The present invention relates to a recombinant process for the production of cytochrome C ₅₅ ι of Pseudomonas aeruginosa in the bacterial system of Pseudomonas putida.

Cytochrome C ₅₅ ι is an electron-transport hemoprotein extracted from the bacterium Pseudomonas (Ps) aeruginosa (Horio et al , 1960) Most likely its physiologic role consists of supplying electrons to nitrite reductase, a key enzyme of dissimilative denitrification which reduces nitrite to NO, but it is also able to very rapidly exchange electrons with azurin, an electron-transport protein containing copper which participates in the same and perhaps other metabolic pathways in Ps. aeruginosa. Cytochrome C ₅₅ ι is well characterized from the structural (tridimensional structure) and functional (transfer of electrons with small redox molecules and physiologic macromolecular partners) standpoint and this makes it a macromolecule most suitable for in-depth studies regarding the role that the protein matrix plays in controlling the reactivity of the prosthetic heme group and velocity and direction of the processes of electron transfer, possibly also through site-specific mutagenic studies

For this purpose, it would be most useful to have an efficient system of protein expression available, which makes it possible to easily obtain relevant quantities of native cytochrome C _55] and which can then be used also to express site-directed mutants The expression of the cytochromes of type c poses several problems i) the heme group is linked to the protein by two covalent bonds whose formation is catalyzed by a specific enzyme, ii) the cytochromes c incorporate heme after having reached a specific cellular compartment (intermembrane space of the mitochondrium for eukaryotic cytochromes, periplasmic space for the prokaryotes), towards which they are translocated by a characteristic signal sequence present in the protein, subsequently removed by a specific protease.

To date expression of this class of proteins has been undertaken in the following cases a) mutant yeast cytochromes expressed in the yeast itself by the substitution of the encoding gene in the chromosome (Margoliash et al , 1990), this approach allows to express only mutants of yeast cytochrome which maintains the function of the protein itself at an acceptable level, since the growth of the cell is linked to the presence and

activity of this cytochrome, b) expressions of cytochrome C ₅₃₀ of Th. versutus in E. coli (Ubbink et al , 1992), this approach has enabled expression of a bacterial cytochrome with reasonable yields in a heterologous system, but it requires an almost anaerobic environment in order that E. coli produce a sufficient quantity of heme This condition is experimentally not defined quantitatively and less simple to obtain compared to aerobic culture conditions, especially on a medium-large scale

These problems are solved by the present invention which relates to the expression of cytochrome C ₅₅₁ of Ps aeruginosa produced in Ps. putida This bacterial species has been selected since it grows naturally in aerobic conditions and presents limited nutritional demands, thereby enabling possible growths in the fermenter with restricted costs, in addition, it normally expresses type c cytochromes and this ensures the presence and efficiency of the systems of heme incoφoration The approach selected proved to be effective, with elevated expression levels of cytochrome C ₅₅ ι with properties indistinguishable from that of the native one and absence of cell toxicity, and which might be applied also to other bacterial cytochromes c and possibly to eukaryotes The physico-chemical characteristics of cytochrome C ₅₅₁ have in addition made it possible to develop a procedure for the purification of the recombinant protein, particularly easy, rapid and economic Thus, it constitutes a first object of the present invention a recombinant process for the production of a hemoprotein having the ability of transporting electrons and comprising the sequence of cytochrome Cj ₅ ι of Pseudomonas aeruginosa, characterized in that this hemoprotein is produced in Pseudomonas putida As mentioned, cytochrome C _55] is a hemoprotein which acts as an electron transport system, it thus finds useful applications in the diagnostic field, for example as chromogenic substrate for peroxidase or in electrochemical studies, in which it is used for the detection, measurement and control of electronic transfer reactions between oxidoreductive proteins and the electrode The hemoprotein obtained by the process of the invention includes the natural form of cytochrome C ₅₅ ι, as well as its precursors characterized by all or a portion, typically an N-terminal part, of a signal sequence which has the function of directing the cytochrome towards the cellular compartment where incorporation of heme takes place and where the protein exerts its functional activities This signal sequence can be both that ofthe native natural protein or it can be an exogenous signal sequence The hemoprotein object ofthe invention can be prepared in the following manner a) providing a host, transformed with an expression vector comprising a DNA sequence which encodes this hemoprotein, in such conditions that said hemoprotein is

expressed, and b) isolating or purifying said hemoprotein

This represents a second aspect of the present invention This approach is typically based on the construction of a nucleotide sequence which encodes the hemoprotein that is desired to be expressed and on the expression of the hemoprotein in a recombinant host organism The culture of the genetically modified organism leads to the production of the desired protein endowed with biologic activity Thus, further objects ofthe present invention consist also of, an expression vector comprising a DNA sequence which encodes a hemoprotein of the invention, a host transformed with a suitable expression vector according to the invention, and a DNA molecule of natural or synthetic origin, comprising a sequence encoding the hemoprotein according to the present invention A host in which a hemoprotein can be expressed according to the invention is prepared by transforming a host with a compatible expression vector according to the invention The expression vector can be prepared by a) enzymatically synthesizing a DNA sequence which encodes the hemoprotein of the invention starting from a part ofthe genome of Ps. Aeruginosa (Silvestrini et al , 1989); and b) inserting said DNA within an expression vector Alternatively, an expression vector can be prepared by a) isolating from the Ps. aeruginosa genome the gene coding for the cytochrome

C ₅₅ ι, and b) inserting said gene in the expression vector

Accordingly, the hemoprotein according to the present invention is prepared providing a transformed host and cultivating this host in such conditions in which the hemoprotein can be expressed

In order to produce a hemoprotein of the invention by means of the recombinant DNA technique, a gene which encodes said hemoprotein is prepared The invention includes a DNA molecule consisting essentially of the following sequence [SEQ ID

NO I]

GAA GAC CCC GAA GTG CTG TTC AAG AAC AAG GGC TGC GTG GCC

TGCCATGCCATCGACACCAAGATGGTCGGCCCGGCCTACAAGGAC GTC GCC GCC AAG TTC GCC GGC CAG GCC GGC GCG GAA GCG GAA

CTC GCG CAG CGG ATC AAG AAC GGC AGC CAG GGC GTC TGG GGC

CCG ATC CCG ATG CCG CCG AAC GCG GTC AGC GAC GAC GAG GCG

CAG ACC CTG GCG AAG TGG GTC CTG TCG CAG AAA TGA This DNA molecule can be immediately preceded by the signal sequence consisting of

ATG AAA CCG TAC GCA CTG CTT TCG CTG CTC GCC ACC GGC ACC CTG CTC GCC CAG GGC GCC TGG GCC [SEQ ID NO. 2] The coding DNA sequence typically does not contain introns

The gene encoding cytochrome C ₅₅₁ can be isolated from the operon in which it is naturally present by means of the PCR technique (Polymerase Chain Reaction, Mullis and Faloona, 1987) For this purpose oligonucleotides complementary to the 5' terminal end and 3' terminal end of the gene coding for cytochrome Cjji, can be synthesized and then used for cloning in the expression vector exploiting the restriction sites present therein Typically, the expression vector includes appropriate transcription and translation control elements, such as a promoter for the gene to be expressed, a transcription terminal site as well as translation start and termination codons The gene is presented in the correct structure so as to enable expression of the hemoprotein in a host compatible with the vector The expression vector typically comprises an origin of replication and possibly a marker gene such as a gene conferring resistance to an antibiotic As mentioned, the expression vector is used to transform a suitable host which is cultivated in such a way as to ensure that the expression occurs. The transformed host can be either a prokaryote or an eukaryote In particular, bacterial hosts can be used. A preferred batcterial host is Ps. putida. The hemoprotein that is expressed can be isolated and purified As mentioned above, the hemoprotein can include a transport signal sequence. The transport sequence is typically present at the N-terminal end ofthe hemoprotein ofthe invention A hemoprotein according to the invention can be typically used for diagnostic applications In particular, an area of intense research activity is represented by studies on metalloproteins electrons transfer reactions A particular approach to these studies consisted of the use of electrochemical methods to investigate the electron transfer reactions which take place at the interface between the electrode and the solution Thus, electrodes able to react with various types of cytochromes have been devised: in particular, the electrochemistry of cytochrome C _55] of Ps. aeruginosa has been studied through the use of gold electrodes modified with polyfunctional organic molecules and it has thus been demonstrated that cytochrome C ₅₅ ι is able to exchange electrons with this electrode (H Allen O Hill, et al ., J. Elletroanal. Chem., Ill (1987). 129-140)

This information has suggested the use of cytochrome C _Si ι in the construction of biosensors able to detect enzymatic reactions which directly involve this molecule These biosensors prove useful in assay methods for the determination of enzymes and substrates or in clinical procedures For example, they have been used for the determination of glucose present in biological fluids, in particular in diabetics (European patent application No EP 125137) or for the detection of H ₂ O ₂ in active or passive systems, in particular to control the activity of redox enzymes such as glucose-oxidase, oxalate-oxidase and cholesterol-oxidase (British patent No GB 2206414) The following examples illustrate the invention In the attached drawings

Figure 1. (A) Sequence of two oligonucleotides [SEQ ID NOs 3 and 4] used to verify the presence of the cit gene in the 3 5 kb genomic DNA fragment of Ps. aeruginosa, previously characteπzed (Siivestrini et al , 1989) Restriction map of the fragment and localization on the fragment itself of the oligonucleotide sequences and ofthe nir and cit genes

(B) DNA sequence obtained by the Sanger method using as primers the oligonucleotides 28 [SEQ ID NO 3] and 29 [SEQ ID NO 4] The zones encoding the end of the nir gene and the beginning of the cit gene are translated into aminoacids Figure 2. Oligonucleotides used for the selective amplification through PCR of cit gene starting from the fragment of 3 5 kb described in Fig 1 A and for the subsequent cloning in vector pNM185 (oligo NM-1 [SEQ ID NO 5] and NM-2 [SEQ ID NO

6])

Figure 3 Agarose gel (1 2%) analysis to verify the amplification through PCR of the cit gene.

A and B. 1 and 10 ng of pEMBL18NR amplified with primers NM-1 and NM-2 nv lambda phage digested with Hindlll as molecular weight marker

The arrow indicates the fragments amplified and their size

Figure 4 Map of the clones used to determine the sequence amplified by PCR The sequence ofthe insert citE is shown in Figure 6.

Figure 5 Map of the vector pNM185 and of the clone derived therefrom used for the expression in Ps. putida amplified by PCR

Figure 6 Sequence of the insert citE [SEQ ID NO 7] containing the gene of cit

C ₅₅₁ The ribosome binding site RBS ( ), the starting codon (ATG) and the only silent substitution detected in position 102 ofthe coding sequence are indicated

Figure 7 Autoradiography of colony hybridization for the isolation of the recombinant plasmid pNM-cit

Figure 8. Agarose gel (1 2%) analysis to verify the isolation of recombinant plasmid pTZ18-citE (1)

Figure 9 Reversed-phase high pressure liquid chromatography analysis of a purified preparation of recombinant C ₅₅₁ cytochrome The separation was carried out on a C 18 column with linear gradient of acetonitrile in water- trifluoroacetic acid and with detection at 220 nm wavelength

Figure 10. Analysis of isoelectric focusing of a purifed preparation of cytochrome

The separation was carried out on polyacrylamide gel (Immobiline dry Plate) with pH gradient from 4 to 7 The recombinant cytochrome C ₅₅ ι focuses in a homogeneous protein band with pi = 4 9 (sample 1 ) The isoelectric point was estimated by comparison with a mixture of standard Biorad proteins (sample 2)

Figure 11 Polyacrylamide gel in SDS-tricine

A and B increasing amounts of purified cytochrome C ₅₅ ι from Ps. aeruginosa; C and D increasing amounts of purified cytochrome C ₅₅₁ from Ps. putida,

M molecular weight markers (range 10-100 kDa)

Figure 12: Spectra of purified cytochrome C ₅₅ ι from Ps. putida, in the oxidized form

(dashed line) and reduced form (continuous line)

Figure 13. Spectra in the visible of purified cytochrome C ₅₅ ι from Ps. putida: the protein in the reduced form was analyzed at the three reported pH values

Figure 14. Spectra of circular dichroism of purified cytochrome C ₅₅ ι from Ps. putida: the spectra ofa solution of oxidized cytochrome are shown

Figure 15. Spectra of circular dichroism of purified cytochrome C ₅₅₁ from Ps. putida the spectra of a solution of reduced cytochrome are shown Figure 16. Spectra of circular dichroism in the deep UV region The spectrum of a solution of oxidized cytochrome is shown

Figure 17. Mass spectrometry analysis of a purified preparation of recombinant cytochrome Cjsi

Peak A with mass of 9309 97 Da corresponds to the mass calculated of cytochrome C ₅₅₁ , peak B corresponds to an adduct cytochrome Cjsi-sodium ion formed in the ionization conditions

Example 1: Identification ofthe cit gene in the operon

Recent literature data (Nordling et al , 1990, Arai et al , 1990) have reported the isolation of the gene coding for cytochrome C ₅₅₁ (cit) in an operon of the Ps. aeruginosa bacterium genome, starting from this information, the presence of this gene was checked in a fragment of genomic DNA 3 5 kb long, previously isolated

(Silvestrini et al , 1989) and containing the gene coding for the nitrite reductase enzyme (nir)

For this purpose specific oligonucleotides were synthesized (designated primers 28 [SEQ ID NO 3]and 29 [SEQ ID NO 4]) complementary to the 3' end ofthe nir gene and to the 5' end of the cit gene, respectively, and the nucleotide sequence of a segment of 200 bases was determined this sequence, compared with that reported in the literature, revealed the presence of the cit gene in the genomic DNA fragments under study

The sequence of the primers used, their localization inside the previously characterized fragment and the sequence of the segment of genomic DNA are reported in Figure 1

Methods employed

Except for the case explicitly indicated with specific references, all the methods of molecular genetics employed herein are described in Sambrook et al (1989)

1) Preparation of DNA for the determination ofthe sequence

A single-stranded DNA (ssDNA) is prepared by infecting a culture of DH5 bacterial cells with the DNA of plasmid pEMBL18-NR (Silvestrini et al , 1989), in the presence of bacteriophage Fl The infected culture produces ssDNA in the medium this is recovered by precipitation with PEG/NaCl and controlled by electrophoresis on 1 % agarose gel in TBE medium

2) Construction of primers 28 and 29

The nucleotide sequence of primer 28 [SEQ ED NO 3] was decided on the basis of previous information (Silvestrini et al , 1989) whereas that of primer 29 [SEQ ID NO 4] was designed after determination of the first segment of the new sequence. The sequences are reported in detail in Figure 1

3) Determination ofthe nucleotide sequence

The sequence was determined by the method of Sanger et al (1977) using Sequenase version 2 0 reagents (USB) both with dGTP and dITP to eliminate problems of compression deriving from the high content in GC of the DNA of Ps. aeruginosa.

The sequence reactions were resolved by means of electrophoresis on 6% urea/polyacrylamide gel

Example 2. Isolation and cloning ofthe cit gene Once the presence of the cit gene was identified in the operon, isolation of the gene from the remaining part of the operon was undertaken This operation was deemed necessary for a series of reasons

the presence of the flanking sequences (including that coding for nitrite reductase) complicates the nutritional requirements of the transformed Ps. putida strain, since the culture medium must be enriched with compounds such as KNO ₃ and lowers the yield in biomass as a result of a toxic effect of overexpression ofthe nitrite reductase; the efficiency of transcription starting from promoter Pm of expression vector pNM185 is reduced due to the position of the cit gene located at the 3' end of the nir gene, which is instead found immediately downstream ofthe promoter; the protocol for purification is also complicated as a result of co-expression of nitrite reductase

In order to obtain better yields of expression and to simplify the protocol of bacterial growth and purification o the recombinant protein, the cit gene was isolated from the context of the operon using PCR (Polymerase Chain reaction, Mullis and Faloona,

1987) Synthetic oligonucleotides (Figure 2) were designed and synthetized, suitable for subsequent cloning of the cit gene in vector pTZ18 (for the determination of the nucleotidic sequence) (Mead et al , 1986) and pNM185 (for the expression in Ps. putida) (Mermod et al , 1986)

In the case of vector pNM185, which possesses only the transcription starting signals, in addition to the sequence recognized by EcoRI, also the RBS (Ribosome Binding

Site) was inserted in the synthetic oligonucleotides ofthe cit gene needed for initiation of translation

For the cloning in the sequence vector, the same oligonucleotides described for cloning in pNM185 were used The result of the PCR reaction is a fragment of approximately 350 nucleotides, subsequently purified and inserted in the above described vectors.

The recombinant plasmids were inserted by transformation in E.coli JM109 and isolated in single colonies by hybridization with a radioactive probe corresponding to the cit gene The restriction maps of the different constructs are reported in Figures 4 and 5: the recombinant plasmids were designated pTZ18-citE and pNM-cit

To verify that the sequence of the fragment of 350 nucleotides actaully corresponded to that of the cit gene, the nucleotide sequence of this fragment was determined starting from the recombinant plasmid pTZ18-citE The sequence reported in Figure 6 [SEQ ID NO 7] is identical to that already published (Nordling et al , 1990, Arai et al , 1990) with the exception of a substitution from T to C in position 102 of the coding sequence

The sequence of this zone of the gene of Ps. aeruginosa was then verified before amplification, and the substitution resulted to be present, excluding thereby an error of polymerase during the amplification process

This mutation, which conversely has no influence on correct translation, can be 5 explained on the basis of the naturally frequent onset of silent mutations, considering that the Ps. aeruginosa strain we used for isolation of the nir and cit genes differed from that used by Nordling et al. (1990) and Arai et al. (1990)

Methods employed

Except for the cases explicitly indicated with specific references, all the methods of 0 molecular genetics employed herein are described in Sambrook et al (1989)

1) Isolation ofthe cit gene by PCR

In order to isolate the citT gene, the oligonucleotides reported in Figure 2 were designed The sequences of the oligonucleotides are reported in this figure [SEQ ID

NOs 5 and 6] and the function of each subsequence is specified (i e cleaving site for 5 restriction enzyme, linker, ribosome binding site or RBS, coding sequence of the cytochrome)

The two oligonucleotides are complementary to the 5'-terminal end and to the 3'- terminal end of the cit gene, respectively and were used for the cloning in vector o pNMl 85 and also for the cloning in the sequence vector pTZ 18

The PCR reaction was performed using 1 and 10 ng of the recombinant plasmid pEMBL18-NR in the presence of 50 pmoles of each of the two specific primers; the

Taq polymerase used is Amplitaq (Perkin Elmer Cetus Corp )

The reaction conditions were as follows 5 a) 5' at 95°C b) l' at 94°C c) l' at 58°C d) 2 ^* at 72°C

The b-d steps were repeated for 30 consecutive cycles and the mixture was 0 subsequently equilibrated at 35°C for 15'

Then, one tenth ofthe reaction was analysed on a 1.2% agarose gel in TBE

The result, shown in Figure 3, reveals the presence of an amplified fragment of approximately 350 nucleotides; this fragment was extracted from the gel and purified using the low melting point agarose procedure 2) Cloning in the expression and sequence vectors

The fragment of 350 nucleotides containing the cit gene was cloned in the following vectors, using the restriction sites described

pNM185 in EcoRI site, pTZ 18 in EcoRI site For this purpose the vectors were digested with the above enzyme (Biolabs) according to the instructions supplied by the manufacturer, digestion was controlled 5 on 1% agarose gel in TBE; in parallel, the same digestions with EcoRI were carried out on the fragment deriving from PCR. Both the DNAs (vectors and inserts) were purified by means of the low melting point agarose procedure The recombinant plasmids were obtained by means of ligation in the presence of T4 DNA Ligase (Biolabs) for 12 hours at 16°C; the constructs were then inserted by transformation of o the E coli JM 109 cells made competent by a treatment with CaCl ₂

For the isolation of the recombinant plasmid pNM-cit approximately 200 colonies deriving from the transformation were replica-plated onto LB plates containing Kanamycin (30 g/ml) and onto nylon filters (Hybond N, Amersham) The colonies were grown at 37°C for 12 hours The filters were treated with a denaturing solution 5 to denature the plasmid DNA and hybridized with cit gene isolated and labelled by the random priming technique with P -ATP The hybridization conditions used are those recommended by the manufacturer Amersham for Hybond N filters The positive colonies (see figure 7) were isolated from the replicated plate and from these colonies the plasmid DNA was prepared; the presence ofthe fragment of 350 bp o was further checked by EcoRI digestion.

For isolation of the recombinant plasmid pTZ18-citE the plasmid DNA was directly prepared starting from the colonies deriving from transformation: the presence of the insert was checked by EcoRI of digestion (see figure 8) 3) Determination ofthe complete gene cit sequence 5 The nucleotide sequence of the cit gene was determined by the Sanger method using the reagents Sequenase version 2.0 (USB); to eliminate the problems of compression deriving from the high content in GC of DNA of Ps. aeruginosa, the sequence reactions were carried out both with dGTP and 7AZA-dGTP The sequence was resolved by means of electrophoresis on 6% urea/polyacrylamide gel 0 Example 3. Expression ofthe cit gene in Ps. putida

The presence of the cit gene in the previously isolated operon (Silvestrini et al., 1989) laid the basis for carrying out the expression of the cytochrome in a bacterial species, Ps. putida, similar to that of the origin (Ps. aeruginosa) and which was already used for the expression ofthe nir gene present in the same operon (Silvestrini et al , 1992) 5 This bacterial species was chosen since, despite being correlated to the native species, it normally grows in aerobic conditions and presents limited nutritional requirements, making it possible its growth in the fermenter with limited costs In addition, the

expression of high levels of type c cytochrome in E. coli was so far carried out only for a different cytochrome c (Ubbink et al., 1992), using conditions of growth in partial anaerobiosis which limit the possibility of expansion on a large scale of the growth itself. The expression vector containing the cit gene (plasmid pNMcit), was introduced by transformation into strain of Ps. putida PaW340: enrichment in type c cytochromes was tested by differential spectra between the oxidized form and the reduced form of the iron atoms present in the hemoproteins on total cell lysed.. To verify whether the protein produced effectively corresponded to cytochrome C ₅₅ ι, a protein of apparent molecular weight of 9-kDa in SDS-PAGE gel and with electrophoretic mobility in denaturing conditions identical to that of the native cytochrome from Ps. aeruginosa, was purified to homogeneity (Parr et al. 1976). This protein was further controlled by determination of the N-terminal sequence (first 35 residues): this sequence perfectly corresponded to the sequence of the mature cytochrome C ₅₅ ι previously published (Ambler, 1963) and shows that the protein, coded at the gene level as a pre-protein with a signal sequence of 22 aminoacids needed for the translocation in the in bacterial periplasma (Nordling et al., 1990), is correctly processed also in the heterologous system of Ps. putida. Methods employed Except for the cases explicitly indicated with specific references, all the methods of molecular genetics employed are described in Sambrook et al. (1989).

1) Transformation of PaW340 with the vector pNMcit.

The above described plasmid pNMcit was purified from the clone of E.coli JM109, in which it was previously inserted, using the method of alkaline lysis. The DNA thus obtained (10-100 ng) was introduced by transformation into PaW340 cells made competent by the CaCl ₂ and MgCl ₂ method (Lederberg and Cohen, 1974); the transformants were selected at 30°C on LB medium containing 30 g/ml of kanamycin. The presence of the plasmid was controlled in the transformants extracting the DNA by the method of alkaline lysis: the clone containing the recombinant plasmid was called PaW340-pNMcit

2) Induction ofthe cytochrome expression.

Plasmid pNMcit contains the cit gene cloned under control of the promoter Pm: the expression can be induced by stimulating the transcription from this promoter with the inductor m-toluate (Mermod et al., 1986). Induction was carried out as is described below.

With a colony isolated from clone PaW340-pNMcit on solid LB medium + 30 g/ml of kanamycin, a 5 ml pre-culture was inoculated into the same liquid medium and

allowed to grow at 30°C for approximately 6-8 hours

The pre-culture was then diluted (1 100) in 100 ml of LB liquid medium + 30 g/ml of kanamycin (non-induced control) and, in parallel, on the same medium containing 0 5 nM m-toluate (induced sample), the culture was grown for 16-18 hours at 30°C The same induction assay was carried out for strain PaW340 containing only the plasmid pNM185 (without the cit gene) as control The cells were collected by centrifugation at 12000 φm for 20' at 4°C

3) Differential spectra

The cells deriving from the above described induction experiment were resuspended in 0 1 M phosphate buffer pH 7 0 (4 ml g cells) and sonicated in ice 5 x 1'

To the supernatant of the sonication recovered by centrifugation (20' at 12000 φm) potassium ferric cyanide was added to oxidize the iron of hemoproteins possibly present, this oxidized extract was divided into equal volumes in spectrophotometric cells ( 1 cm optical path) and a baseline between 400 and 500 nm was recorded In one of the samples sodium dithionite (solid) was subsequently added and the spectrum at the same wavelength was recorded

4) Purification ofthe cytochrome

The cells of the strain PaW340-pNMcit are grown on a large scale according to the methods described above for the induced samples typically, from 1 5 litre cultures ( 6 x 250 ml in 2-litres beakers) approximately 10 g of wet cells are obtained.

The purification protocol is identical to that reported by Parr et al (1976) up to gel filtration chromatography through a Sephadex G-75 column At this point the fractions with slow chromatographic mobility (in which the presence of type c cytochromes are checked spectrophotometrically) are collected and pooled, these fractions are brought to pH 3 9 with the addition of acetic acid and centrifuged at 12000 φm for 20' to eliminate any precipitates The supernatant is recovered and loaded onto a CM52 ion exchange column (Whatman) equilibrated with 50 mM ammonium acetate, pH 3 9, the column is subsequently washed with the same buffer and the protein is eluted with 50 mM ammonium acetate, pH 4.45. The eluted fractions are analysed spectrophotometrically between 250 and 650 nm to determine the purity of the sample obtained, generally, for the native cytochrome this index of purity is determined from the ratio between Abs (550-570 nm) ofthe reduced form and the Abs (280 nm) of the oxidized form this ratio must have a value of 1 14 for a 100% pure protein (Parr et al , 1976) 5) Determination of the N-terminal sequence

The analysis of the sequence was carried out using an Applied Biosystems model 470 A sequencer in gas phase supplied by an Applied Biosystems model 120A PTH

analyser for the determination of phenylthiohydantoin derivatives of aminoacids The sample is applied to glass fibre filters treated with trifluoroacetic acid, coated with polyprene and pre-washed according to the instructions given by the manufacturing company Example 4. Production of cytochrome C ₅₅₁ from Ps. putida PaW340

This example describes the procedure set to produce the cytochrome C ₅₅₁ starting from the expression system pNMcit in PaW340 applying an optimized fermentation protocol The subsequent purification procedure allows to obtain preparations of cytochrome C ₅₅₁ with a high degree of purity in only two steps The fermentation protocols of the strain PaW340-pNMcit and subsequent purification were optimized in order to be applicable on large scale and obtain a higher production yield In order to obtain significant amounts of biomass and high levels of specific expression the fermentation procedure was optimized in preliminary experiments in which the following parameters were considered composition of the culture medium, temperature, glucose concentration and partial pressure of oxygen The optimized conditions were applied later on to 10-litre fermenters and the cellular biomass obtained was used for the extraction of cytochrome Cjsi For purification, a new procedure was developed, characterized by a limited number of steps and an elevated overall yield, easily applicable on industrial scale The recombinant protein obtained according to the new purification procedure resulted to be pure from the physicochemical standpoint and functionally active Methods employed

1) Optimization ofthe fermentation conditions The best conditions for the growth of strain PaW340-pNMcit were studied by evaluating the effect of the growth conditions on the levels of biomass and on the expression of the recombinant protein so as to ensure the achievement of significant levels of volumetric productivity and specific expression (mg of cytochrome C ₅₅₁ /gram of cells) As reported in Table 1 , the best results are those obtained in the conditions of test 6, in which approximately 20 grams of wet biomass/litre of fermentation were recovered with levels of cytochrome C ₅₅ ι of approximately 1 milligram/gram of cells

Table 1. Fermentation ofthe recombinant strain PaW340-pNMcit

Operating conditions Results

Test Medium Temp Glucose % Oxygen Biomass cyt Cssi cyt c ₅₅₁

No °C % satur mg/l mg/g biomass

1 SAEM 30 - 40 118 11.8 0.10

2 TBM 30 - 0 6 4 2 0 70

3 TBM 30 0 2 5 31 4 4 0 14

4 TBM 30 0.2 0 7 0 8 0.11

5 F120 30 0 2 2 41 17 3 0 42

6 F120 32 0 4 2 21 19 3 0 92

7 F120 32 0 4 2 19 19 5 1 03

8 F120 32 0 4 2 18 17 0 0 94

The optimized conditions selected for the production are characterized mainly by the following parameters

- culture medium F120 at pH 7 - temperature of 32°C

- O ₂ level maintained at 2% of saturation

The reproducibility of the optimized fermentation protocol, which is a condition essential for its application on a preparative scale, was confirmed by replications carried out in 10-litre fermenters (tests 7 and 8 reported in table 1) and on a higher scale

2. Fermentation a) Composition ofthe culture medium

LK medium (Luria-Bertani with kanamycin)

Bactotryptone 20 g/1 Yeast extract 10 g i

NaCl 10 g/1

Kanamycin 50 mg/l

Medium F 120 Hydrolyzed casein 18 g i

Yeast extract 36 g/i

KH ₂ PO ₄ 0 58 g/1

K ₂ HPO ₄ 3 14 g/1

Glucose 4 g/1 Toluic acid 0 68 g/1

Kanamycin 50 mg/l

b) Vegetative phase

To two 1 -litre beakers containing 250 ml of LK medium, 0 5 ml of a suspension ofthe recombinant strain PaW340-pNMcit was added and the cultivation was carried out at 30°C on an oscillating shaker at 160 φm for 16 hours The bacterial culture obtained was used as inoculum for the productive phase c) Productive phase

10-litre fermenters were used containing 5 litres of F 120 medium at pH 7 inoculated with the product of the vegetative phase Fermentation was carried out at 32 1°C, with stirring adjusted to 300 φm and an airflow of 0 25 litres/litre of culture During fermentation stirring was automatically modified so as to maintain the level of oxygen at 2% saturation The productive phase of fermentation lasted for approximately 9 hours, until a final cellular growth corresponding to an optical density measured at 600 nm of approximately 20 units was obtained At the end of fermentation the cells collected by centrifugation typically corresponded to approximately 100 grams of wet weight

3. Purification

The purification operations were carried out at 4°C The biomass obtained from a 5-litre fermentation (approx 100 grams of wet weight) was resuspended in 1200 ml of 0 1 M tris-1 mM phenyl-methylsulfonylfluoride-1 mM EDTA buffer, pH 7 and the cells were disintegrated by two passages in a mechanical homogenizer (Type APV-Rainin) at the pressure of approximately 800 bar Alternatively, cell rupture could be obtained by sonication The cellular homogenate was centrifuged at 7000 g for 30 minutes, the supernatant was recovered, brought to pH 4 0 by the addition of diluted acetic acid and centrifuged again To the pink coloured supernatant 2 ml of 5% K ₂ Fe (CN) ₆ was added to oxidize cytochrome Cj ₅ ι and the solution was dialysed for approximately 4 hours against a 20 mM acetate buffer solution pH 4 (buffer A) The dialysed solution was loaded onto a chromatographic column containing a 200 ml CM-Sepharose Fast Flow resin bed (Pharmacia Biotech company) and pre-equilibrated with buffer A The column was washed with 1 litre of buffer A and subsequently eluted at the following conditions linear gradient from 100% buffer A to 35% of buffer B (20 mM sodium acetate-0 5 M NaCl - pH 4 0) in 60 minutes followed by an isocratic elution for 30 minutes with a phase consisting of 65% buffer A and 35% buffer B and a linear gradient from 40% to 100% buffer B in 30 minutes The fractions containing cytochrome C _5J ι, characterized by a slight pink colour were controlled by spectrophotometric analysis and reversed-phase high pressure hquid chromatography (RP-HPLC) and pooled on

the basis of the results of the analysis. The preparations of cytochrome C ₅₅ ι were stored at +4°C or, alternatively, lyophilized after dialysis against 10 mM ammonium acetate buffer, to obtain a preparation ofthe recombinant protein in the solid form. A typical example of the results obtained in the purification procedure is reported in the following scheme:

Purification stage Cytochrome C ₅₅₁ Yield mg %

Cellular homogenate 90 100

Supernatant after acid 76.5 85 precipitation

Pool after CM-Sepharose 58.5 65 colony

The preparations of cytochrome C ₅₅₁ obtained according to the purification protocol described presented typically a purity greater than 90% when they were examined by reversed-phase high pressure liquid chromatography (figure 9), isoelectric focusing (figure 10) and polyacrylamide gel electrophoresis (figure 1 1). Example 5. Characterization of recombinant cytochrome C ₅₅ [ in the native form Recombinant Cytochrome Cj ₅ ι obtained by the purification described in Example 4 was characterized to establish whether the protein obtained was effectively identical to the native form purified from Ps. aeruginosa.

Some of the main characteristics of this hemoprotein are common to other type c cytochromes: firstly, the prosthetic group, a poφhyrin containing an atom of iron, that is covalently bound to the protein component by means of two thioether bonds with two cysteine residues of the protein (C12 and C15, Ambler, 1963). The presence ofthese specific covalent bonds between heme and protein, missing in the other types of cytochromes (a, b, d, etc.) is in itself symptomatic of the correct conformation assumed by the protein.

In addition, the experimental measure of the mass obtainable with high precision by means of mass spectrometry techniques, constitutes a direct confirmation of the molecular structure of the recombinant cytochrome C551 with regards to the integrity ofthe polypeptide chain as well as formation ofthe covalent bond with heme. A correct tridimensional structure influences also the spectroscopic characteristics of the chromophor (heme); it, in fact, depends not only on the intrinsic properties of light

absoφtion of this part ofthe heme but also on the interaction of this chromophor with the surrounding environment, i.e the protein component

Also the information obtained in circular dichroism experiments in the visible zone can be correlated with the integrity ofthe molecule around the chromophor, which also in 5 this case is the heme

Thus the characterization of the recombinant cytochrome was carried out by electrophoresis on gel as well as through extensive spectroscopic characterization, optical and circular dichroism The following were analysed

• the presence of the covalent bond between the protein and heme, by means of o electrophoresis in denaturing conditions and heme-specific staining,

• the spectra of the recombinant cytochrome in both the oxidized and reduced form (figure 12),

• the presence of a typical spectroscopic pH-dependent variation, already largely characterized in native cytochrome C _{55 !} purified from Ps. aeruginosa (Silvestrini et 5 al , 1981 ) (figure 13),

• the spectra of circular dichroism in the visible zone, by near and deep UV for the recombinant cytochrome in the oxidized form (figures 14 and 16),

• the spectra of circular dichroism in the visible zone, by near UV for the recombinant cytochrome in the reduced form (figure 15) 0 This characterization has made it possible to establish that cytochrome C ₅₅ ι produced by the recombinant DNA in Ps. putida is in the native form and that, as regards the characteristics of the molecule analysed by us, it is indistinguishable from the native cytochrome Csji purified from Ps. aeruginosa

Methods employed 5 1) Electrophoresis

The recombinant cytochrome was analysed by electrophoresis in denaturing conditions according to the method of electrophoresis in Tricine-SDS (Schagger and Von Jagow, 1987). The gel was stained with Coomassie Blue and with a heme- specific stain according to the benzidine method (Thomas et al , 1976) 0 The result of this experiment indicates that the recombinant cytochrome possesses a heme group covalently bound, since it does not dissociate from the protein in denaturing conditions 2) Optical spectroscopy Spectroscopic analysis was performed using a Cary 219 double beam spectrophotometer (Varian) The sample of recombinant cytochrome, in 0 1 M Na/phosphate buffer, pH 7 0 was analysed between 260 and 600 nm (in the oxidized form) and between 380 and 600 nm (in the reduced form) The protein deriving by

purification is found in the oxidized form, the reduced form is obtained by the addition of solid sodium dithionite

Figure 10 shows the spectra of the oxidized and reduced form of recombinant hemoprotein both the spectra show the characteristic peaks of absoφtion at 280, 410 and 530 nm (oxidized C ₅₅ ι) and at 417, 520 and 551 (reduced C ₅₅ already described for the native C ₅₅₁ purified from Ps. aeruginosa (Horio et al , 1960) The presence of pH-dependent spectroscopic variation of the absoφtion peak at 551 nm , characteristic of native C ₅₅₁ purified from Ps. aeruginosa (Silvestrini et al , 1981), was also verified in this case the sample of recombinant cytochrome, initially at pH 5 5, was brought to pH 7 2 and to pH 10 0 by addition of 4 M NaOH To each pH value the spectrum between 500 and 600 nm was recorded- figure 13 indicates that at alkaline pH, the absoφtion peak at 551 nm shifts towards longer wavelengths, with a simultaneous decrease of the maximum value of absoφtion This characteristic spectroscopic variation (alkaline shift), absent in other type c cytochromes, is most likely correlated with the variation of the ionization state of a residue of the native cytochrome C ₅₅ ι in proximity to the prosthetic group, which is recorded by heme itself.

3) Spectroscopy of circular dichroism The experiments of circular dichroism were performed on a Jasco J500 spectropolarimeter complete with data analyser (Jasco model DP SOO N) In these experiments a solution of the recombinant cytochrome in 0 1 M Na/phosphate buffer, pH 7 0 was analysed between 200 and 600 nm (oxidized cytochrome) and between 380 and 600 nm (reduced cytochrome) Also in this case the reduction of heminic iron was obtained by the addition of solid sodium dithionite To improve the quality of the signal, the spectra in the 200-450 nm zone were accumulated 4 times and those in the 450-600 nm zone twice The result of these experiments, shown in Figures 14-16, indicate that the recombinant cytochrome is identical to native C ₅₅₁ purified from Ps. aeruginosa; also in this case the recombinant protein has the characteristic of native

4) Determination ofthe molecular mass by mass spectrometry

The preparation of cytochrome C ₅₅ ι, dissolved in methanol-water-acetic acid (50:50 0 1) at the concentration of 0 2 mg/ml was injected at a flow rate of 2 icrolitres/minute into a single quadrupole Hewlett-Packard model 5989A mass spectrometer connected to a Hewlett-Packard model 59987A electro-spray interface The experimental value ofthe molecular mass (figure 17) gave a value of 9309 97 Da corresponding, within an interval of variation of 1 Da, to the value calculated

summing the molecular weights of the apoprotein and heme covalently bound with two thioether bonds and containing an iron atom

Bibliographic references Ambler R P (1963) Biochem J 46, 1289-1301

Arai H , Sanbongi Y , Igarashi Y and Kodama T (1990), FEBS Letters 26 196-

198

Horio T , Higashi T , Sasagawa M , Kusai K , Nakai M and Okunuki K (1960),

Biochem J , 77, 194-201 Lederberg E M and Cohen S N (1974), J Bacteriol , JJ9, 1072-1074

Mead D A et al (1986), Prot Engin 1, 67

Mermod N , Ramos J L , Lehrbach P R and Tirnmis K (1986), J Bacteπol, 167.

447-454

Mullis and Faloona (1987), Meth Enzymol 155, p 335 Nordling M , Young S , Karlsson B G and Lundberg L G (1990), Febs Letters 259,

230-232

Parr S R , Barber D , Greenwood C , Phillips B W and Melling J (1976), Biochem

J , 157, 423-430

Sambrook J , Fritsch E F and Maniatis T (1989), "Molecular Cloning a Laboratory manual", second edition, Cold Spring Harbor Laboratory Press

Sanger F , Nicklen S and Coulson A R (1977), Proc Natl Aca Sci USA, 78, 5463-

5467

Schagger H and von Jagow G (1987), Anal Biochem , 166, 368-379

Silvestrini M C , Galeotti Barra D , Gervais M , Schinina E , Barra D , Bossa F and Brunoπ M (1989), Febs Letters, 254, 33-38

Silvestrini M C , Cutruzzola F , D'Alessandro R , Brunori M , Fochesato N and

Zennaro E (1992), Biochem J , 285, 661-666

Silvestrini M C , Brunori M , Wilson M T and Darley-Usmar V (1981), J Inorg

Biochem , 14, 327-338 Thomas P E , Ryan D and Levin W (1976), Analyt Biochem , 75, 168-176

Ubbmk M , Van Beeumen J and Canters G W (1992), J Bacteriol 174, 3707-3714

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT:

(A) NAME: Ministero dell'Universita e della Ricerca

Scientifica e Tecnologica

(B) STREET: Piazzale Kennedy, 20

(C) CITY: Roma

(E) COUNTRY: Italy

(F) POSTAL CODE (ZIP) : 00144

(G) TELEPHONE: +39-6-59911 (H) TELEFAX: +39-2-59912153

(ii) TITLE OF INVENTION: Recombmant process for the production in Pseudomonas putida of the cytochrome c551 of Pseudomonas aeruginosa

(iii) NUMBER OF SEQUENCES: 7

(iv) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentin Release #1.0, Version #1.30 (EPO)

(vi) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: IT MI96/A 000515

(B) FILING DATE: 15-MAR-1996

(2) INFORMATION FOR SEQ ID NO: 1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 249 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Pseudomonas aeruginosa

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: GAAGACCCCG AAGTGCTGTT CAAGAACAAG GGCTGCGTGG CCTGCCATGC CATCGACACC 60 AAGATGGTCG GCCCGGCCTA CAAGGACGTC GCCGCCAAGT TCGCCGGCCA GGCCGGCGCG 120

GAAGCGGAAC TCGCGCAGCG GATCAAGAAC GGCAGCCAGG GCGTCTGGGG CCCGATCCCG 180

ATGCCGCCGA ACGCGGTCAG CGACGACGAG GCGCAGACCC TGGCGAAGTG GGTCCTGTCG 2 0

CAGAAATGA 249 (2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 66 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "DNA leader sequence"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: ATGAAACCGT ACGCACTGCT TTCGCTGCTC GCCACCGGCA CCCTGCTCGC CCAGGGCGCC 60 TGGGCC 66

(2) INFORMATION FOR SEQ ID NO: 3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "synthetic oligonucleotide 28"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: ACGACAAGAC CCTGAAGCT 19

(2) INFORMATION FOR SEQ ID NO: 4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "synthetic oligonucleotide 29'

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: TACGCACTGC TTTCGCTGCT 20

(2) INFORMATION FOR SEQ ID NO: 5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 42 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "synthetic oligonucleotide NM-1"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: CGGCGCGAAT TCGAGGAACC GTGATGAAAC CGTACGCACT GC 42

(2) INFORMATION FOR SEQ ID NO: 6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 40 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "synthetic oligonucleotide NM-2"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: CGGCGCGAAT TCTCATTTCT GCGACAGGAC CCACTTCGCC 40

(2) INFORMATION FOR SEQ ID NO: 7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 338 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

23

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:

GAATTCGAGG AACCGTGATG AAACCGTACG CACTGCTTTC GCTGCTCGCC ACCGGCACCC 60

TGCTCGCCCA GGGCGCCTGG GCCGAAGACC CCGAAGTGCT GTTCAAGAAC AAGGGCTGCG 120

TGGCCTGCCA TGCCATCGAC ACCAAGATGG TCGGCCCGGC CTACAAGGAC GTCGCCGCCA 180

AGTTCGCCGG CCAGGCCGGC GCGGAAGCGG AACTCGCGCA GCGGATCAAG AACGGCAGCC 240

AGGGCGTCTG GGGCCCGATC CCGATGCCGC CGAACGCGGT CAGCGACGAC GAGGCGCAGA 300

CCCTGGCGAA GTGGGTCCTG TCGCAGAAAT GAGAATTC 338