# Patent

## Title
Recombinant negative strand RNA virus expression-systems

## Abstract

This is a division of application Ser. No. 404,326, filed Sept. 7, 1989, now U.S. Pat. No. 5,701,023 which is in turn a divisional application of Ser. No. 07/333,184 filed on Apr. 4, 1989, now abandoned.

## Background
The present invention relates to recombinantly produced trichosanthin and DNA coding sequences therefore. Biochem Biophys Acta, 696:239 (1982). 1982), Chapter 3.

Trichosanthin (TCS) i a plant protein which is obtained from the Trichosanthes kirilowii root tuber. The protein, which is also known as alpha-trichosanthin (Law) and Radix trichosanthis (Kuo-Fen), is a basic, single-chain protein having a molecular weight of about 25,000 daltons. An incorrect protein sequence of TCS has been reported (Gu; Wang), and a molecular model has been derived from X-ray analysis (Pan).

It has been shown that TCS is a potent inhibitor of protein synthesis in a cell-free lysate system (Maraganore). This activity is consistent with the observed homology in amino acid sequence between TCS and the A chain of ricin, a ribosome-inactivating protein (RIP) which shows amino acid homology with a number of other RIPs, including abrin A chain (Olnes, 1982, 1987) and modeccin (Olsnes, 1982), and various single-chain ribosome-inactivating proteins, such as pokeweed anti-viral protein (PAP) (Irvin), RIPs from a variety of other plants (Coleman; Grasso; Gasperi-Campani) and the A subunit of Shiga-like toxins from E. coli (Calderwood).

TCS, or plant extracts containing TCS, have been used in China as an abortifacient agent in humans, particularly during midtrimester (14 to 26 weeks). As such, the drug has been administered by intramuscular, intravenous, or intraaminotic routes, typically at a single dose of between about 5-12 mg. The phenomenon of mid-term abortion has been attributed to the selective destruction of placental villi. Other studies indicate that the syncytiotrophoblast is preferentially affected (Hsu; Kao) and that secretion of hCG may be impaired (Xiong). TCS has also been shown to have a suppressive effect on human choriocarcinoma, and the protein appears to be able to pass the blood/brain barrier (Hwang).

It has recently been shown that TCS has a selective inhibitory effect on viral expression in human T cells and macrophages infected with human immunodeficiency virus (HIV). This is evidenced by nearly complete inhibition of HIV-derived antigen in infected cells treated with the protein, as well as selective inhibition of protein and DNA synthesis in the infected cells. Similar results were also discovered for momorcharin, a basic glycoprotein obtained from the seeds of the bitter melon plant (Falosia; Spreafico; Lin; Barbieri). These findings, and applications of the two proteins for the treatment of HIV infection, are detailed in U.S. Pat. No. 4,795,739 for "Method of Selectively Inhibiting HIV".

Particularly in view of the ability of TCS to inhibit viral expression in HIV-infected human T cells and macrophages, it would be desirable to produce a relatively pure, invariant preparation of TCS, for use as a human therapeutic agent. Methods of preparing TCS from the roots of T kirilowii have been reported (Yueng). Analysis of the purified TCS produced by earlier-disclosed known methods indicates that the protein is only partially purified, and in particular, contains hemaggultinating contaminant protein(s). A more recent purification method described in co-owned patent application for "Method of Purifying Trichosanthin", filed on even date herewith, yields a highly purified TCS preparation which is substantially free of protein contaminants, including hemaggultinating proteins.

Additionally, it would be desirable to produce TCS by means of recombinant DNA technology. Synthesis of the protein by recombinant methods would avoid the difficulty of obtaining T. kirilowii roots in fresh form, since at present the tuber roots are available only from certain regions of the Orient. Recombinant production of TCS would also avoid the problem of variations in primary amino acid sequence in TCS obtained form natural root material from different geographic areas.

Recombinant production of TCS would also facilitate the production of peptide derivatives of TCS, including bioactive peptide portions of TCS, and bioactive portions of the protein fused with functional peptides which confer, for example, enhanced target-cell specificity.

It is therefore one object of the present invention to provide a recombinant TCS protein capable of selectively inhibiting viral expression in HIV-infected human T cells or macrophages.

It is a related object of the invention to provide the coding sequence for TCS from T. kirilowii.

Still another object of the invention is to provide sets of degenerate primers corresponding to spaced amino acid regions of TCS which are homologous to spaced amino acid regions of RIPs, for use in selectively amplifying plant-derived genomic sequences which code for such RIPs.

In one aspect, the invention includes a cloned nucleic acid molecule which encodes a trichosanthin protein having the functional properties of Trichosanthes-obtained trichosanthin. The nucleic acid molecule is included in the sequence: ##STR1## where basepairs 411 to 1151 encode the mature form of TCS isolated form Trichosanthes kirilowii.

The nucleic acid of the invention may include:

(a) basepairs 411 to 1151 which encodes mature TCS from T kirilowii,;

(b) in addition to (a), basepairs 342-410, which encodes an amino terminal extension of the mature form of TCS from T. kirilowii;

(c) in addition to (a), basepairs 1152 to 1208 which encodes a carboxy terminal extension of the mature form of TCS from T. kirilowii; and

(d) a TCS coding sequence joined with a ligand peptide coding sequence, encoding a fused protein having a ligand peptide which confers cell-surface recognition properties on the fused protein.

The invention also includes the coding sequence for TCS from T. kirilowii in combination with an expression vector. One preferred expression vector construction contains a promoter, a ribosome binding site, an ATG start codon positioned adjacent the amino-terminal codon of TCS, and a stop codon positioned adjacent the carboxy terminal codon of mature TCS.

In another aspect, the invention includes a primer mixture for use in selectively amplifying a genomic fragment coding for first and second spaced regions of TCS from T. kirilowii, by repeated primer-initiated strand extension. The primer mixture includes a first set of sense-strand degenerate primers, and a second set of anti-sense primers, where each set contains substantially all of the possible coding sequences corresponding to the first and second region of known trichosanthin amino acid sequence, respectively. That is, each degenerate primer set includes at least one primer species which is effective to hybridize with the coding sequence of the corresponding amino acid region.

In a preferred embodiment, the primers in the first and second primer sets are designed to hybridize to first and second coding regions, respectively, which encode TCS amino acid sequences that are homologous in amino acid sequences to first and second amino-acid sequences in a variety of RIPs, such as ricin A chain, abrin A chain, pokeweed antiviral protein, and barley ribosome inhibitor. The two primer sets may be used to obtain genomic coding sequences for the corresponding RIPs, by repeated primer-initiated strand extension.

Also forming a part of the invention is a recombinant trichosanthin protein having the functional properties of mature trichosanthin (a) derived from T. kirilowii and (b) having the sequence: ##STR2##

The recombinant TCS protein may further include an amino-terminal extension having the sequence: ##STR3## and/or a carboxy-terminal extension having the sequence: ##STR4##

The invention further includes a recombinant process for the production o a trichosanthin protein having the functional properties of Trichosanthes-obtained TCS. This recombinant process involves inserting a DNA sequence encoding the TCS protein into an expression vector, transforming a suitable host with the vector, and isolating the recombinant protein expressed by the vector.

These and other objects and features of the invention will become more fully apparent when the following detailed description of the invention is read in conjunction with the accompanying drawings.

FIG. 1 shows the amino acid sequence of mature TCS isolated from T. kirilowii as determined herein (upper line) and as reported previously (lower line);

FIG. 2 illustrates the steps in the method used to obtain cloned TCS coding sequences;

FIGS. 3A and 3B show the DNA sequence from an amplified genomic fragment containing a portion of the TCS coding sequence, and the corresponding amino acid sequences in the three possible reading frames in both directions;

FIG. 4 shows the nucleotide sequence of the TCS coding region from T. kirilowii and adjacent 5'- and 3'-end sequences;

FIG. 5 illustrates the steps in the method used to express mature TCS in a bacterial system;

FIG. 6 shows plots of percent inhibition of HIV antigen (p24) production as a function of culture concentration of plant-derived TCS (open-boxes) and rTCS (closed boxes);

FIG. 7 shows plots of percent inhibition of .sup.3 H-leucine incorporation into trichloroacetic acid precipitable protein as a function of concentration of plant derived TCS (open boxes) and rTCS (closed boxes) in a cell free rabbit retriculocyte lysate protein synthesizing system.

FIG. 8 illustrates the steps in a method for producing a fused TCS protein containing a CD4+ peptide moiety; and

FIG. 9 compares the amino acid sequence of TCS with those of exemplary RIPs.

The terms below have the following meanings as used herein:

A "trichosanthin protein" is a protein having at least about 90% amino acid sequence identity with alpha-trichosanthin obtained from T. kirilowwii.

A trichosanthin protein has the functional properties of Trichosanthes-obtained trichosanthin if it has (a) the ability to selectively inhibit expression of HIV-antigen in HIV-infected T-cells or monocyte/macrophages, and/or (b) protein-synthesis-inhibitory activity.

This section describes methods for obtaining a genomic region containing the coding sequence for TCS from T. kirilowii, and for expressing mature TCS protein in a bacterial expression system.

TCS was purified by a novel method which is detailed in co-owned patent application for "Purified Trichosanthin and Method of Purification", filed on even date herewith, and outlined in Example 1. The protein was at least about 98% pure as judged by HPLC and gel electrophoresis analysis.

The primary amino acid sequence of the purified trichosanthin was determined under contract with the Protein Chemistry Services at Yale University School of Medicine. The sequence is shown in FIG. 1 (upper line) along with the previously published sequence (lower line) of TCS (Gu; Wang). Variations between the two sequences are noted by double underlining.

As seen from FIG. 1, the present sequence differs substantially from the published sequence. Most significant, as compared to the published sequence, the present TCS sequence lacks a block of 10 amino acids at position number 70 and contains an additional sequence of 21 amino acids at position number 222. The present sequence agrees closely with X-ray diffraction data on crystalized TCS, and resolves inconsistancies between X-ray diffraction data and the previously published TCS sequence. The new sequence, particularly including the 21-amino acid addition, also provides greater sequence homology with a number of RIPS, such as ricin A chain and abrin A chain (see below) than the earlier published sequence.

FIG. 2 outlines the steps described below for obtaining the complete coding sequence of TCS from T. kirilowii. The actual procedure used is given in Example 2.

With reference to the figure, genomic DNA isolated from T. kirilowii is mixed with at least two sets of degenerate primers in a reaction mixture designed for carrying out selective amplification of a TCS coding sequence.

In preparing the sets of degenerate primers, two spaced amino acid regions of TCS were selected for coding sequence targeting. The two amino acid sequences which were selected are overlined in FIG. 1 and relate to a 35-mer degenerate primer for the sequence denoted A and to a 32-mer degenerate primer for the sequence denoted B.

Each set of degenerate primers were designed such that at at least one primer sequence is effective to hybridize with the DNA sequence coding for the corresponding amino acid sequence. Deoxyinosine nucleotides were incorporated in order to generate probes longer than 20 nucleotides of manageable complexity (Ohtsuka; Takahashi). One of the two primer sets is designed for hybridization with the anti-sense strand of one coding region, and the other primer set, for hybridization with the sense strand of the second coding region.

The primer set corresponding to the 35-mer includes 128 isomers and is of the general sequence: ##STR5## where bases placed in parantheses indicate a mixture and I is inosine. This set is designated MPQP-1, and was designed for binding to the anti-sense strand of the TCS coding region. The other two primer sets, designated MPQP-2 and MPQP-3, each consist of 128 isomers and together comprise all potential coding sequences of the 32-mer and are of the general sequences: ##STR6## respectively. They were designed for binding to the sense strand of the TCS coding region, and were typically used in a primer mixture designed MPQP-2/-3.

A DNA amplification reaction was carried out by repeated primer initated strand extension, using a commercially supplied kit (Perkin-Elmer/Cetus) and according to methods supplied by the manufacturer as outlined in Example 2. The product of the DNA amplification step was isolated by agarose gel electrophoresis, and by polyacrylamide gel electrophoresis, with detection by ethidium bromide fluorescence and/or autoradiography. A major product of about 255 base paris was detected.

FIGS. 3A and 3B show the DNA sequence of the amplified material, and the amino acid sequences corresponding to all three reading frames in both directions. The underlined translation shows a sequence that is homolgous to amino acids 128 through 163 in TCS. This sequence is within the region predicted to be amplified and configured that a TCS or TCS-like coding region was amplified.

Southern blot analyses were performed on the DNA prepared from the plant tissue to assess the organization and the complexity of TCS genes in the total DNA background. The Southern blots were probed separately with .sup.32 P-labelled MPQP-1 and MPQP-2/-3. The results (not shown) suggested that there might be several TCS-related genes, and that the overall complexity of the plant genome is on the order of a mammalian genome and could be effectively screened using standard lambda-phage banks.

With continued reference to the method outlined in FIG. 2, the amplified coding sequence from above was used as a probe to identify one or more T. kirilowii genomic library clones containing TCS coding sequences. The genomic library clones were prepared and probed conventionally, as outlined in Example 2. Two clearly positive plaques were picked, amplified and converted to plasmids, according to protocols supplied by the manufacturer of the cloning system. One clone, designated pQ21D, contained an approximate 4kb insert; the other, designated pQ30E, contained an approximate 0.6 kb insert. The pQ21D vector has been deposited with The American Type Culture Collection, 12301 Parklawn Drive, Rockville, Md., 20852, and is identified by ATCC No. 67907. Partial sequence analysis showed that the 4 kb insert contained sequences that coded for a protein having substantially the same amino acid sequence shown for plant-derived TCS in FIG. 1. The 0.6  kb insert was found to contain sequences encoding a peptide homologous to, but not identical with, plant-derived TCS.

The complete sequence for the insert of pQ21D was determined and is shown in FIG. 4, along with the corresponding amino acid sequence of TCS. As seen in the figure, the sequence encodes a protein that contains a continuous amino acid sequence identical to that of plant-derived TCS except for two conservative changes--a Thr for a Ser substitution at amino acid position 211 and a Met for a Thr substitution at position 224.

The minor differences between the two sequences are presumably related to variations between different T. kirilowii strains. The purified TCS was obtained from T. kirilowii roots from the Canton region of China; the genomic DNA was obtained from T. kirilowii leaves from Korea.

These conservative sequence variations illustrate strain-related DNA sequence variations which result in functionally equivalent trichosanthin proteins.

A comparison of the amino acid sequence of mature plant-derived TCS (FIG. 1) and that encoded by the DNA in FIG. 4 shows that TCS is likely produced as a secreted protein that undergoes post-translational processing at both the amino and carboxy ends. Specifically, nucleotides 342 through 410 code for a putative secretory signal peptide having the sequence: ##STR7## and nucleotides 1152 through 1208 code for a putative carboxy terminal extension that is not present in the mature protein, and which has the sequence: ##STR8##

Although the role of the carboxy-terminal extension has not yet been determined, it is possible that this peptide functions to neutralize the ribosome inhibiting activity of the peptide prior to cellular secretion.

According to one aspect, the invention includes a nucleic acid which encodes for a trichosanthin protein which has the functional properties of Trichosanthes-obtained TCS. The nucleic acid preferably has the sequence shown in FIG. 4, where basepairs 411-1151 of the sequence code for mature TCS from T. kirilowii. The nucleic acid of the invention may include:

(a) basepairs 411 to 1151 which encodes mature trichosanthin from T. kirilowii;

(b) in addition to (a), basepairs 342-410, which encodes a putative amino terminal extension of the mature form of trichosanthin from T. kirilowii;

(c) in addition to (a), basepairs 1152 to 1208 which encodes a putative carboxy terminal extension of the mature form of trichosanthin from T. kirilowii; and

(d) a TCS coding sequence joined with a ligand coding sequence, encoding a fused protein having a ligand peptide which confers cell-surface recognition properties on the fused protein.

Recombinant TCS was produced using the above TCS coding sequence, following the steps outlined in FIGS. 4 and 5, and described in Example 5. With reference to FIG. 5, plasmid pQ21D from above was digested with EcoRI and NcoI, releasing a 1.2 kb fragment insert containing the complete coding sequence for TCS. This TCS-coding fragment was cloned into plasmid pKK233-2 which was previously digested with EcoRI and NcoI. After replication the recombinant plasmid, designated pQ21D/pKK233-2, was divided into two samples. One sample was digested with EcoRI and SalI, and and the second sample with SalI and NcoI to generate an EcoRI/SalI amino portion fragment and a SalI/NcoI carboxy portion fragment. The two fragments were cloned into M13 phage vectors for site specific mutagenesis, to place a NcoI site containing an ATG start codon at the amino terminal end of the mature TCS coding sequence, and a double TAA translation stop sequence plus a HindIII cloning site after the carboxy end of the mature sequence, as illustrated in FIG. 5.

The modified sequences were excised, and cloned into a pKK233-2 expression vector (Pharmacia) which contains a synthetic trp/lac promoter positioned appropriately ahead of a ribisome binding site that is also positioned appropriately ahead of an ATG start codon contained within an NcoI site. Several clones were characterized and verified to contain the modified insert in the correct orientation. The DNA sequences of the modified regions were directly verified for one clone, designated pQR19.

More generally, the pQR19 expression vector is exemplary of a TCS coding sequence operatively placed in an expression vector for TCS expression in a suitable host. In a preferred embodiment, and as exemplified by pQR19, the expression vector construction contains a promoter, a ribosome binding site, and an ATG start codon positioned before and adjacent the amino terminal codon of mature TCS, and a stop codon positioned after and adjacent at the carboxy terminal codon of mature TCS.

For expression of recombinant TCS (rTCS), plasmid pQR19 and similar clones were propagated in an appropriate E. coli host strain that carries a lacIq gene for regulation of the synthetic trp-lac promoter. The host strain XL-1 Blue (Bullock) was employed. Its relevant genotype is recAl, endAl, gyrA96, thi, hsdR17 (rk--, mk+), supE44, relAl, .lambda.-, lac-[F', proAB, lacIqZ.DELTA.M15, Tn10 (tet.sup.R)]. Induction of promoter activity may be achieved by adding 5 mM IPTG (isopropylthio- galactoside). Under culture conditions described in Example 3, cells carrying pQR19 and similar plasmids were induced and, at a selected cell density, the cells were harvested and disrupted by sonication. Aliquots of total cell material, of material pelleted at 15,000.times.g for 5 min, and of material remaining in solution at 15,000.times.g for 5 min were analyzed by polyacrylamide gel electrophoresis and subsequently by Western blot analysis. The Western blot was probed with rabbit anti-TCS sera.

The results showed an immunoreactive product that comigrated with authentic TCS in the total cell and soluble cell fractions from pQR19/XL1-blue induced cells, but not in the insoluble fraction from the same cells, nor in any fraction from pKK233-2 (vector)/XL1-blue induced cells, i.e., cells containing the pKK233 expression vector without the TCS coding insert.

The pQR19 expression vector which contains the TCS coding sequence, and which expresses rTCS in a suitable bacterial host has been deposited with The American Type Culture Collection and is identified by ATCC No. 67908.

Clarified cell extract material was fractionated using the steps described in Example 1, yielding rTCS with a purity, as judged by gel band staining with Coomassie blue on SDS polyacrylamide gels, of greater than 90%. About 70 mg of purified rTCS were obtained from nine liters of culture.

The rTCS protein produced is exemplary of an rTCS protein derived from the amino acid sequence shown in FIG. 4. More generally, the rTCS protein of the invention includes a recombinant protein containing the entire amino acid sequence for mature TCS, as described above, and a recombinant TCS protein containing an amino-terminal extension having the sequence: ##STR9## and/or a carboxy-terminal extension having the sequence: ##STR10##

The invention thus further includes a recombinant process for the production of a trichosanthin protein having the functional properties of Trichosanthes-obtained trichosanthin. The method includes the steps of inserting a DNA sequence encoding said protein into an expression vector, transforming a suitable host with the vector, and isolating the recombinant protein expressed by the vector.

In one preferred embodiment, the expression vector is pQR19 and the host is E. coli.

As previously described in above-cited U.S. Pat. No. 4,795,739, TCS obtained from T. kirilowii is a potent and selective inhibitor of HIV antigen expression in HIV-infected T cells and monocyte/macrophages. The inhibitory effect of rTCS on expression of HIV-specific antigens in HIV-infected T cells can be demonstrated as follows: Acutely HIV-infected human T cells were treated with varying concentrations of rTCS. After four days culture, the amount of HIV p24 antigen present in cell free culture supernatants was quantitated using a commercially available antigen capture immunoassay (Coulter). Inhibition was determined by comparison of results for treated cultures and untreated cultures.

The viral inhibition studies detailed in Example 4A compared the inhibitory activity of plant-produced TCS with the above rTCS protein. The plots in FIG. 6 show percent inhibition of p24 HIV antigen production as a function of culture concentration of plant derived TCS (open boxes) and rTCS produced as above (open boxes). As seen, both proteins gave substantially the same level of inhibition at higher protein concentrations, although the plant-derived protein was more effective at the lowest protein concentrations.

Also, as mentioned above, it has been shown that plant-produced TCS is a potent inhibitor of protein synthesis in a cell-free lysate system. The protein-synthesis inhibitory properties of both plant-produced TCS and rTCS were compared in a reticulocyte lysate system, as outlined in Example 4B. The plots in FIG. 7 show percent inhibition of .sup.3 H-leucine incorporation as a function of concentration of plant-derived TCS (open boxes) and rTCS (closed boxes) in the reticulocyte system. The plots show that both plant-produced and recombinant TCS have substantially the same specific protein synthesis inhibitory activity.

In another aspect, the invention includes TCS fused at its amino or carboxy end with a ligand peptide to form a fused ligand/TCS protein. The TCS making up the fused protein is preferably rTCS or bioactive portion thereof, as described above.

Where TCS is used to inhibit viral expression in HIV-infected human cells, the protein may be advantageously fused with a soluble CD4 peptide, which shows specific binding to the HIV-related gp120 antigen present on the surface of HIV-infected cells (Till), or with a monoclonal antibody specific against an HIV-specific cell surface antigen.

The fused TCS protein may be formed by chemical conjugation or by recombinant techniques. In the former method, the peptide and TCS are modified by conventional coupling agents for covalent attachment. In one exemplary method for coupling soluble CD4 to TCS, recombinant CD4 (rCD4) is derivatized with N-succinimidyl-S-acetyl thioacetate (Duncan), yielding thiolated rCD4. The activated CD4 compound is then reacted with TCS derivatized with N-succinimidyl 3-(2-pyridyldithio) propionate (Cumber), to produce the fused protein joined through a disulfide linkage.

As an alternative method, recombinant TCS (rTCS) may be prepared with a cysteine residue to allow disulfide coupling of the rTCS to an activated ligand, thus simplifying the coupling reaction. The TCS expression vector used for production of rTCS can be modified for insertion of an internal or a terminal cysteine codon according to standard methods of site-directed mutagenesis.

In a preferred method, the fused protein is prepared recombinantly using an expression vector in which the coding sequence of the fusion peptide is joined to the TCS coding sequence. FIG. 8 illustrates the construction of an exemplary expression vector for a fused TCS/CD4 protein.

Briefly, an EcoRI-StuI DNA fragment containing the coding region for the first 183 amino acids of mature CD4 peptide (Maddon) is inserted into an M13MP19 phage between SmaI and EcoRI sites and the vector, in a single-strand form, is then subjected to primer mutagenesis. Specifically, the amino-terminal portion of the CD4 gene is modified with primer MP101 (5'-CCAGCAGCCATGGAGGGAAACAAAG -3'); and the carboxy portion of the gene is modified with primer MP102 (5'-CATCGTGGTGCTAGCTCCACCACCACCACCACCACCACCACCACCCATGGAGGC ATGCAAGCTTG -3'). These modifications place an NcoI site containing an ATG start codon at the beginning of the mature CD4 peptide coding sequence, and a string of proline codons terminating at an NcoI cloning site after amino acid 180 in the CD4 sequence, as illustrated in FIG. 8.

The NcoI fragment from the phage vector is inserted into the pQR19 expression vector from above previously cut with NcoI. Successful recombinants are confirmed by restriction analysis for proper orientation of the CD4 sequence insert.

An expression vector formed as above, and designated pQR19/CD4 in FIG. 8, contains (a) a synthetic trp/lac promoter positioned appropriately ahead of a ribosome binding site that is also positioned appropriately ahead of an ATG start codon contained within an NcoI site, (b) the CD4 coding sequence, (c) a spacer coding sequence coding for 10 proline residues, which spaces the CD4 and TCS protein moieties, (d) the coding sequence for mature TCS and (e) a stop codon positioned adjacent the carboxy-terminal codon of mature TCS. The method generally follows that used in fusing a soluble CD4 to domains 2 and 3 of pseudomonas exotoxin A, as described previously (Chaudhary).

Plasmid pQR19/CD4 is analysed for expression of fused TCS protein as above. Briefly, the expression vector is cultured in a suitable bacterial host under IPTG induction conditions to a desired cell density. The cells are harvested, ruptured by sonication, and the cell material is clarified by centrifugation. The clarified material is tested for (a) binding to gp120 antigen, to confirm CD4 ligand binding activity, and (b) for ribosome inhibition activity, to confirm TCS enzymatic activity.

The protein may be purified by molecular-sieve and ion-exchange chromatography methods, with additional purification by polyacrylamide gel electrophoretic separation and/or HPLC chromotography, if necessary.

It will be appreciated from the above how other ligand/TCS-containing fusion proteins may be prepared. One variation on the above fusion is to exchange positions of the CD4 and TCS molecules in the fusion protein.

As described above, the coding sequence of TCS was obtained by selective amplification of a TCS coding region, using sets of degenerate primers for binding to spaced coding regions of a TCS coding sequence in genomic DNA. This section describes the use of such sets of degenerate primers for selective amplification of coding sequences for a variety of RIPs.

In selecting suitable primer sets, the amino acid sequences of TCS and one or more RIPs are examined for regions of sequence homology, i.e., regions where the amino acids sequences are identical or differ at most by one or two amino acid residues. Typically, the length of the regions being examined should contain at least about 7 amino acids, i.e., at least about 20 nucleotides, although it is appreciated that longer oligonucleotide primers are preferred, even though overall complexity is increased.

FIG. 9 shows the complete amino acid sequences of TCS (top line), and three RIPs whose sequences have been published. The RIPs are ricin A chain (Lamb), abrin A chain (Funatsu) and barley ribosome inhibitor (Asano). The amino acids are indicated by conventional one-letter codes. Amino acid matches among the four proteins are shaded.

As seen from the figure, there are several regions, each containing at last seven amino acids, which show a high degree of amino acid sequence homology among the proteins, i.e., sequence matching in at least about 4 of the 7 amino acid positions. The relatively greater homology among TCS, ricin A chain and abrin A chain, as compared with barley ribosome inhibitor, presumably reflects evolutionary divergence since TCS, ricin A and abrin A chain are all derived from dicotyledons, and barley inhibitor is obtained from a monocotyledon.

Considering the sequences from amino acids 8-14 in the upper line in FIG. 9, it is seen that the amino acid sequence for abrin A chain --GATSQSY-- differs from the corresponding TCS and ricin A chain sequences by only 1 amino acid each, and therefore is a likely choice for design of the primer set. The disadvantage of the GATSQSY sequence is that the presence of two serine residues (S) introduces a six-fold degeneracy at two points in the sequences. However, this problem is not prohibitive if inosine (I) is used in the third and/or first codon position, to reduce degeneracy (down to as little as twofold).

The abrin sequence in the same 8-14 amino acid region differs from the corresponding TCS by a proline-to-serine substitution in the fifth position. Since an ICI sequence will hybridize with both the proline and four of the serine codons, and an additional AGI sequence will hybridize with the other two serine codons, the primers can be made two fold degenerate at this position to encompass both TCS and abrin coding sequences.

Likewise, the abrin sequence in this region differs from the corresponding ricin A chain sequence by a serine-to-valine substitution in the fourth amino acid position. Since an III sequence is needed to bind to both the serine and proline codons, this position can be made completely neutral. The other five amino acid positions are preferably made degenerate, to optimize the specificity of primer binding to corresponding genomic coding regions. The total number of primers in the final primer set is preferably between about 16-128 although more complex mixtures can be used. The primers are synthesized conventionally using commercially available instruments.

A second set of degenerate primers from another region of TCS which is homologous in amino acid sequence to RIPs is similarly constructed.

The two primer sets are useful in a method for selectively amplifying RIP coding sequences present in genomic DNA from selected plant sources, employing repeated primer-initated nucleic acid amplification. As an example, to amplify coding sequences for abrin A chain protein, genomic DNA from Abrus precatorius is isolated, and mixed with the primer sets, all four deoxynucleosides triphosphates, and polymerase, as outlined in Example 2. After repeated cycles of primer binding and strand extension, the material is fractionated by gel electrophoresis and amplified fragments are identified, for example, by ethidium bromide staining or by autoradiography, according to procedures described in Example 2. Fragments amplified from an RIP gene can be identified by size, as the selection of specific primer sets would predict the size range of the fragment that is amplified. Genes for RIPs are not believed to contain any introns (Halling and the present application).

The amplified material is then used as a (radiolabeled) probe for detecting genomic library clones prepared from genomic DNA from the plant source, e.g., Abrus precatorius. The identified library clones are analysed, as above, for fragments containing a complete RIP coding sequence. Alternatively, overlapping genomic library fragments containing amino and carboxy portions of the coding sequence can be combined to produce a complete coding sequence.

More generally, this aspect of the invention includes a primer mixture and method of using the mixture for selectively amplifying RIP coding sequences. The primer mixture includes a first set of sense-strand degenerate primers, and a second set of anti-sense primers, where each set contains at least one primer sequence which is effective to hybridize with the corresponding coding sequence in TCS which encodes the region of amino acid homology with RIPs, particularly RIPs from dicotyledon plants.

Once the amplified genomic sequence is obtained, as described, the sequence can be used as a probe for isolating genomic library fragments containing the desired RIP coding sequence.

It will be appreciated that the method can be used to obtain the coding sequence from plants which produce known RIPs, and also to screen other plants for the presence of genes encoding as-yet-unknown RIP or RIP-like proteins.

The following examples illustrate various methods used to obtain and verify the nature of the coding sequence and recombinant proteins described above. The examples are intended to illustrate, but in no way to limit, the scope of the invention.

T. kirilowii root tubers were obtained from the Canton region of the People's Republic of China. Leaves of T. kirilowii were obtained from Korea and were collected and immediately frozen on dry ice for shipment. Samples were than stored at -70.degree. C.

QAE Zetaprepp.TM. anion exchange cartridges and SP Zetaprep.TM. cation exchange cartridges were supplied by AMF Cuno Cor. (Meridan, Conn.); and Pellicon ultrafiltration membranes (10,000 MW cutoff), from Millipore Corp. (Bedford, Mass.). M13/MP18 and M13/MP19 were obtained from New England Biolabs (Beverly, Mass.). Lambda-Zap II.TM. cloning vector system was supplied by Stratagene (La Jolla, Calif.). Expression vector PKK233-2 and its IPTG-inducible E. coli host strain, XLI-blue, were obtained from Pharmacia (Piscataway, N.J.) and Stratagene (La Jolla, Calif.), respectively. Restriction enzymes were obtained from New England Biolabs (Beverly, Mass.) or Promega (Madison, Wis.). DNA primer-initiated amplification reagents were obtained from Perkin-Elmer/Cetus (Norwalk, Conn.).

Synthetic oligonucleotide primers were prepared by conventional, automated phosphoramidite methods using either a Biosearch Cyclone or an Applied Biosystems Model 380B instrument.

The methods for preparation and manipulation of nucleic acids, and the recombinant DNA techniques employed herein are broadly accepted and applied and are generally referenced by Ausubel, F. M. et al. (eds) "Current Protocols in Molecular Biology" Vols. 1 and 2, John Wiley & Sons, New York (1988) and Maniatis, T., et al., "Molecular Cloning. A Laboratory Manual," Cold Spring Harbor Laboratory, 1982.

A clarified extract of the roots of T. kirolowii was obtained by overnight extraction of homogenized tubers of T. kirilowii. The extract was clarified by centrifugation, and the clarified material was passed through a QAE Zetaprep.TM. anion exchange resin, which is supplied commercially in cartridge form. The ion exchange step was carried out at low ionic strength, i.e., low conductivity, which has been found effective to enhance TCS purification, and in particular, to remove hemagglutinin contaminants. The low-conductivity buffer was 20 mM phosphate, pH 8.0.

The flowthrough from the anion exchange resin was adjusted in pH and ionic strength, and preferably concentrated, preparatory to further protein purification by chromatography on a cation exchange resin. The concentration step was carried out by ultrafiltration using a 10,000 molecular weight filtration membrane, yielding a solution which is largely free of low-molecular weight contaminants.

The treated flowthrough material equilibrated with 50 mM phosphate, pH 8.0 buffer was applied to an equilibrated SP Zetaprep.TM. cation exchange resin, and the column was washed extensively with buffer (15-20 volumes) until the elution profile reached a baseline value. The extensive washing removed loosely bound material, including, particularly, endotoxins and high molecular weight lipopolysaccharides (LPS), and is necessary for achieving high purity TCS.

TCS was now eluted from the column in highly purified form by elution with 50 mM phosphate buffer, pH 6.0 containing 60 mM NaCl, to release bound TCS from the resin. The purified TCS protein was at least about 98% pure, as evidenced by HPLC profile and staining patterns on SDS gel electrophoresis.

Genomic DNA was isolated from frozen T. kirilowii leaves by a modification of published methods (Taylor). Briefly, frozen tissue was ground to a fine powder using a mortar and pestle kept on dry ice. .beta.-mercaptoethanol was then added to 2% of the initial volume followed by an equal volume of hot 2.times.extraction buffer (2% cetyltrimethylammonium bromide (CTAB), 100 mM Tris-Cl, pH 8.0, 20 mM EDTA, 1.4 M NaCl).

This slurry was gently stirred in a 55.degree. C. water bath until the temperature reached 50.degree. C. The slurry was then transferred to appropriate centrifuge bottles and extracted twice with an equal volume of chloroform:isoamyl alcohol (24:1). Phase separation was achieved by centrifugation. A 1/10 volume of 10% CTAB was added and the extraction repeated.

The upper aqueous phase was removed to another container and the DNA precipitated by adding an equal volume of precipitation buffer (1% CTAB, 50 mM Tris-Cl, pH 8.0, 10 mM EDTA) to lower the sodium concentration to 0.35 M. The DNA was collected and washed with cold 70% ethanol, 0.1 M sodium acetate to convert the DNA to a sodium salt, followed by a wash by 95% cold ethanol. The DNA could then be dried and redissolved in 10 mM Tris-Cl, pH 7.5, 1 mM EDTA. To further eliminate contaminants, the DNA was re-precipitated from CTAB by adding an equal volume (original) of 2.times.extraction buffer, followed by two volumes of (original) TE buffer (10 mM Tris-HCl, 1 mM EDTA), pH 8.0. The DNA was once again converted to the sodium salt, washed with ethanol as above, dried, and dissolved in TE buffer, pH 8.0. Greater than 5 mg of high molecular weight DNA was obtained from approximately 35 g of tissue.

Three degenerate sets of probe sequences were synthesized, corresponding to two separate coding regions. The first DNA sequence is a 35-mer and encompasses the protein sequence overlined and denoted A in FIG. 1, and the second sequence is a 32-mer and encompasses the protein sequence overlined and denoted B in the figure.

The probe sets were prepared by conventional automated methods using instruments commercially available and following the manufacturers' instructions. (Biosearch, San Rafael, Calif., and Applied Biosystems, Foster City, Calif.). Deoxyinosine nucleotides were incorporated in order to generate probes longer than 20 nucleotides of manageable complexity (Ohtsuka; Takahashi). The sense-strand probe set corresponding to the 35-mer, designated MPQP-1, included 128 isomers. The anti-sense-strand second and third sets, designated MPQP-2 and MPQP-3, each included 128 isomers and were 32-mers.

A DNA amplification reaction was carried out by repeated primer initated strand extension, in a reaction mixture containing (a) 1-2 micrograms of the T. kirilowii DNA isolated as above, (b) .sup.32 P-labeled [.sup.32 P]MPQP-1 and an equimolar mix of .sup.= P]MPQP-2 and -3, as primers, (c) all four deoxynucleoside triphosphates, and (d) Tag polymerase. About 20 rounds of thermal cycling were performed, employing conventional DNA amplification reaction conditions, as outlined in instructions from the manufacturer (Perkin Elmer-Cetus, Norwalk, Conn.). A similar DNA-amplification reaction was carried out using unlabeled primer sets.

The product of the DNA amplification step was fractionated on 3% Nusieve, 1% ME agarose (Seakem.TM., FMC Bioproducts, Rockville, Md.) and stained with ethidium bromide. A major product of about 255 base pairs was detected. The material was also fractionated on 5% polyacrylamide gel electrophoresis and the bands detected by autoradiograpy, with similar results. In both cases, very little DNA other than the amplified material was detected.

Amplified DNA was recovered form polyacrylamide gels by elution followed by ethanol precipitation. A portion of one such preparation, approximately 100 nanograms, was taken for DNA sequence analysis. The DNA sample plus 30 ng of unlabeled MPQP-1 were taken up in 10 .mu.l of TE (10 mM TrisNcl, pH 7.5, 1 mM EDTA) and heated to 100.degree. C. for 5 minutes to denature the double-stranded fragment. The mixture was quick frozen on dry ice to prevent the template from annealing. Two .mu.l of 5X Sequenase sequencing buffer (USB Biochemicals, Cleveland, Ohio) was added and the primer allowed to anneal to the template for 5 minutes at 37.degree. C. The standard sequencing protocol supplied by the manufacturer was then followed.

The DNA sequence obtained and its translation into all three reading frames is shown in FIGS. 3A (for the sense strand) and in FIG. 3B (for the complementary strand).

Genomic DNA obtained as above was digested to completion with EcoRI and cloned into a standard library cloning vector, in this case, the Lambda-Zap II.TM. system of Stratagene (La Jolla, Calif.). For use as a probe, the amplified 255-bp fragment from above was radiolabeled by random priming (Boehringer-Mannheim kit, Indianapolis, Ind.).

Approximately 0.5-1.0.times.10.sup.6 plaques were probed with the .sup.3 P-radiolabeled 255 -bp probe. Two clearly positive plaques were picked, amplified and converted to plasmid, according to protocols supplied by Stratagene. One clone, designated pQ21D, contained an approximate 4 kb insert which included the complete TCS coding sequence.

The region containing the TCS coding region was sequenced by standard double-strand sequence methods, using universal sequence primers as well as unique synthetic oligonucleotide primers as needed. A smaller subclone containing only the TCS coding region was generated by subcloning the 1.2 kb EcoRI to NcoI fragment (FIG. 4) from pQD21D into pKK233-2. The resulting recombinant plasmid was designated pQD12D/pKK233-2.

The pQ21D/pKK233-2 cloning vector from Example 2 was divided into two samples. One sample was digested with EcoRI and SalI, to release an EcoRI to SalI fragment containing the amino portion of the TCS gene. A second portion of the DNA was digested first with NcoI, and treated with Klenow to generate a blunt end. The DNA was then digested with SalI to release a SalI to NcoI (blunt) fragment containing the carboxy portion of the gene. After isolating the two fragments by gel electrophoresis, the EcoRI to SalI fragment was cloned into M13MP19 (EcoRI to SalI), and the SalI to NcoI (Klenow repaired) fragment was cloned into M13MP18 (SalI to SmaI). Fragment insertion and production of single-strand phage DNA was performed according to known methods.

The phage single-strand DNA's were subjected to primer mutagenesis using standard methods. The amino portion of the gene (in the M13MP19 vector) was modified with primer ONcoN (5'-CCTGCTGTGGCCATGGATGTTAGC -3'); and the carboxy portion of the gene was modified with primer QTer01 (5'-CGAAACAATATGGCATA ATAAAGCTTCCGAGCTCG -3'). These modifications placed an NcoI site containing an ATG start codon at the beginning of the mature TCS protein sequence and a double TAA translation stop sequence plus a HindIII cloning site after the carboxy end of the mature sequence, as illustrated in FIG. 5.

The modified sequences were excised from purified phage DNA as an NcoI-SalI and an SalI-HindIII fragment, respectively, and cloned together into NcoI-HindIII digested pKK233-2. pKK233-2 is a plasmid containing a synthetic trp/lac promoter positioned appropriately ahead of a ribosome binding site that is also positioned appropriately ahead of an ATG start codon contained within an NcoI site. It is supplied commercially (Pharmacia).

Several clones were characterized and verified to contain the modified insert. The DNA sequences of the modified regions were directly verified for one, designated pQR19.

The plasmid pQR19 and similar clones were propagated in the E. coli host strain, SLl-blue. The significant feature of the strain is that it carries the lacIq repressor gene on a F' episome (discussed above). LacIq protein controls expression from the lac operator and is blocked from repression by the addition of IPTG to 5 mM.

Plasmid pQR19 and another isolate were analyzed for expression of TCS. Cultures were first grown in Luria broth medium supplemented with 100 .mu.g/ml ampicillin, to select for maintenance of the plasmid, to a density of 0.7 measured at .sub.600 nm before adding IPTG, then allowed to grow for 4 hours. These conditions did not result in high levels of expression.

Cultures were then inoculated in Luria broth plus 100 .mu.g/ml ampicillin containing 5 mM IPTG, and allowed to grow to saturation density overnight (pQR19-XLl-blue induced cells). The induced cells were collected by centrifugation, resuspended in 100 mM Tris-HCL, pH 8.5, 5 mM EDTA at a concentration of about 10 A.sub.600 units/ml and disrupted by sonication. Aliquots were taken and centrifuged at 15,000.times.g for 5 minutes to separate soluble from insoluble components.

The insoluble, pelleted material was resuspended in sonication buffer to the same volume as the original aliquot. Samples of each fraction were run on 10% SDS-PAGE. One set of samples was stained for total protein with Coomassie Blue; another set of samples was blotted for Western analysis, with the results discussed in Section II.

The ability of rTCS to mediate selective inhibition of HIV replication in infected T-cells was evaluated in parallel with purified plant-derived material. Cells of the CD4+ T-cell line VB (Lifson, 1986) were inoculated with HIV-1 by incubation at 37.degree. C. for one hour with an aliquot of a titered cryopreserved HIV-1 virus stock (virus isolate HIV-1.sub.DV (Crowe, 1987)). After washing, the cells were resuspended to 1.11.times.10.sup.5 per ml, and 0.9 ml of this suspension plated in replicate wells of 24 well culture plates. 0.1 ml volumes of serial dilutions of purified plant-derived TCS and rTCS were then added at 10X the desired final concentrations to yield 1.0 ml cultures containing 1.times.10.sup.5 cells in 1.0 ml of culture medium containing the desired concentration of TCS. After culturing for 4 days at 37.degree. C. in a humidified 5% CO.sub.2 /air atmosphere, culture supernatants were harvested and viral replication in treated and control cultures was assessed by measuring HIV p24 antigen content using a commercially available capture immunoassay kit according to manufacturer's instructions (Coulter, Hialeah, Fla.).

As shown in FIG. 6 (open boxes), in accord with observations reported elsewhere (U.S. Pat. No. 4,795,739), plant-derived TCS purified to apparent homogeneity from the root tubers of T. kirilowii inhibited HIV replication in a concentration-dependent fashion in this acute infection assay system. The biological activity of rTCS produced in E. coli and purified to apparent homogeneity (closed boxes), was essentially indistinguishable from that of the native product when tested in parallel in an assay system for inhibition of HIV replication at TCS concentrations above 0.005 .mu.g/ml (FIG. 6). At lower concentrations, rTCS appears to show slightly less specific activity than the plant-derived protein.

The ability of TCS to irreversibly inactivate ribosomes, thereby inhibiting protein synthesis, is conveniently measured in standardized assays of in vitro translation utilizing partially defined cell free systems composed, for instance, of a reticulocyte lysate preparation as a source of ribosome and various essential co-factors, mRNA template(s) and amino acids. Use of radiolabelled amino acids in the reaction mixture allows quantitation of incorporation of free amino acid precursors into trichloroacetic acid precipitable proteins.

As shown in FIG. 7, the protein synthesis-inhibitory activity of rTCS produced in E. coli and purified to apparent homogeneity, is indistinguishable from that of plant-derive TCS.

Although the invention has been described with reference to specific methods and compositions, it will be apparent to one skilled in the art how various modifications and applications of the methods may be made without departing from the invention.

## Claims

1. A recombinant process for the production of a trichosanthin protein having the functional properties of Trichosanthes-obtained trichosanthin comprising into an expression vector suitable for expression of the trichosanthin protein in a selected host,

2. The process of claim 1, where the expression vector is pQR19 and the host is E. coli.

3. The process of claim 1, wherein said DNA sequence is included in the sequence: ##STR11## which encodes the mature form of trichosanthin isolated from Trichosanthes kirilowii.

4. The process of claim 3, wherein the expression vector contains a bacterial promoter, a ribosome binding site, and an ATG start codon positioned before and adjacent the amino-terminal codon at position 411, and a stop codon positioned after and adjacent the carboxy terminal codon at position 1151.

5. The recombinant process of claim 1, wherein said DNA sequence encodes a polypeptide including the sequence: ##STR12##

6. The process of claim 5, wherein said DNA coding sequence further includes, attached to the amino or the carboxy terminus of the coding sequence for said polypeptide, coding sequences for a ligand peptide effective to bind specifically to a cell-surface antigen.

7. The process of claim 5, wherein said DNA coding sequence further includes a DNA sequence encoding a carboxy terminal extension of said polypeptide, wherein the carboxy terminal extension has the sequence ##STR13##

8. The process of claim 5, wherein said DNA coding sequence further includes a DNA sequence encoding an amino terminal extension of said polypeptide, wherein the amino terminal extension has the sequence ##STR14##