were made in complete (TNM-FH) medium. 5x105 Sf-9 cells (taken in 5 mL of medium) were seeded into 100 mm petri dishes for each viral dilution and incubated at 27°C for one h to allow the cells to attach. 1 mL of the diluted virus was added to the cells carefully and incubated at 27 °C with gentle shaking every 5 min inorder to ensure uniform spreading of virus over the monolayer. For overlaying the agar was prepared by mixing molten sea-plaque agarose (2%) and medium at 1:1 ratio, with X-Gal to a final concentration of 150 µg/ mL and maintained at 42°C. Medium containing the virus was removed from the plates and the agarose overlay was carefully applied over the monolayer. After the agarose is solidified, the plates were incubated at 27
°C under humidified conditions for 5-10 days until visible blue plaques were observed.
Baculovirus amplification
Blue plaques were visually identified and the agar plug was picked using a glass Pasteur pipette and bulb, dissolved in 1 mL medium to produce P1 viral stock.
This P1 plaque lysate was used to infect Sf-9 cells in 25 cm2 flasks with 2x106 log- phase Sf-9 cells in 5 mL complete TNM FH medium. After 6 days the supernatant containing the amplified virus and lysed cells (P2 viral stock) was collected and stored at 4 °C for further infection and expression.
2.3.20.3 Expression in mammalian systems Mammalian cell cultures and transfections
H1299 and HEK Mammalian cell lines were maintained in DMEM medium supplemented with 10% heat-inactivated fetal bovine serum in a humidified atmosphere of 5% CO2 and 95% air. The mammalian expression constructs encoding CRLAAO with or with out native signal sequence were transfected using lipofectamine 2000 (Invitrogen Cat. 11668-027) according to the manufacturer’s instructions. 24 h after transfections, the conditioned media and the cells were harvested separately. Cells were lysed by sonication and the lysates were centrifuged for 10 min at 15,000 rpm to prepare membrane and soluble fractions.
Immunohistochemistry
Cells were grown on a chamber slide glass fixed with 1:1 mixture of acetone and formaldehyde and ethanol at -20 °C. After washing with PBS for three times the slide was incubated with a 1 µL of primary antibody (α-CRLAAO or α-tubulin Ab) in 5mg of skim milk, 198 µL of PBS for 20 min at room temperature. The slide glass was washed with PBS three times and incubated with 2µL of αRabbit-IgG antibody conjugated with an appropriate fluorophore, 198 µL of PBS for 1 h at room temperature and washed with PBS three times. The slide glass was photographed through the microscope with the proper excitation wavelength.
Invitro-translation
Mammalian expression vector pCDNACRLAAO S- was added to Rabbit reticulosite lysate (Promega Cat. No. L4960) in a 50 µl reaction following the manufacturers instructions and incubated at 30 0C for 90 min. 10µL of the translation products were separated by SDSPAGE fixed by Soaking the gel in a mixture of 7% acetic acid, 7%
methanol, 1% glycerol for 5 min. Gel was dried at 80 0C under vacuum for 1 h. Gel was exposed on a X-Ray film for 4 h and developed.
2.3.21 General protein related methods
2.3.21.1 Determination of protein concentrations
Most proteins show maximal absorption at 280 nm, mainly due to their content of tyrosine and tryptophan (contaminating nucleic acids may be present which absorb strongly at 280 nm). Warbung and Christian have measured the optical density of the protein sample at 280 nm and 260 nm, and they found the next dependency:
Protein (mg/ mL) = F 5 1/d 5A280nm, where F= A280nm/A260nm
For estimating the concentration of CRLAAO immediately after the purification of CRLAAO from the acrod side fraction, the following formula was employed for the estimation of the protein concentration.
[LAAO] (mg/ml)= (Mwt 5 A460nm 5 dilution factor) / ε
Mwt: 60,000 Da for CRLAAO; A460nm: Absrorption maximum of obtained from the wavelength scan; ε: Molar absorption coefficient of protein bound FAD.
Other than the above-mentioned methods, Bradford reagent was used as the standard colorimetric assay. BSA was used for generating standard plots.
2.3.21.2 Preparation of soluble and insoluble cell fractions
For lysing cell pellets in large scale (1-20 g), the cells were harvested by centrifugation (Heraeus Cryofuge) at 6000 rpm for 30 min at 4 °C. and the wet cell paste (100-200 g) was stored at –20 °C until use. E. coli cell paste was thawed and resuspended in 30 mL 0.1 M Tris/HCl pH = 7.8 (or appropriate buffer). The cell suspension was sonicated three times for 5 min. (50 % pulsed /10 min break), and the slurry was centrifuged at 12000 rpm for 30 min at 4 °C. The pellet was resuspended in 300 mL of 0.1 M Tris and sonicated again for three times for 5 min. (50% pulsed) and centrifuged for 30 min at 12000 rpm. Supernatant was separated from the insoluble fraction, pellet. For small-scale preparation, cell pellet was thawed and resuspended in appropriate lysis buffer. Sample was then frozen in liquid nitrogen and thawed at 37°C. This freeze thawing was repeated for three times. Samples were
further sonicated (30 s each of 3 cycles) to achieve complete lysis. The cell lysates were centrifuged at 12000 rpm for 5 min and the insoluble pellet was separated from the supernatant.
2.3.21.3 Preparation of unglycosylated CRLAAO
Standard CRLAAO prepared from snake venom was used as a positive control during the experiments. 100 µL of the enzyme (100 µg/ µL) was deglycosylated a first denaturation in 11 µL 10x denaturing buffer and incubation for 10 min at 100 °C. After cooling to room temperature 14 µL NP40, 14 µL of G7 buffer and 1 µL PGNase F were added. This reaction mixture was kept at 37 °C for 1 hr, then 166 µL 6x SDS-loading buffer and 694 µL water were added (total volume 1 mL). The final concentration of deglycosylated LAAO is 10 ng/µL. CRLAAO untreated with PNGase-F was prepared in similar way but without treating PNGase- F. 10 µL (100 ng) and 1 µL (10 ng) of these standards were loaded per lane for SDS- PAGE and Western blotting analysis respectively.
