STREPTOMYCES VECTORS FOR PRODUCTION OF HETEROLOGOUS PROTEINS

Abstract: Nucleic acid sequences and DNA vectors useful for the production of CD4 chimeric proteins, as well as other heterologous proteins, in Streptomyces are disclosed. The nucleic acid sequences of the invention comprise the coding sequence for the signal peptide of the Streptomyces longisporus tyrosine inhibitor (LTI) gene operatively linked with a propeptide sequence consisting essentially of an amino acid sequence coding for from one to about 6 amino acids, the sequence of said amino acids selected to result in the formation in Streptomyces of a protein product having a homogeneous amino terminus after processing to remove said signal peptide formed on the protein product during synthesis of the protein product. In an alternative embodiment of the invention, the propeptide is omitted and the LTI signal peptide is operatively linked with a nucleic acid sequence coding for a heterologous protein which has been modified to code for the sequence lys-ala- at the 3' end. The invention also provides cells transformed with the nucleic acid sequences or vectors of the invention, and methods of using the nucleic acid sequences and vectors of the invention to produce heterologous proteins in Streptomyces.

Description: STREPTOMYCES VECTORS FOR PRODUCTION OF HETEROLOGOUS PROTEINS Field of the Invention The present invention relates to production of heterologous proteins in microorganisms. More particularly, the present invention relates to nucleic acid sequences and expression vectors for expression of heterologous proteins, particularly soluble CD-4 proteins, in Streptomyces. Background of the Invention The principal route of infection of HIV viruses is mediated through attachment of the virus to the CD4 protein. CD4 (also referred to as T4) is a T cell surface glycoprotein that associates with the major histocompatibility complex class II molecules. However, CD4 is also the target for the HIV-1 envelope glycoprotein, gpl20. Once the HIV-l virus binds to CD4 on the target cell surface, the viral PNA is then introduced into the target cell by direct fusion of the SUBSTITUTE SHEET apposing viral and plasma membranes (Maddon et al., Cell, 5 4. - 8 65-874 (1988). The DNA sequence of the CD4 has been disclosed by Maddon et al. (Cell 4.2:93-104 (1985)). From this it was deduced that the mature CD4 protein consists of an extracellular region containing four immunoglobulin-like domains (V1-V4) , a transmembrane domain, and a charged intracellular region of approximately 40 amino acids (Maddon et al. , supra) . Recombinant soluble derivatives of CD4, such as sT , comprise proteins in which the transmembrane and cytoplasmic domains which have been deleted, yet retain the ability to bind gpl20. sT4 has also been shown to inhibit both viral infectivity and HIV mediated syncytia formation (Deen et al., Nature 331: 82-84 (1988)). This inhibition results from the association of the sT4 protein with the HIV gpl20 envelope protein, thereby competing with the native CD4 protein on the cell surface. The mechanism by which CD4 and soluble CD4 inhibits viral infectivity and syncytia formation is not completely understood. In vitro, it has been shown that the addition of soluble CD4 to HIV-1 or HIV-l infected cells appears to induce the release of gpl20 without a concomitant increase in other viral proteins. This implicates CD4 as an active rather than passive inhibitor of HIV-l. However, in vivo, the sT4 protein and similar soluble CD4 proteins are rapidly eliminated from serum. This rapid elimination severely limits the clinical application of soluble CD4 as an inhibitor of HIV function. To prolong the serum clearance time, several soluble CD4 chimeras have been constructed. Traunecker et al. (Nature 331:84-86 (1988)) disclosed CD4 chimeras (V1V2 and V1V2V3V4) in which the carboxy terminal portion of the protein consists of a murine immunoglobulin light chain constant region. Traunecker et al. further disclosed that these constructs were expressed in myeloma cells. Traunecker et al. (Nature 339:68-70) (1989) subsequently disclose CD4 chimeras (V1V2) fused to murine IgM or IgG2 heavy chain constant regions. These constructs were also disclosed to form pentamers. Seed (EP-A-325,262, published July 26, 1989) and Capon et al. (WO89/02922, published April 6, 1989) disclose CD4 chimeras fused to human IgG heavy and light chain constant regions. The proteins disclosed by Seed and Capon et al. were all expressed in mammalian systems, i.e. COS, BHK (baby hamster kidney) and CHO cells. As a result, the disclosed CD4 chimeras are expensive to produce and the overall production capacity is limited, relative to alternate expression systems. It is thus an object of the present invention to produce CD4 chimeras in a bacterial host system to reduce the cost of production as well as to produce CD4 chimeras which have an increased serum half-life and/or potency against HIV infections relative to soluble CD4. It is common for proteins produced by recombinant DNA techniques to be fused with a portion of a protein produced by the host cell or other protein. These extra portions typically do not add to the function of the desired protein but their presence can hinder folding or processing of the desired protein. It is thus desirable to be able to produce proteins which are as near as possible to the native protein so these effects can be avoided or reduced. Accordingly, it is also an object of the invention to produce CD4 derivatives, chimeras and other proteins in bacterial host systems that do not contain amino acids derived from host system proteins or that contain only a minimal number of amino acids added to the authentic protein sequence. It is a further object of the invention to provide vectors for producing CD4 chimeras and proteins and other proteins that result in chimeras that do not contain amino acids derived from host system proteins or that contain only a minimal number of amino acids added to the authentic protein sequence. Summary of the Invention The present invention provides nucleic acid sequences and DNA vectors useful for the production of CD4 chimeric proteins, as well as other heterologous proteins, in -4- Streotomyces. The nucleic acid sequences of the invention comprise the coding sequence for the signal peptide of the Streptomyces lonoisporus tyrosine inhibitor (LTI) gene operatively linked with a propeptide sequence consisting essentially of an amino acid sequence coding for from one to about 6 amino acids, the sequence of said amino acids selected to result in the formation in Streptomyces of a protein product having a homogeneous amino terminus after processing to remove said signal peptide formed on the protein product during synthesis of the protein product. In an alternative embodiment of the invention, the propeptide is omitted and the LTI signal peptide is operatively linked with a nucleic acid sequence cpding for a heterologous protein which has been modified to code for the sequence lys-ala- at the 3'end. In other aspects the present invention provides Streptomyces cells transformed with a nucleic acid sequence or vector of the invention, and methods of using the nucleic acid sequences of the invention to produce heterologou£_ proteins in Streptomyces. This invention is more particularly pointed out in the appended claims and is described in its preferred embodiments in the following description. Detailed Description of the Invention The present invention provides nucleic acid sequences and DNA vectors useful for the production of CD4 chimeric proteins, as well as other heterologous proteins, in Streptomyces. The nucleic acid sequences of the invention comprise the coding sequence for the signal peptide of the Streptomyces lonoisporus tyrosine inhibitor (LTI) gene operatively linked with a propeptide sequence consisting essentially of an amino acid sequence coding for from one to about 6 amino acids, the sequence of said amino acids selected to result in the formation in streptomyces of a protein product having a homogeneous amino terminus after processing to remove said signal peptidε formed on the protein product during synthesis of the protein product. Preferably the amino -5- acid sequence of the propeptide is selected to cause processing of the protein product at a position between the end of the nucleic acid sequence coding for the signal peptide and the beginning of the propeptide seguence. In preferred embodiments of the invention, the amino acid seguence of the propeptide is thr-, thr-pro-ala-ala- (SEQ ID NO: 1) , or thr- pro-ala-ala-ala- (SEQ ID NO: 2) . The propeptide sequence thr- is more preferred, as it is only one amino acid and provides good yield of protein product. In these embodiments of the invention^ the nucleic acid sequence of the propeptide preferably comprises the sequence ACC (coding for thr) ; ACC CCG GCC GCT (SEQ ID NO:3) (coding for thr-pro-ala-ala, SEQ ID NO:l) or ACC CCG GCC GCT GCT (SEQ ID NO: ) (coding for thr- pro-ala-ala-ala, SEQ ID NO:2). The nucleic acid sequences of the invention are preferably operably linked with other DNA sequences to form an expression vector which may then be inserted in Streptomyces for production of the heterologous protein. Heterologous proteins thus formed will have a homogeneous N-terminus comprising the sequence of the propeptide. In an alternative embodiment of the invention, the propeptide is omitted and the LTI signal peptide is operatively linked with a nucleic acid sequence coding for a heterologous protein which has been modified to code for the sequence lys-ala- at the 3'end. The nucleic acid sequence is preferably operably linked with other DNA sequences to form an expression vector which may then be inserted into Streptomyces for production of the heterologous protein. In this embodiment of the invention, signal sequence is cleaved from the heterologous protein at the end of the signal sequence such that the heterologous protein is formed having an N-terminus of lys-ala-X, where X is the remainder of the heterologous protein. This embodiment of the invention is particularly advantageous for production of CD4 derivatives and chi eric proteins. Applicants have surprisingly and unexpectedly found that by altering only one amino acid at position 2 near the -6- N-terminus of CD4 (in the VI region) , a heterologous protein could be formed that is efficiently secreted and correctly processed to remove the entire LTI signal sequence, but which still retained substantial gpl20 binding capacity. By altering the nucleic acid sequence coding for the CD4 derivative or chimera such that it codes for lys-ala- at the amino terminus, the product coded for contained an N-terminus amino acid sequence similar to the sheep L3T4 (CD analogue) receptor, which has an N-terminus of lys-ala-. When it is desired to use this embodiment of the invention to prepare heterologous proteins other than CD4 derivatives or chimeras, the nucleic acid seguence coding for the heterologous protein may be modified to code for lys- ala-at the amino terminus by deleting the bases coding for the two amino acids at the N-terminus and substituting a sequence coding for lys-ala for the deleted bases, or a sequence coding for lys-ala may simply be added to the 3 'end of the coding sequence for the LTI signal peptide. Thus heterologous proteins having a modified N-terminus having the amino acid sequence lys-ala- will be formed in Streptomyces. Because of the possible deleterious effects of additional amino acids on the function of heterologous proteins, Applicants desired to produce heterologous proteins that have the sequence of the native, protein (or portions of it) with few or no additional amino acids derived from host system proteins, as is not the case when heterologous proteins are formed as fusion proteins. Applicants have surprisingly found that the pro-peptide of the LTI gene can be deleted or modified and heterologous proteins formed that have nearly authentic N-termini and are also efficiently secreted from the Streptomyces host cell. It became apparent the amino acid sequence surrounding the signal peptide cleavage site can have dramatic effects on. signal peptide processing. There are at least two parameters which can influence processing and consequently, secretion and production levels: the physico-chemical nature -7- of amino acids and the net charge within the region surrounding the cleavage site. A statistical analysis of eukaryσtic and prokaryotic species signal peptides has shown clear preferences for specific classes of amino acid at specific residues within the region surrounding the signal peptide cleavage site (VonHeijne, FEBS Letters 2^ _:439-446 (1989)). For example, amino acids residues at positions -3 and -1 are generally small, uncharged residues. Alanine is the most frequent amino acid at these positions. Positions -3 and -1 also show a clear bias against certain classes of amino acids; for example, aromatic, charged, hydrophobic residues and proline were not found at position -1 within 78 eukaryotic signal peptides. Within the amino terminus of the mature protein, proline and glycine are seldom found at position +1. Both experimental data and statistical analysis indicate a clear preference for a net neutral or negative charge within the region surrounding the cleavage site (Li, et al., Proc. Natl. Acad. Sci. USA 8J5:7685-7689 (1988), ϊanane and Mizushima, 263:19690-19696 (1988), Summers, et al., J. Biol. Chem. 264:20082-20088 (1989), Von Heijne, J. Mol. Biol. 192:287-290 (1986) of bacterial signal peptides. Li, et al. supra have shown that when the amino terminus of E_ t . coli alkaline phosphate was mutagenized such that the net charge increased from 0 to +2, the total production of alkaline phosphatase dropped 50 fold. In addition, the time required for the precursor processing into the mature product changed from undetectable for the wild type protein to 30 minutes for the mutant with the net charge of +2. The severity of this defect was somewhat decreased by reducing the net charge to +1. Summers, et al. supra described a variety of hybrid proteins between the B-lactamase signal peptide and chicken muscle triσsephosphate isomerase. The native triosephosphate iso erase is not secreted into the periplasm of E. coli. This protein could be exported into the periplasm, however, when the arginine residue at position 3 of the triose phosphate isomerase was changed to a proline or serine residue. These -8- observations lend credence to Von Heijne's statistical analysis which showed a clear preference for a net negative charge within the cleavage site region. It has been found that the first two amino acid residues of CD4 (in the VI region) lys-lys- are absolutely required for gp 120 binding activity. In attempts to provide CD4 derivatives and chimeras having authentic N-termini (e.g. KK-V1V2) , it was found that CD4 derivatives and chimeras could not be produced. When the CD4 V1V2 regions are fused to the LTI signal seguence, the resulting product is processed in at least two forms. Applicants surprisingly and unexpectedly found that moving the positively charged lysine residues away from the signal peptide cleavage site by inserting up to five amino acids resulted in a CD4 heterologous protein that has a homogenous N-terminus, was efficiently transported into the culture medium, and still retained gpl20 binding capacity. Even more surprisingly, Applicants found that by changing lysine at position 2 of the CD4 molecule to alanine, a homogeneous CD4 protein having a nearly _uthentic N-terminus could be produced despite the presence of lysine at the N- terminus end. In addition, this heterologous protein was also efficiently secreted into the medium, and retained gpl20 binding capacity. Heterologous proteins produced using the nucleic acid sequences and vectors of the invention have homogeneous N-termini, i.e., the N-terminus of the product has the same amino acid seguence which gives them several advantages over heterologous proteins that are produced as a mixture of forms. Production costs are lower since fewer separation steps are needed in order to obtain the desired product in a purified form. A single product rather than a mixture of heterologous proteins is preferred for regulatory approval. Additionally, natural folding and function of the protein product is encouraged by the absence of amino acids derived from host system proteins. In other embodiments the invention is directed to DNA vectors for expressing heterologous proteins in Streptomvces which comprise a coding sequence for the heterologous protein operatively linked to a promoter and a nucleic acid sequence coding for the signal sequence of the Streptomyces lonσisporus tyrosine inhibitor gene operatively linked with a propeptide seguence consisting essentially of an oligonucleotide coding for from one to about 6 amino acids, the sequence of said amino acids selected to result in the formation in Streptomyces of a heterologous protein having a homogeneous amino terminus after processing to remove the signal peptide formed on a heterologous protein during synthesis of the heterologous protein. Preferably the coding sequence for the heterologous protein comprises a sequence coding for a HIV gpl20 binding region. In other embodiments, the invention is directed to DNA vectors for expressing heterologous proteins in Streptomyces which comprises a coding sequence for the heterologous protein operatively linked to a promoter and the Streptomyces lonqisporus trypsin inhibitor gene signal sequence, wherein the sequence coding for the heterologous protein is modified at its 5' end by adding bases coding for the amino acid sequence lys-ala, or by deleting bases coding for the two amino acids at the 5' end and substituting for the deleted sequence a sequence coding for the amino acid sequence lys-ala. Preferably the coding sequence for the heterologous protein comprises a sequence coding for a HIV gpl20 binding region. Additional embodiments of the invention are directed to methods for using the DNA vectors of the invention, which comprise the steps of introducing a vector of the invention into a Streptomyces host cell, and growing the host cell in a suitable culture medium. Further embodiments of the invention are directed to Streptomyces cells transfected with the nucleic acid sequences or DNA sequences of the invention. The CD4 T lymphocyte receptor protein, herein referred to as "CD4", is a surface glycoprotein that interacts with the HIV envelope protein, gpl20. This high affinity interaction occurs between gpl20 and the extracellular domain of CD4. The extracellular domain of CD4 comprises 4 regions of limited homology with the immunoglobulin variable (V) regions. The approximate boundary domains for the CD4 variable-like regions (V1-V4) are, respectively, amino acids 100-109, amino acids 175-184, amino acids 289-298, and amino acids 360-369. Thus, as used herein, VI refers to amino acids 1-183 (approximately) and so on. Removal of the transmembrane and cytoplasmic domains of CD4 results in a soluble receptor protein. Such soluble CD4 proteins, consisting of all or portions of the external region of the human CD4 receptor, have been shown to inhibit HIV-l infection and virus-induced cell fusion (see e.g., Deen et al., Nature 331: 82-84 (1988). As used herein, the term "HIV gpl20 binding domain" refers to any soluble human CD4 molecule (i.e., lacking the transmembrane and cytoplasmic domains) that binds HIV gpl20 with essentially the same or greater affinity than the full-length CD4 receptor protein. The binding affinity of CD4 for gpl20 can be assayed as described by Arthos et al., (Cell 52:469-481 (1989). The CD4 molecule of the present invention comprises the minimal HIV gpl20 binding region found in domain VI (amino acids 41-55) . Preferably, it includes the V1V2 domains of CD4 or substantially the same sequence (i.e., differs by no more than 15 amino acids) . Hence a preferred embodiment of the present invention is V1V2 joined to a human immunoglobulin constant region. The DNA coding sequence for CD4 is disclosed by Maddon et al. (Cell 2 :93-104 (1985) and incorporated by reference herein with the noted exception that the mature amino terminal of human CD4 is Lys-Lys and not Gln-Gly-Lys- Lys as reported by Maddon et al. (see, Littman et al., Cell 5J5:541 (-1988) . DNA encoding for CD4 is available from various sources, -for example, plasmid pT4B (Maddon et al. , supra) . Alternatively, cDNA molecules encoding the CD4 sequence can be derived from mRNA of T lymphocytes which express CD4 using known techniques (e.g., polymerase chain reaction) or can be synthesized by standard DNA synthesis technigues. In addition to those proteins exemplified below, one of skill in the art can readily construct DNA molecules which encode further CD4 derivatives. For example, it is within the art to construct amino acid additions, substitutions, deletions, rearrangements (e.g., V1V3, V1V4) and chemical modifications thereof. Such derivatives, however, must retain the ability to bind HIV gpl20. An inherent advantage of the CD4-Ig chimeras of the present invention is the potential to increase the potency for inhibition/inactivation of an HIV infection relative to soluble CD4. This can be accomplished through the acquisition of Fc effector functions as described more fully below. The effector functions, which lie primarily within the Fc portion of the immunoglobulin, include: prolonged serum clearance time (Nakamura et al., J I mun 100:376-383 (1968); protein A binding (Deisenhofer et al. Biochem 20:2361-2370 (1981); Fc receptor binding (and Antigen-dependent cellular cytotoxicity (ADCC) : mediated through the binding of antibody to Fc receptors on cytotoxic T-cellε) ; complement fixation (e.g., Clq binding); dimerization (Davies et al., Annu Rev Immunol j.:87 (1983); and placental transfer (Morgan et al., Adv Immunol 10:61-134 (1987). However, when expressing portions of the immunoglobulin heavy chain, some of the effector functions will be lessened or eliminated. An advantage of the present invention, therefore, is that effector functions which are desired may be conferred back to the molecules of the present invention by the addition of amino acids which are associated with those functions. For example, crystallographic studies of Fc fragments of human IgG indicates that dimerization occurs within the hinge region and CH3 domain (see, e.g., Deisenhofer et al., J Biochem 2^:2361-2370 (1981) . Therefore, by adding amino acids from the hinge region or CH3 domain, one may produce IgG fragments that have the ability to di erize. -12- Furthermore the Fc effector functions disclosed above may be derived from any appropriate immunoglobulin constant region or isotope. For example, IgGl, IgG2, IgG3, IgG4, IgM, IgΛ or IgE, but preferably IgGl. Human IgGl is preferred since this immunoglobulin subclass has been shown to be the most efficient at mediating cell killing by both complement and ADCC (Bruggeman et al., J Exp Med 166:1351- 1361 (1987) . The IgGl constant region is comprised of several domains: the CHI, Hinge (h) , CH2 and CH3 as disclosed by Ellison et al. (Nar .10: 4071-79 (1981)), and incorporated by reference herein. The immunoglobulin constant region of the present invention may include all or a portion of a human immunoglobulin heavy chain in which the variable region has been deleted and replaced with CD4 or an HIV gpl20 binding fragment thereof. In addition, the gpl20 binding fragment thereof. In addition, the gpl20 binding domain can be joined directly (i.e., synthesized or expressed as a single polypeptide) or indirectly (i.e., coupled after synthesis) to the immunoglobulin constant region. One embodiment is a human immunoglobulin IgG constant region lacking most (i.e., at least 51%) or all of the CH3 domain. Preferably the immunoglobulin constant region comprises most or all of the CH2 domain, that is the CH2 domain plus or minus the hinge (h) region. Preferred embodiment include -CHlhCH2, -hCH2 and -CH2. However, the present invention need not be limited to a single constant region domain. Another embodiment of the present invention is an aglycosylated human immunoglobulin constant region, comprising at least one.domain. Preferred embodiments include, but are not limited to, -CHlhCH2CH3, -hCH2CH3, -CH2CH3, -hCH2, -CH2 and -CH3. The most preferred embodiments include, but are not limited to, -hCH2 and -CH2. Preferably the protein of this particular embodiment is produced in a bacterial host system. It is more preferable that the host cell is from the genus Streptomyces. Methods for the preparation of DNA which encodes the heavy or light chain constant region of immunoglobulins are taught, for example, by Robinson et al., PCT Application Publication No. W087/02671, published May 7, 1987. Moreover, one skilled in the art can prepare a DNA molecule encoding the human IgGl sequence from any cell line expressing IgGl, for example, ARH-77 (from the ATCC) using known techniques (e.g., polymerase chain reaction) . Alternatively, the DNA molecule can be synthesized by standard DNA synthesis techniques. In addition to those proteins specifically exemplified herein, one of ordinary skill in the art can readily construct DNA molecules which encode further Ig derivatives. For example, amino acid additions, substitutions, deletions, rearrangements and chemical modifications thereof. Other examples include, the substitution of deletion of cysteine residues, utagenesis to increase Fc receptor binding and/or Clq binding, or the introduction of Fc regions from other immunoglobulin molecules. Such derivatives possess some or all of the Fc effector functions as disclosed below. Depending on which regions of IgG are expressed, the resulting chimeric protein may bind to antibodies to human IgG, Protein A, complement (specifically Clq) , and Fc receptors on appropriate cells of the immune system, e.g. acrophages. Furthermore, the binding of complement (and other effector functions) appears dependent on other factors than simply the presence of relevant sequences on the heavy chain constant region, since different subclasses of human IgG which contain a complement binding sequence differ in their complement binding capacity. Thus, other structural features may be involved. The recombinant DNA molecule and nucleic acid sequences of this invention may comprise additional DNA sequences,, for example, a vector comprising a regulatory element, one or more selectable markers, and sequences that code for replication and maintenance functions. The regulatory region typically contains a promoter found upstream f rom t h e coding sequence of this invention, which functions in t h e binding of RNA polymerase and in the initiation of transcription. In other words, the regulatory element or region is operatively linked to the recombinant DNA molecule o f this invention. It will be appreciated by one of skill in the art that the selection of regulatory regions will depend upon the host cell employed. The selectable marker system can be any of a number of known marker systems such that the marker gene confers a selectable new phenotype on the transformed cell. Examples include Streptomyces drug resistance genes such as thiostrepton resistance ribosomal methylase (Thompson et al., Gene <J0:51 (1982)) and neo ycin phosphotransferase (Thompson et al. , supra) . DNA sequences coding for replication and maintenance functions include, for example, those derived from the Streptomyces. pIJlOl derivatives (see, e.g., Keiser et al., Mol Gen Genet 185:223 (1982)) or the Streptomyces SLP1 derived vectors (see, e.g.. Bibb et al., Mol Gen Genet 184: 230 (1981)). The vector of this invention may also contain a marker which permits gene amplification. Such markers which serve to amplify gene copy number in Streptomyces include the gene for spectinomycin resistance (Hornemann et al., J Bacteriol 169:2360 (1987)) and arginine auxotrophy (Altenbuchner et al., Mol Gen Genet 195:134 (1984). The present invention also relates to a host cell transformed with the recombinant DNA molecule of this invention. Such host cell is capable of growth in a suitable culture medium and expressing the protein encoded by the recombinant DNA molecule of this invention. Such host cell is prepared by the method of this invention, i.e., by transforming a desired host cell with the plasmid of this invention. Such transformation is accomplished by utilization of conventional transformation techniques. Preferred host cells belong to the genus Streptomyces. For example such hosts include, but are not limited to, S. lividans f S. coelicolor. S. albus, and S. lonqisporus. Preferred e bodiments include S. lividans and S. lonoisporus. Other host cells which may be suitable include, but are not limited to, mammalian cells, insect cells, yeast and other bacterial cells (e.g., E. coli. Salmonella. Bacillus) . Thus, this invention and the products thereof need not be limited to any specific host cells. For expression of heterologous proteins in Streptomyces. there are a variety of promoters available. Examples include the galactose-inducible promoter of the Streptomyces galactose operon (Fornwald et al., _ oc. Natl. Acad. Sci. USA 8£: 2130 (1987)), the constitutive promoter of the S. lividans J-galactosidase gene (Eckhardt et al., J. Bacteriol 169:4249 (1987) or Brawner et al., U.S. Patent 4,717,666), the S. lonoisporus trypsin inhibitor gene (see EP-A-264,175, published April 20, 1988), or a temporarily regulated promoter such as that reported in M. echinosporsa (Baum et al., J. Bacteriol 170:71 (1988)). Regions for transcription termination in Streptomyces are derived from the 3 1 end of several Streptomyces genes, for example the termination signal at the end of the Streptomyces galactose operon or that found at the end of the S. fradiae neomycin phosphotransferase gene (Thompson et al., Proc Natl Acad Sci USA £0:5190 (1983)). Sequences for protein export in Streptomyces include those isolated from the S. lividans LEP- 10 gene and the S. lonqisporus trypsin inhibitor (LTI) gene (see EP-A-264, 175, published April 20, 1988). Preferably the export or signal sequence is derived from but not limited to the LTI gene. More preferably, the signal sequence is modified (e.g., additions, substitutions, deletions and/or rearrangements) as described in the Examples section. For the initial expression studies, the CD4-lg chimeras as more fully described in the Examples, were fused to the LTI prepro sequence. The Streptomyces replication functions for this expression vector were provided by plasmid pIJ351, which is a derivative of PIJ101 (Keiser et al., Mol Gen Genet 185:223 (1982)). The host strain used was wild- type S. lividans 1326 (Bibb et al., Mol Gen Genet 184:23 (1981)). The present invention also relates to a method o producing the protein encoded by the recombinant DNA molecul of this invention which comprises culturing the transforme host of the invention in an appropriate culture media and th isolation of such protein. By "appropriate culture media" i meant that media which will enable such host to express th coding sequence of the invention in recoverable quantity. I will be appreciated by one of skill in the art that the appropriate culture media to use will depend upon the host cell employed. The isolation of the protein so produced is preferably from the host's culture medium, that is, the protein of the invention is preferably exported into the culture medium. The protein(s) of the present invention may be isolated and purified in accordance with standard techniques. For example, extraction, selective precipitation, column chro atography, affinity chromatography or electrophoresis. For example, the IgG chimeric proteins may be purified by passing a solution containing said chimeric protein through a column which contains immobilized protein A or protein G which selectively binds the Fc portion of the fusion protein. See for example, Reis et al., J Immunol 132:3098-3102 (1984). The chimeric protein may then be eluted by treatment with a chaotropic salt or by a change in pH (e.g., 0.3M acetic acid) . Alternatively, the protein of the invention may be purified on anti-CD4 antibody columns, or anti-immunoglobulin antibody columns. The protein and protein product of the present invention may be used for the treatment of HIV viral infections. As a prophylactic, the CD4-Ig chimeras are administered to individuals at high risk to AIDS or individuals who show exposure to HIV by the presence of antibodies to HIV. Administration of an effective amount of the chimeric protein at an early stage of the disease or prior to its onset would act to inhibit infection of CD4 + lymphocytes. As a therapeutic, administration of CD4-Ig chimeras to individuals infected with HIV would inhibit extracellular spread of the virus. The dosage ranges for the administration of the chimeric protein of the invention are those to produce the desired effect whereby symptoms of HIV or HIV infection are ameliorated. This amount administered is selected so as to maintain an amount which suppresses or inhibits secondary infection by syncytia formation or by circulating virus throughout the period during which HIV infection is evidenced such as by presence of anti-HIV antibodies, presence of culturable virus and presence of -24 antigen in patent sera. The presence of anti-HIV antibodies can be determined through use of standard ELISA or western assays for example, anti- gpl20, anti-gp41, anti-tat, anti-p55, anti-pl7 antibodies, etc. The dosage will generally vary with age, extent of the infection, and counterindications, if any, for example, immune tolerance. The dosage can vary from 0.01 mg/kg/day to 50mg/kg/day, but preferably 0.01 to 1.0 mg/kg/day. The chimeric molecule can be administered intravenously, intraperitoneally, intramuscularly, or subcutaneously. if administered parenterally, it can be by single injunction (e.g., bolus) or by gradual perfusion over time. The proteins of the invention may be used in combination with other agents, for example, in association with agents directed against other HIV proteins, such as reverse transcriptase, protease or tat. An effective therapeutic agent against HIV should prevent virus medicated as well as cell to cell transmission of infection. The proteins may also be used in combination with other anti¬ viral agents, for example, azidothymide (AZT) . The proteins of this invention can also be used as reagents to identify natural, synthetic or recombinant molecules which act as therapeutic agents or inhibitors of CD4 + cell interactions. For example, the proteins _an be used in screening assays, such as assays for protein interaction measured by ELISA based methodologies to assay for competitors of the CD4 receptor surface domain. Based on in vitro data showing that soluble CD4 proteins bind to cells expressing HIV env proteins, the proteins of the present invention can also serve as selective targeting molecules for HIV infected cells in vivo. As a target specific carrier protein, CD4-Ig proteins can serve, for example, as the carrier protein for delivery of cytotoxic agents to the infected cells, including the delivery of liposome formulations. The examples which follow are illustrative, and not to be construed as limiting of the present invention. EXAMPLES Enzymes used in the genetic manipulations were obtained from commercial sources and were used substantially in accordance with the vendor's instructions. Except where otherwise indicated, procedures were carried out substantially as described by Maniatis et al.. Molecular Cloning. Cold Spring Harbor Laboratory, 1989. Example 1 Preparation of CD4-IqG Chimeras Using recombinant DNA manipulations, the coding sequence for the V1V2 domains of CD4 are followed by the Hinge-CH2 or Hinge-CH2-CH3 regions of human in various plasmids, which function in various, host cells. The V1V2 region contains amino acids 1-183 (see Maddon et al.. Cell 42.: 93-104 (1985) and Littman et al.. Cell, 55:541 (1988)). The Hinge-CH2 and Hinge-CH2-CH3 regions comprise IgGl amino acids. 97- 228 and 97-330, respectively (see Ellison et al., NAR 10::4071-79 (1982). A) VlV2-hCH2DHFR and VlV2-hCH2CH3DHFR (CHO cells, : The expression vector ST4184.DHFR, disclosed in EP- A-331,356 (published September 6, 1989), was modified to create pl-asmid V1V2183DHFR: Plasmid ST4184.DHFR was cut with EcoRI and Nhel. and a 682 base paid fragment, containing nucleotide 1-682 of the CD4 DNA was isolated. This fragment was ligated to a synthetic linker encoding CD4 amino acids 177-183 followed by a TAΛ termination codon. In addition, the synthetic linker created a Hindlll site by changing nucleotides 693 (G to A) and 696 (C to T) of the CD4 sequence. These nucleotide changes do not alter the amino acid sequence. The resulting fragment, which is flanked by EcoRI and Xbal ends, was ligated into the EcoRI and Xbal site of another sT4184.DHFR plasmid. The sequence of the synthetic linker is substantially as follows: 5' CTAGCTTTCCAGΛΛAGCTTCCTΛAT 3 ' 3' GΛAAGGTCTTTCGΛAGGATTAGATC 5' The resulting plasmid, V1V2183DHFR, contains the mouse 3-globin promoter, DHFR gene, SV40 poly A region and the CD4 coding sequence for amino acids 1-183. V1V2183DLHFR was subsequently linearized by Hindlll. which cuts after the V1V2 coding sequence. Next, a Banll-Aval fragment which encodes the Hinge- CH2 region (nucleotides 299-677, Ellison et al., supra) was isolated from human IgGl cDNA. (The Hindlll site was introduced by PCR mutagenesis.) Each fragment was ligated into the Hindlll site of a different V1V2183DHFR plasmid with synthetic linkers: i) A Hindlll-Banll linker; to ligate the 3' end of the V1V2 gene with the 5 1 end of Hinge CH2 or Hinge-CH2CH3. 5' AGCTTCCΛAGGTGGΛGCC 3' 3' AGGTTCCACC 5' ii) An Aval-Hindlll linker; used to introduce a stop codon after the CH2 coding sequence and to join the 3* end of the Hinge-CH2 frage ent with the 5'end of the V1V2 sequence. 5' CCGΛGAGTAGTGACTGCΛGA 3' 3' CTCATCΛCTGACGTCTTCGA 5' These constructs maintain the correct reading frame without the addition of non-CD4 or non-IgGl amino acid residues at the Hindlll junction and are herein referred to as VlV2-hCH2DHFR and VlV2-hCH2CH3COS, respectfully. B) VlV2-hCH2COS and VlV2-hCH2CH3COS (COS Cells. : The vectors VlV2-hCH2DHFR and VlV2-hCH2CH3DLHFR were digested with Nhel. Fragments of 1,817 and 2,147bp, respectfully, were isolated which comprises the carboxy end o f V2 the complete Hinge-CH2 or Hinge-CH2CH3 coding region, the bovine polyA region, the mouse .-globin promoter, and part of the mouse DHFR coding region. The vector V1V2183COS2 (described below) contains the Rouse sarcoma virus LTR, the SV40 early promoter, VIV2, the SV40 poly A early region, and the mouse 3-globin promoter operatively linked to the mouse DHFR coding region. When V1V2183COS2 was digested with Nhel, a 1,723 bp fragment containing the carboxy end of V2 to the mouse DHFR coding region was removed. The Nhel fragments from VlV2-hCH2DHFR and VlV2-hCH2CH3DHFR were then ligated into the Nhel sites of vector V1V2183COS2, which restores, in the correct reading frame, the carboxy end of V2 and the DHFR gene, respectively. The resultant vectors are herein referred to as VlV2-hCH2COS and VlV2-hCH2CH3COS, which was used to transect COS cells. Construction of V1V2183COS: To create plasmid V1V2183COS2, plasmid V1V2183COS2, plasmid V1V2183DHFR was digested with EcoRI and Xbal. The EcoRI site was filled in, and a 707 bp fragment consisting of the V1V2 coding region was isolated. This fragment was ligated to plasmid Rst4COS2 (described below) , which had been cut with Smal and Xbal to delete the sT4 coding region. The resulting plasmid was V1V2183VOS2. Construction of Rst4COS2: To create plasmid Rst4COS2, plasmid st4DHFR (Maddon et al., PCT/WO88/01304, published February 25, 1988) was digested with Smal and EcoRI. The EcoRI site was filled in and a 338 bp fragment containing the SV40 early promoter was isolated. This fragment was ligated into Rst4DHFR (see below) , which had been cut with BamHI and the sticky ends filled in. The resulting plasmids were screened for orientation, and one in which the SV40 early promoter is in the opposite orientation to RSV LTR was selected as " plasmid Rst4COS2. Construction of RST4DHFR: Plasmid TND (Connors et al., DNA. 1_: 651-661 (1988) was digested with Bglll and Hindlll. A 600 bp fragment containing the Rous Sarcoma Virus (RSV) LTR was isolated. Using a commercially available linker (New England Biolabs, Beverly, MA) consisting of a Hindlll "sticky end", a Smal site, an a EcoRI "sticky end", the RHV LTR fragment was ligated to plasmid ST4DHFR (Maddon et al., supa) which had ben digested with Bglll and EcoRI to delete the SV40 early promoter. C) 0mpAylV2-hCH2 and OmpAVlV2-hCH2CH3 fE. coli) The vectors VlV2-hCH2DHFR nd VlV2-hCH2CH3DHFR were digested with Afllll and Xbal. Isolated were fragments of 746 and l,041bp; respectfully which contains part of VI (approximately 73 amino acids) and the complete Hinge-CH2 or Hinge-CH2CH3 coding regions. These Aflll-Xbal fragments replaced the Aflll-Xbal fragments of plasmid OmpΛVlV2, disclosed in EP-A-331,356 (published September 6, 1989), to create a vector containing the lambda p L promoter and the ompA signal sequence fused to either VlV2-hCH2 or VlVl-hCH2CH3. The vectors, which function in E. coli are here in referred to as 0mpAvlV2-hCH2 and 0mpAVlV2-hCH2CH3. D) VlVl-hCH2-Tkk, VlV2-hCH2-KA, VlV2-hCHLStrept and VIV2-hCH2CH3strent (Stre tomyces_ The vectors VlV2-hCHL2DHFR and VlV2-hCH2CH3DHFR were digested with Afllll and Xbal. Isolated were fragments of 746 and l,041bp, respectfully which contains part of VI and the complete Hinge-CH2 or Hinge-CH2CH3 coding regions. These Afllll and Xbal. fragments replaced the AFIII -Xbal fragments of plasmid 12B1. Plasmid 12B1 contains V1V2 operatively linked to the Streptomyces lividans longisporus trypsin inhibitor (LTI) promoter and signal sequence (see, EP-A- 264,175, published April 20, 1988), as well as Streptomyces replication functions as found on plasmid pIJ351 (Keisert et al., Mon Gen Genet 185:223 (1982)). The resultant vectors herein referred to as VlVl-hCH2strept and VlV2-hCH2CH3strept, comprise the LTI signal sequence fused or linked to V1V2- Hinge-CH2 or VlV2-Hinge-CH2CH3. These vectors are designed to function in Streptomyces and export VlV2-hCH2 or V1V2- hCH2CH# into the culture medium. However, the naturally occurring LTI protein i expressed as a prepro protein, that is, the signal sequenc is cleaved upon secretion and the pro-sequence is cleave extracellularly. As a result, the LTI protein isolated ca have heterologous amino terminal ends. The expression o VlV2-hCH2 or VlV2-hCH2CH3 from VlV2-hCH2strept and V1V2 hCH2CH3strept, respectively, produce this anomaly; i.e proteins with heterologous amino terminal ends. The rati of pro-protein to mature protein i.e. lacking all signa sequence amino acid residues can be shifted by variations i culture conditions, e.g. pH, dissolved 0 2 etc. is an alternative, the wild-type LTI prepro sequenc i.e. signal sequence plus pro-peptide) has been modified t produce proteins with homogeneous amino terminal ends. Th wild-type prepro sequence comprises the following amin acids: Met-Arg-Asn-Thr-Ala-Arg-Trp-Ala-Λla-Thr-Leu-Ala-Leu-Thr-Λla Thr-Ala-Val-Cys-Gly-Pro-Leu-Thr-Gly-Ala-Ala-Leu-Ala-i-Thr Pro-Ala-Ala-Ala-Pro-Ala-Ser; wherein i denotes the cleavag site between the signal sequence and pro-protein. In on construct, the pro-protein sequence was deleted and replace with Thr (T) . (The amino terminus of the mature CD4 protei is KK) . Hence the amino terminus of this chimeric CD4 Immunoglobulin protein is Thr-Lys-Lys (TKK) . In anothe construct, the pro-peptide sequence and the first two amin acids of the mature chimeric protein were deleted and replace with Lys-Ala (KA) . The vectors that encode VlV2-hCH2 ar hereby referred to as VlV2-hCH2-TKK and VlV2-hCH2-KA, respectively. These vectors encode VlV2-hCH2 proteins havin a homogeneous amino terminus of Thr-Lys-Lys or Lys-Ala, respectively. The complete nucleotide sequence for vector V1V2 hCH2-KA and the corresponding amino acid sequence for th chimeric ' protein VlV2-hCH2-KA is disclosed as follows: 1 GOG30CAAΪA CGCAAACCGC CTC1XCGGC GCGTIG3XG AITCAITAAT GCAβCIOGCA 61 03 GG_7IT CCCG___DGGA ASGOGGGCAG ICΞAGOQCAAC GCAAITAAIG T_2_3_T__3CT 121 C_ '1___ 1__3 GC_fiCCCC____ L i , J_A_3CIT TA_ _C_TCCG GCiU_i__IGT IGIGTQGSAT 181 Η3K_M3CQ3A TSACAATTTC ACACAGSAAA C__ -_-_G_C C_l__-___CG __A_π___GCT 241 CQ3XAGCIC TXICGGGGA GXG3C0GQ3 AAOCSGCCCG eAGL iUL 3oι α J ι_JCiuι_; TOGΓAAICAT GIBAIEKCGA ΠACIΠΞACG G-BCCAIGAA OGCASGQGGG 361 IGOKΞGGGSG TCEOXGACA GCTCAACOGG AAIUI IIJAG GXIAITAAC TAAGOGCSGG 421 AAAΪCGGCCA CTI GCTGCT ICSOSCGAIC AAGΪ-AGOGCT (.AGITOCAGS G3TCAIG0GG 481 rosAACiciG TcscπcGOG OΏC KΓI-; AACACGCAAG GΠACIGAAA CACAIΌGGGΓ 54i αaG3___rrr ITOGCGSCG GE IΌOGT GCGACTCOOG σcocosσic CGGCACCAAA 601 COGSaCGGG T03G3 0C CTOGAAI-CT G0GGA__33_T GCSCSCA A G 033 ASC A l>Met Arg Asn Thr 660 GX OX -333 GCA. GX A_X OC GX C1C A03 OX AX GX GIC IGC G_Α OX 5>Ala Arg Trp Ala Ala Thr Leu Ala Leu Thr Ala Thr Ala Val Cys Gly Pro 711 CDC ACC G__. GX G33 CIC GX AAG GX GIG GIG CIG GX AAA AAA G33 GAT 22>Lβu Thr Gly Ala Ala Lau Ala Lye Ala Val Val Lau Gly Lys Lys Gly Asp 762 KΑ GIG (ΞAA CIG AT T33T ACA _CT ΕX CSG AG AAG AX AIA. C_SA TIC OC 39>Thr Val Glu Lau Thr Cys Thr Ala Sβr Gin Lys Lys Ser Me Gin Phβ His 813 103 AAA ABC TX ASC <3G __IR. AAG AIT CIG GGA. ART CAG GX IOC TIC TEA 56>Trp Lys Asn Sar Asn Gi Ma Lys Ma Lau Gly. Asn Gin Gly Ser Phe Leu 864 ACT BA G3T 0_A Ε AAG CDS AST ©T OX GCT ( 3C TEA A_Ξ& AGA. AQC CIT 73>Thr Lys Gly Pro Sar Lys Lau Asn Asp Arg Ala Asp Sar Arg Arg Ser Leu 915 TQ3 (____. CSA G3A AAC TIC OX CIG A1C AIC AAG AAT CIT AAG __ _. GAA CSC 90>Trρ Asp Gin Gly Asn Pha Pro Lau Ma Me Lys Asn Leu Lys lie Glu Asp 966 TEA GffiT ACT TBC AIC TGT CΞAA GIG GSG QC CSG AAG GSG GSG GIG <3A TIG 107>Sβr Asp Thr Tyr Me Cys Glu Val Glu Asp __ln Lys Glu Glu Val Gin Leu 1017 CT__ GIG TIC G_Α TEG ACT GX AAC ICT OC ACC OC CIG CIT CSG G33 CAG 124. Leu Val Phe Gly Leu Thr Ala Asn Ser Asp Thr Hi Leu Leu Gin Gly Gin 1068 AX CDS ACC CBS ____ TIG G_G AGC OX _Σ_T GGT AGT AGC OX ICA. GIG CAA 141- Sβr Leu Thr Leu Thr Leu Glu Ser Pro Pro Gly Ser Ser Pro Ser Val Gin 1119 TGT AG3 AGT OCA A03 G3T ASA AAC A_Α CSG G33 G33 AAG ACC CIC TCC GIG 158>Cys Arg Sβr Pro Arg Gly Lys Asn Me Gin Gly Gly Lys Thr Leu Ser Val 1170 ICT CSG CIG GRG CX CSG GST 2GΣ GX AX TQ3 ACA TX ACT GIC TTG CAG 175>Ser Gin Leu Glu Leu Gin Asp Sβr Gly Thr Trp-Thr Cys Thr Val Leu Gin i22l AAC CSG AAG AAG GIG GG TIC ASA AIA ®C AIC GIG GIG CIΑ GCT TIC CSG 19? ► Asn Gin Lys Lys Val Glu Phe Lys Me Asp Me Val Val Leu Ala Phe Gin 12- ASA GCT IOC AAG GIG GSG OX ASA ICT TGT GSC AAA ACT CSC ACA. IX CCA. 2C_ Lys Ala Sβr Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr C s Pro 1323 033 TX OCA GCA CCT CΞSA CIC CIG G3303. CCG TCA GX TIC CIC TIC OX 226>Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro 1374 OCA ASA OX AAG GSC ACC CIC AIG AIC TX 033 ACC CCT CΞSG GIC ACA IGC 243>Pro Lys Pro Lys Asp Thr Leu Met Me Ser Arg Thr Pro Glu Val Thr Cys SUBSTITUTE SHEET -24- 1425 GIG G_G GIG (-AC GIG _-X C_C ____ _AC ____ -__; -_C A_^ 260>Val Val Val Asp Val Sβr His Glu Asp Pro Glu Val L s Phe Asn Trp Ty 1476 GIG C_C -_C -__3 G_G -__- C_ff -AT -_r __^ 277^Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gi 1527 ^ ^MZKE σ£ GIG GI£IG GlZ CI KJ2 GI CIS CK: C & 294>Tyr Asn Sβr Thr Tyr Arg Val Val Sβr Val Leu Thr Val Leu His Gin As 157 8 TOS -__- AAT _Σ£ AAG G_G T_C A_G T_E AAG G_Γ ΗX -^ 311>Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val S β r Asn Lys Ala Leu Pr 1629 GX OX i_JC GSG AAA A AIC TX AAA GX AAA G33 CBG OX CCA GC TP 328>Ala Pro lie Glu Lys Thr lie Ser Lys Ala Lys Gly Gin Pro Arg Glu •• 1680 CaOOGASGC TT-X-IAAlCr A__AGTCGaOC TGCBGCCGGG GGT_n_GC2G GGOOGGAGCG 1740 GGCGGASDCG TGCICIGACC TOCGGOOCGA GI___CGK2C GIGAOGGAAT GGAAGGCIGC Q 1800 ___. ι___i' ca___iG_cσr AJECTOGGOGA. CCGACTOCOG AGGOCBCGGC GGACACGATC i860 GC XCGCT GGOGCCGGGC CICGIACQX CGCTO30GGC AGGAG03GCG GCAGTAGICC 1920 CGGCIDCGGC CG_CQ00GGA TJXi- l__flC TOCGAGCOGC ACCSG3CGCA GAGCTICGCG 1980 CGGiαSGCGT α3CT53GGGT GGIGGIGCIC AIGGCCaOG AOCGIACGCG GCACGICTCG 2040 TaGCGAGGCG AGIO333CGC GSGGERCOGC CTOCAOGAAG TOOCQSCGGG GCCGACCCCG 2100 GGαaβER T COCAGGAΠΆ CTCOOQOGGC TTOGAOOOOG GOCGCCGICG GCQOGIACGΓ 2160 CSCOGACOX CGOXIACGT C_ACCGQGA_D3 AO3IACG30G GS3GG3AG03 AGTIAGIGCG 2220 AAGTG3G0CC A IUUGAGC CGGQOSECT GCCGGG03GC OCGCICCIGG 2280 CGiCGlCGIC C1GSE0ΞDQC3 TCCTQCIC_C GOOGTOGGOG • JJ LauriO. 2340 GC1GGΞGGAG GG0G30GAGC AIGICG9CG ACGCC'ICGGC CAOCICCCOC GCCGTGAGCA 24oo CCSCCACΠSΓ giαagcπaos TOGGOCAGOG ccfiGGsocx OXCROCCGΓ TΠΞCCCSOGG 2460 COQOOGGATC CTOGTπXCG UTlUJLTIT CGGCGGCCCG GGTCQOCTCG AGGKX_AGGG 2520 CGCG3CGGGT GδOOQOGTGC CAKCGHXT CQGICACGGC GSCOCOGQX ' CGCAGCICCC 2580 CGOOGTOGGC GKQXOGCC AGGAGCAGAT a_agflU_fl GGOCTOGGIG ___3C0GC0GT 2640 CGSGOOGGSG CAICTGOCQC AGGTAGCGGG TCCAΪTCGAT GQOCCG3CG XCCGGGI G 27oo cocGCTOG ciαsroαcas CGCGSGAGG TOOCTOCSG CGAGOCGADC CCGGCGGOGΓ 2760 αSTOCTOGGT CA GOCGCCG GΠ^GGTCCC CXS^CCGICC GδGGδGTTCG AAOQGGGCGA 2820 αsraxπxc GGTOQCCGTC TIGSGGTOGG CGCGGGCGRG TICGSGGGOG GGCGCCΓΓCC 2880 CGTOCEGGSΓ CTTQSCGAIG TACTCGQCGA GGTCGT GGC GICGCGCTCG GICICCAGCC 2940 GCTTGAAGIC GaCQ GTGC CQGTOGIDGG GOGIGAAGGC GGGGπGSDC TIGCGCAGGG 3000 CQ30GGICCA CAOGGACCGC CAGΗ3C00CT G0C3 0G1C GSGCGCGGCG 0CGGICG3CT 3060 CGAAGGIGGC G_ _AICTGC TTCGCGGSCC GCTCCOO IC GGICOQGOCG OCGACCSGGA 3120 CEATOGOGIG GAICTGCGGG ΕXC_AGCCGT TCA__CTGCCC CA_XGTG____ ICGGICGCGC 3180 GGAΪCAKΞCC GBGGISOCGG ATCCGGTCTC GGATCOOCTC GCQGIOGGCG GCCOGGTGCC 3240 CG'IULTIGGC CCGGCGXCG G0CCCG1GC OGCOCGIGAT CAGIHXTGG TAGGCGCOCG 3300 GOOGCCGGGG GCTGTCOGQC G1CT1CGGGG T_CCC_GG__G GGCGTCCAIG AGGICCGOGA 3360 GCOGGTOCG 5300CAIOG CGGQCCGIGA AGGTCACCAG G__AGGCGGIC CCCCCGCGCT 3420 ΕΞATOCSCTC GACCACGGCG GOGGESflCr OCTOGGOCOG CTIGIGCCGG ATCGTGGCGG 3480 OGCAGACCGG GCAGAGXAG AICCGCOCGC ACCGCAICAG GOCCAGGACC ACGGACGTTC 3540 OGGCCGOCGT CTOGQOGACG AICACGCCGG AGGCAGG3TC CAICSGGGCG CGGCOGCAGC 36oo ccriσcscQC GGCG CCG CIGAICOQOC ACAGCGICCG GCGGOGGCIG TACOGGGCGG SUBSTITUTE SHEET l_ι in in o in ϋi cπ Ui ui in n ui n tn in * _> __. ι>> (_ι ι<_( t_t ω ω o o to oo oo ω to t-» o to to oo oo -J σ> <_. In in to o _. ω ω to to (-• o o to to co j -i σi oo to _> oo ro β o _. o s o o o o oo M φ o ._ c 8 s _. oo o o _> _ θ . oo to __ o o o s o o o o o o- o- o o o o o o o o o o o o o ' 6180 CGGΏGCGSC AGCA IC Γ TICCAICIGA G ΓGCIGGAΓ CIGIGCGGGC GGCAGAACAT 6240 ACCGGTOCGC CTTAICGACT CXTOGAICGT CAACOGTIG TICAAGGGGG AGCGGAAGX 6300 CAAGACAI C GGCAICGCCC GCGICCCTCG CCCGGCCAGG TT__3GCGAIA CQCGAGCCG 6360 G0GIQ3GGAC yiCb'lCTl TOGACGGGGT GAAGAICGIC GGGAACATCG GCGOGAΪAGT 620 ACGCfi σiCG C_X__ GCTOG GSGCGCGGG GAICAICCIG GIO_____G-__ ACAICACCAG 6480 CAICQCGGAC CGGCGICICC AAAQGGCCAG CCG GGTISC GTCTICRCCC TTCOCGTCGT 6540 'LCLCLLXJGSP CGCGAGGAGG CCATOGOCIT CATDCGGGAC ____Α_3IA!IGC __GCIGAI___C 6600 GCSCAAGGOG GAIQGCGACA TTT0OGTGAA -__-ACTCG3G GSCAATC093 ATCGGCIGGC • 6660 CTIU 'IG I GQCAGOGAAA AGGGTGGGX ITXGAOCIG TICGAGGAGG CGTCTICCGC 6720 CTOGGΓΠΌC AIΠXCATGA TGAGOCBGAC CG_GTCI__-_ AAO^ΠTCCG -_ΠCCCICGG 6780 AA2DGCGCIG CACS GGA TCS -GGAA T-TECGCGGOC AACOGAΪAAG CGO 'L MT 6840 cci GGffiGC CGGΠXXTC GSCCICGATΓ CGICAGIGM. GAICSCCCOG A__AGCG___TC 6900 AAGGGGTΠG CGGGΓCCCGG TCGGOGOCGG GOGQGGGSGG CAGGf&XGC α__ _iGcc 6960 TCTGGGSCGG GC0G3SCGGC AGGGGGMCG GOGGCCGGGC CS-GCIGCSGG CATCCAAGCT 7020 TOGCZ GGC CG'I GTI IΑ CAACGTOGTC AOGGGAAAA OCdOQCGI ACCCAACITA 7080 A1U3X 1 C AGCA__A_CCCC (JLTl'llJUU A GC'iGGCG AA T_GCG_AG£G GCCC_3C_CCG 7140 Aicmiiunc RS__EU_____M I_OCA_ GΓT GCGCAGCCIG AAIGGOGAAT GGCGCCIGAT 72oo uuub'jm - ciu riACGC ATOΠSΓGOGS ramcαc OGCAIATQGΓ GCZ CICAG 7260 Z AICΓGC CIGAIGCCG 2_I_¥31___AG CXSGCCOCGA CAOCOQCCAA CACCOGCIGA 7320 OJJUUX'1-A CϋJU lGC 'IGdϋOCGGC ATOOGCπSC AG AGCTO TGADCGITC 7380 C_3GG__JClGC lG'mi GA GGTI K_ADC GaX_A____00G AAACGOGOGA GACGAAAGGG 7440 CCTOGTOAIA CUULJIAL TAIA TΓAA I__XA_K_A_CA AIAATOGTIT -_7_I_AG_OG_C 75oo AGGΓGGCRCΓ ΓΠΌQGQGAA ATOTOCGOGG AACOCCTAΓΓ GΓΠAΠTT TCTAAAΠACA 7560 TΪAAAIATG TATCCGCICA TGAGδCAAIA AQXEGftTAA AIGCITCAAT AA__A_TIGAAA 7620 AAG3AAGSGT A_K_A____-_ C AACATIUJG I CGCIXTI' Al'lOJLTlTr TKXGGCAIT 7680 uj riυ GTΓΓΓIU 'I A_X AGAA_C GCΓGGIGAAA GIAAAAGAIG CTCAAGAICA 7740 G ΠSGGTOCA CGAGIGGGΓΓ A__A_DOGA__CT GGAICTCASC AGCG3E_AGA TCCΠGAG^G 78oo I ΓIUJOJU: G BCS-AGGΓΓ TΓCCAAIGAΓ G__3CACITIT AAAGΠCTX ΈAIGTGGCGC 7860 GGTAΓEATOC OSIAΓΓGACG CCGGGCA_«_A. GCAACTCGST CGCCGCAIAC ACTATTCΓCA 7920 GAATOACTTG GΓIGRGEACΓ CZC PGΣ&C AGAAAAGCAΓ CΓIACQGAIG G____X__CAG 7980. ASG_______A TQCAGTGCIG CCAIASOCAT GAGIGA ASC ACIGOQGOCA __CT__ACΓICT 8040 GSCAACGATC GGBGGAOCGA AGGAGCTAAC' U TITITI CACAACAIGS GGGAICAIGT 8100 AAL'LT JLT- GA UGTIGQ3 AACOQGAGCT GAAIGAAGX AIACCAAACG ACGAGOGTGA 8160 OCCBCGAIG CCT3IAGCAA T3GCAACAAC GTTGCOCAAA -____TAAC___ GCGAACIACT 8220 LACTCESG TCCCGGCAAC AATTAAΪAGA CCGGAIGGAG GCGGAIAAAG TTGCAGGACC 8280 ACΠCΓGOGC TCGGCOCΓΓC CGGCTQGCIG G ΓIAI- CΓ GAIAAAICTG GSVGCOGGIGA 8340 GULJI GG CGOGGTATCA TIXΞCMΞCACR GGGQCCAGAT QGTAAGCOCT CCCGIAICG 8400 AG_ ATCIAC ACGADQGGGA GTCAGGCAAC TAIGGAIGAA CS^AAΪAGAC AGAICGCIGA 8460 GAIAGGTGX TCZ GATTA AGCAI'lGGiA AC_X_TCA____C CAA i'i'lAC (3_I3ffi^IAC 8520 ιτrø_πx_AT TTAAAACΓTC AITITIAAΓΓ TAAAAGGAIC TAGGTGAAGA TCCTITΓΓGA 8580 TAATCΓCATC ACCAAAAIXX CΓΓAACGIGA GTTΓΓΌGΓIC CACIGAGOGT CAGACCCCG 8640 AGAAAAGAIC AAAGGAICTT C__IXSGA_OC TTIT TTCIG CGCGEAAICT GCTGCTIGCA SUBSTITUTE SHEET 8700 AACAAAAAAA CCAOC3CTAC CAG03_IG3T TIGITnX G GAΪCA&3AGC TAOCAACICT 8760 TTTTOCGAAG GEAACIGSCT TCAGCAGAGC GCAGATA CA AAIACIGICC TICIAGIGLA 8820 G SEAGITA GGOCAOCACT I __^GAACTC TSTAGCAOCG 0_πA_A_ACC TCGCTCIGCT 8880 AAICOGI A CXSGTGSCTG CTXΞCCAGIGG CGATAAGICG 'IG'lLTiACOG GGTIGGACTC 8940 AAG&CGAΪAG TTaCCGGAΪA. AGGCGCAGCG GTO33GCIGA ACGG3G3GIT CGΣG 2CKΑ 9ooo GCCCAGCΠG GSGCGASCGA O TACACCGA ACIGAGAIAC 03 GCGTG AGCTAIG GA 9060 AAGCGCCACG CTTOCOa G GGAGAAAG3C GGACAGGEAT C033TAAGCG GCAG3GI03G 9120 AACAGGAGSG CQCACGAGG3 AGCTTOCAGG GGGAAAOGX IGGTAICTTT __T_GIOCIGT 9180 CGSSΓΠCGC aaxzxsc TTCAGCGICG AI ΓΠGTGA QC-TCGTCSG GGGSGOGGAG 9240 CCTAIGGAAA AACGCCAGCA A___CGSCCTT TTisαsGirc I GU TΠ GCTGGOCITΓ 93oo TGCiCAC iG 'ΓI ΠU- IG CGΓΓAΪCOOC IGA CTGΓG GAIASCCGTA TTACOGOCTT 9360 TG?_ΠG_GCΓ GAISCCGCTC GCCGOGOOG Aaα_aooG G CΏCAGOGAGΓ CAGIGAGOGA 9420 GGAAGCGGAA GA Wherein the signal seguence begins at nucleotide 648 and ends at nucleotide 731; VI begins at nucleotide 732 an ends at nucleotide 1286, the Hinge region begins at- nucleotid 1287 and ends at nucletotide 1331, CH2 begins at 1332 an continues to the end of the coding seguence. Example 2 Expression of CD4-IqG Chimeras A) V1V1--.CH2DHFI. and VlV2hCH2Cr_.3DHFR in ClfO cells: VlV2-hCH2 was transfected into CHO cells vi electroporation. 15μg ofVlV2-hCH2 DNA suspended in 15 μ phosphate buffered sucrose (PDSucrose) (272mM sucroe, 7 m sodium phospate pH7.4, 1 mM MgCl2, sterile filtered) was mixe with 1.0 x 107 CHO cells (0.8ml) and incubated on ice for 15 min in a Gene Pulser cuvette (Dio-Rad, Richmond, Calif.). Th cuvette was then placed in the electrode chamber of the Gen Pulser (Dio-Rad) and one pulse of 700 volts/3 / xFd (tim constant=0.7) was delivered to the cells. The cells wer placed on ice for 10 minutes and then diluted to 10 ml wit Growth medium (containing lxITS [insulin, transferrin, selenium; Collaborative Research, Bedford, MAJ , lx Lipide [Gibo, Grand Island, NY]). Cells were then plated onto 9 well microtiter plates at 3 x 10 3 cells/well on a total of plates. After 48 hours the medium was changed to Selectio SUBSTITUTE SHEET -28- medium (i.e., nucleoside-free Growth medium) for DHFR selection. Cells were maintained on Selective medium for approximately 4 weeks. Western blot analysis was performed using 15% SDS-PAGE gel electrophoresis and anti-sT4 polyclonal antibodies. The VlV2-hCH2 gene product was identified by a single positive band at a molecular weight of approximately 4 0 ,000 daltons. Cells from positive wells were moved to 24 well plates and maintained on Selective medium, until ready for scale up. The two clones (3F8 and 2F6) were chosen for scale up. Clone 3F8 was amplified in Selection medium containing 50 nM methotrexate. Cells were replated onto 96 well microtiter plates at 3 x 10 3 cells/well. Amplification was started at methotrexate levels of 50 nM and were to be increased on each successful round of amplification according to standard protocols. Similarly, the VlV2-hCH2CH3 vector was transfected into CHO cells by electroporation. Positive clones were selected by growing the cells on nucleoside-free medium and screening the supernatant of healthy wells by Western blot analysis using anti-sT4 antibodies. The resultant positive clones were then amplified using increasing amounts of methotrexate. B) VlV2-hCH2COS and VlV2-hCH2CH3COS in COS cells: COS cells were obtained from the ATCC (Rockville, MD) and grown according to ATCC recommended protocols. The. cells were then transfected with 10 μg of plasmid DNA in 2.5% Nu serum (Collaborative Research, Cambridge, MA) in DMEM (Dulbecco's minimal essential medium) )GIBCO Grand Island, NY with 400 g/ml DEAE-dextran (Pharmacia) and lOOuM chloroquine (Seed et al., Proc Natl Acad Sci 84:3365-3369 (1987)). Following a 4 hour incubation at 37°C, the medium was removed and the cells were treated with 2ml of 10% dimethylsufoxide in PBS (phosphate buffered saline) for 3 min at 25°, the medium was removed and replaced with DMEM. Both VlV2-hCH2 and VlV2-hCH2CH3 were expressed and exported into the culture media. In a standard transfection -29- in a 6 mm dish (about 10 6 cells) , 20μg of VlV2-hCH2 had accumulated int he medium at 70 hrs post-transfection. Approximately 0.4 / ιg of material was present in the nuclei- free cell lystate. For comparison, the corresponding values for sT4, a soluble CD4 protein which had been expressed at high levels in several mammalian cell types were 20μg in the supernatant and lμg in the cell lysate. The VlV2-hCH2 protein in the medium migrated as two closely spaced fragments. Under nonreducing conditions, the VlV2-hCH2 migrated principally a monomer although approximately 10% of the sample appeared to migrate as a dimer. The VlV2-hCH2 protein in the medium bound to gP120 coupled to a sepharose resin. VlV2-hCH2CH3 was analyzed in the same manner as described above. This protein also expressed well as a secreted product, accumulating to 20-25μg in the medium and l-2μg in the cell lysate. The protein in the medium migrated as a single fragment of approximately 50 kD by SDS/PAGE under reducing conditions, while the cell lysate sample migrated as two equivalent bands of approximately 40 and 50 kD. Under nonreducing conditions the VIV2-hCH2CH3 migrated principally as a single fragment with a molecular weight consistent with dimer formation. This protein bound to gpl20 coupled to sepharose. C) OmpAVlV2-hCH2 and OmpAVlV2-hCH2CH3 in E. coli The E. coli lambda lysogens, AR58 (Debouck et al., EP-A- 0,216, 747, published Aril '1, 1987) and Λrl20 (Mott et al., Proc Natl Acad Sci 82: 88-92 (1985)) were transformed with OPMAVlV2-hCH2 and OmpAvlV2-hCH2CH3, respectively, using standard procedures. Expression of VlV2-hCH2 and V1V2- hCH2CH3 in strain AR58 was accomplished by raising the temperature of the culture media from 32*C to 42*C (see for example, Rosenberg et al., Meth Enzyπtol 101:123 (1983)). The expression of VlV2-hCH2 and VlV2-hCH2CH3 in strain ARI20 was accomplished by the addition of nalidixic acid (Nal) to the culture media (see for example, Mott et al., Proc Natl Acad Sci 82:88-92 (1985) as follows. A culture of ΛR120 was grown at an optical density (at 650nm) of 0. 4 -30- absorbance units at 37"C whereupon nalidixic acid was added to a final concentration of 50μg/ml. The culture was maintained at 37*C in a shaker incubator for approximately 5 hours at which point the cells were centifuged and subsequently chilled to stop the induction. For both the heat and Nal inductions, the cell pellets and the clarified culture media (i.e. centrifuged media) were assayed for VlV2-hCH2 and VlV2-hCH2CH3 expression. Both chimeric proteins expressed well, however only a small percentage of VlV2-hCH2CH3 was detected in the culture media (i.e. exported). D) VlV2-hCH2-TKK, VlVl-hCH2-KA, VlV2-hCH2 strept and VIV2-hCH2CH3strept in Streptomyces Plasmids VlV2-hCH2-TKK, VlV2-hC2strept and VlV2-hC3strept were transformed into S. lividans strain 1326 (Bibb et al., Mol Gen Genet 184:230 (1981)) using standard procedures (see Hopwood et al. , Genetic Manipulation of Streptomyces - A Laboratory Manual. F. Crowe & Sons, Ltd., Norwich, England (1985)). Transformants were selected by overlaying the transformation plates with agar (0.4%) containing lOOμg/ml thiostrepton. Colonies which express the protein(s) of interest are then grown in trypticase soy broth media supplemented with 5 μg/ l thiostrepton. All of the chimeric proteins were secreted into the culture media, however the VlV2-hCH2 constructs were expressed at a higher level than the VlV2-hCH2CH3 construct. VlV2-hCH2-KA was expressed at approximately 14 mg/L. Example 3 Characterization of CD4-IqG Chimeras A) Subunit Structure: The native molecular form of secreted VlV2-hCH2- KΛ and VlV2-hCH2CH3 were analyzed by electrophoresis on a 15% SDS-polyacrylamide gel under non-reducing conditions. The protein bands were identified by Western Blot analysis and the results are shown in Table I. B) Antibody Recognition: All of the chimeric proteins made by the present invention are recognized by both anti-CD4 antibodies (Deen -31- et al., Nature, 331:82-84 (1988)) and anti-human IgG F receptor antibodies (Cappel, Malvern, PA.) See Table I. C) gpl20 Binding: The chimeric proteins made in S. lividans. COOS an CHO cells all exhibited gpl20 binding (refer to Table I) either by the immunoprecipation assay as described by Arthoo et al. -Cell. 57:469 (1989)) or by binding to gpl2 immobilized to sepharose as described below: Samples are diluted to a total volume of 300μl wit precipitation (ppt) buffer (phosphate buffered salin containing 0.5% dry milk and 0.1% NP40) . A 50% slurry o gpl20 sepharose beads (100μl, described below) of wa adde to the diluted sample and incubated at 4'C for 60 min. Th sample was spun (30 sec) in a microfuge to precipitate th chimeric protein/gpl20-Sepharose complex. The complex wa washed 5 times with 400μl of ppt buffer and once with ic cold phosphate buffered saline. The complex was -pun dow again and resuspended in 60μl loading buffer (Laemmli, Nature. 227: 680 (1970)), boiled for 5 minutes and loaded onto a 15 polyacrylamide gel followed by Western blot analysis. Th chimeric proteins expressed in E. coli were not tested fo gpl20 binding. gpl20 was obtained from in-house sources. It i also available commercially, e.g. , American BioTechnology, Cambridge, MΛ. The sepharose beads were prepared as follows: 1.0 liter of Sepharose C1-6B (Pharmacia Fine Chemicals, Piscataway, NJ) was reacted with 52.5 ml epibro ohydrin in basic solution of 0.5N NaOH/30% tetrahydrofuran (THF) at 40" for four hours. The activated sephrose was collected b filtration on a sintered glass funnel and was washe extensively with 30% THF to remove unreacted epibromohydrin. The product was further washed with water until the washin was neutral. This gel was then resuspended wit ethylenedia ine (50ml) overnight at room temperature. The gel was filtered and unreacted ethylenediamine was removed b washing the gel with 0.1M acetic acid followed by water. Th gel was resuspended in 1.0 liter of water, then was reacte with succinic anhydride (25 g ) at pH 6.0. The gel was f urt h er washed with 1.0 liter sodium carbonate solution (0.2M) followed by water until the washing was found to be neutral. This gel was finally washed with isopropyl alcohol and stored at 4'C for further use as a moist powder. D ) Protein A & Protein G Binding: The VlV2-hCH2 chimeric proteins expressed in S. lividans and COS cells do not bind to protein A or protein G . In contrast, the VlV2-hCH2CH3 chimeric protein expressed in S . lividans and COS cells do exhibit binding affinity to both proteins A and G. albeit at different affinities. The VlV2-h CH2CH3 produced in Streptomyces has a lower affinity for proteins A and G compared to the VlV2-hCH2CH3 produced in COS ce411s. The results are summarized in Table I. Table I. Characterization of CD4/I G C hin-eric Prot_->_n<= Subunit Antibody Protein A & Structure Recognition anti-CD- V l V2-hCH2s. lividans monomer anti-Fr- gp 120 Protein G Binding Binding + + VlV2-hCH2 COS monomer and dimer 10 ScH_- UVidanS mθn °™ « (weak) V1V2- COS dimer hCH2CH3 -34- Examole 4 - Construction of fusion plasmids TKK-VIV2. TPAA AA-VIV2 AND and KA-VIV2. Fusion plasmids TKK-VIV2, TPAΛ-VIV2, TPAΛΛ-VIV2 AN KA-VIV2 were constructed as follows from plasmid 12Bl. Construction of plasmid 12B1. Plasmid 12B1 contains VIV2 operatively linked to the Streptomyces lonqisporus trypsi inhibitor (LTI) promoter and signal sequence (see EP-A-264, 175, published April 20, 1988) as well as Streptomyces replication functions as found on plasmid pIJ351 (Keiser et al. Mol Gen Genet 185:223 (1982). The parental plasmid 12B1 was constructed as follows. The Bbvl cleavage site within the coding sequence for the CD4 signal peptide (between nucleotides 148 and 149 of the CD4 DNA sequence; Maddon, et al. Cell 4.2: 93-104 (1985)) was moved by site directed mutagenesis (Kunkel, Proc. Natl. Acad. Sci. USA 82: 488-492)) such that the Bbvl cleavage site was placed between nucleotides 150 and 151. This mutation (named 1478) was inserted into a sCD4 minigene which contained the coding sequence for amino acid residues 1 - 129. An EcoRI + Hindlll fragment containing the 1478 mutation was transferred from M13mpl8 into pUC18 to generate pUCVlpV2(1478) . Following Bbvl digestion of pUCVlpV2(1478) , it was treated with the Klenow fragment of DNA poly erase I to fill-in the 5' single-stranded sequence, then digested with Hindlll. The Hindlll-blunt end fragment resulting from these manipulations was cloned into pLTI450 which had been digested with AccI, treated with the DNA polymerase I Klenow fragment and digested with Hindlll. The resulting plasmid, 12B1/1477, contains a sCD4 minigene (amino acid residues 1-129) fused to the coding sequence of the LTI signal sequence such that the expressed V1V2 protein will contain at its amino terminus the 6 amino acid LTI pro peptide plus residues 1 and 2 of the mature LTI protein. A Streptomyces replicon and selectable marker were cloned into 12B1/1477 by inserting pIJ351 (Kieser, et al., Mol. gen. Genet. 185:223-238 (1982) using the unique PstI site within both plasmids. To create a complete V1V2 minigene (amino acid residues 1-183) in the 12B1/1477 plasmid -35- an Aflll + Xbal fragment from DHFR V1V2 18 #7 in Example 1 was inserted into 12B1/1477 which had been digested with Aflll and Xbal. The resulting plasmid was 12B1. Construction of plasmid PLTI450: A 0.92 kb Sacl-Kpnl fragment containing the LTI gene was inserted into pUClβ which had been digested with Sad and KpnI. This plasmid, pLTI520, was partially digested with EagI and totally digested with Sail then ligated to a synthetic linker (2x stranded) which had EagI and Sail ends: 5•-GGCCGCCGCCCCCGCG (SEQ ID NO:5) CGGCGGCGGGGGCGCAGCT-5• (SEQ ID NO:6) Plasmids obtained from this ligation where screened for insertion of the synthetic linker into the EagI site located approximately 0.5 kb from the Sad site. This EagI site is located at base pair 86 with respect ot the 5' end of the LTI gene. The resulting plasmid contains the LTI promoter and the coding seguence for the signal peptide and signal peptide cleavage site. To create the fusion plasmids TKK-VIV2, TPAA-VIV2, TPAAA-VIV2 and KA-VIV2 oligonucleotide mutagenesis was used to delete the LTI pro peptide coding sequence and create the amino acid coding seguence of choice following the signal peptide cleavage site. The M13 single stranded DNA used for these mutagenesis was mpl8VlV2/12Bl which was created by inserting a 1.1 kb ffcoRI-Xbal fragment from 12B1 into M13mpl8 digested with EcoRI and Xbal. Summarized in Table 3 are the oligonucleotides used for site directed mutagenesis. Table 3 - Oliqonucleotides used to create V1V2 derivatives Derivative Oliαo- Oligonucleotide Nucleotide Sequence 5'GCCCAGCACCACTTTCTTGGTGGCGAGCGCGGCTCC-3• 5'-GCCCAGCACCΛCTTTCTTAGCGGCCGGGGTGGC-3 ' 5'-GCCCAGCΛCCACTTTCTTAGCAGCGGCCGGGG-3 ' 5 • -GCCCAGCΛCCΛCGGCCTTGGCGAGCGCGGCTCC-3• In Table 3, nucleotide sequence 2214 is SEQ ID NO:7; nucleotide sequence 2253 is SEQ ID NO:8; nucleotide seuqence 2254 is SEQ ID NO:9; nucleotide sequence 2216 is SEQ ID NO:10. -36- A 0.7kb EcoRI-AflU fragment isolated from the RF form of the mutagenized mpl8VlV2 was exchanged with a 0.75kb EcoRI- Aflll fragment from 12B1 to generate plasmids pVlV2-2214 (or PTKK-V1V2), pVIV2-2253 (or pTPAA-VIV2) , pVlV2-2254 (or pTPAΛA- VIV2) and pVlV2-2216 (or pKA-VlV2) . pVlV2-2214, also named PTKK-V1V2, was created using oligonucleotide 2214 as shown in Table 3 and contains the LTI signal sequence linked to the modified propeptide sequence threonine, and the modified propeptide sequence threonine is linked to V1V2. pVlV2-2253, also named pTPAA-VlVl was constructed using oligonucleotide 2253 as shown in Table 3 and contains the LTI signal sequence linked to the modified propeptide sequence thr-pro-ala-ala. PV1V2-2254, also named pTPAAA-VlV2 was constructed using oligonucleotide 2254 as shown in Table 3 and contains the LTI signal peptide sequence linked with the modified propeptide sequence thr-pro-ala-ala-ala, which is in turn linked with V1V2. pVlV2-2216, also named pKA-VlV2 was constructed using oligonucleotide 2216 as shown in Table 3 and contains the LTI signal peptide sequence which is in turn linked with V1V2. Example 5 - Construction of ggal *KK-V1V2 fusion plasmid øgal *KK-VIV2 was constructed by mutagenesis from plasmid pøgal sT4/7. P/?gal sT4/7 was constructed as follows. Construction of pβgalsT4/7: Plasmid p*galsT4/7 was constructed from plasmids pUCst4, pIJ702 and p3SSXMCP. p3SSXMCP is a pUC9 (Viera and Messing, Gene .19:259(1982) derivative which contains the -galactosidase promoter and signal sequence. Twenty-six base pairs downstream of the coding sequence for the signal peptide cleavage site is an XmnI site (Eckhardt, et al.. J. Bacteriol. 16_9:4249(1987) ) . Inserted into this site was a synthetic linker which contains a BamHI recognition sequence (New England Biolabs, Beverly, Massachusetts) to generate plasmid p3SSX10. This vector was treated with BamHI and reverse transcriptase followed by Xhol digestion. Ligated to this vector was a fragment which had an Ncol end treated with reverse transcriptase to generate a blunt end and a Sail end. Ligation of the filled-in BamHI -37- site with the filled-in Ncol site recreated both the BamHI and Ncol sites. The resulting plasmid was p3SSXMCP. pUCsT4 was treated with Xbal and reverse transcriptase followed by Ncol digestion. This 1.1 kb Ncol-Xbal (RT) fragment was then inserted into p3SSXMCP which had been treated with Sad and T4 DNA polymerase I followed by Ncol digestion. The Streptomyces replication functions were provided by pIJ702 which was inserted into p3SSXsT4 via the unique Bglll site on both plasmids. The resulting plasmid was p/3galsT4/7. A l.6kb EcoRI-Afllll fragment from p3gal sT4/7 was exchanged with the 0.75kb EcoRI-Aflll fragment from mpl8VlV2- 2214 to generate mpl8BgalVlV2. Site directed mutagenesis was used to delete the coding sequence for amino acid residues - 9 to -1 of the CD4 signal peptide, change the arginine residues at positions -3 and -4 of the ϊgal signal peptide to asparta ate and glutamate, respectively, and delete the coding sequence for residues 1 to 8 of the mature ø-galactosidase protein. These mutations were made using oligonuclotide 2256 whose sequence is 5 » -GCCCAGCACCΛCTTTCTTCGCCGCGTCCTCTACGGCGCCTG-3• (SEQ ID NO: 11) . A 1.6kb -EcoRI-Aflll fragment from the RF form of mpl8.galVlV2-2256 was exchanged with the 0.75kb EcoRI-Aflll fragment from plasmid 12B1. The resulting plasmid, pVlV23gal- 2256 (jSgal *KK-V1V2) has the following characteristics: (1) VIV2 expression is directed by the Bgal promoter, (2) the coding sequence of the βgal signal peptide has been altered such that amino acid residue -4 is a glutamate and -3 is an aspartamate, and (3) V1V2 is fused to the 3gal signal sequence such that the predicted N-terminal amino acid sequence of V1V2 is Lys-Lys. Example 6 - V1V2 Expression in S. lividans Plasmids pVlV2-2214, pVlV2-2253, pVlV2-2254, pVlV2-2216, and pVlV2Bgal-2256 from Example 4 were transformed into S. lividans 1326 (Bibb et al., Mol. Gen. Genet. 184: 230-240 (1981) using standard procedures (Hopwood, et al., Genetic Manipulation of Streptomyces - A Laboratory Manual, F. Crow & S ons Ltd., Norwich, England (1985). Transformants wer selected by overlaying the R2YE transformation plates with 3m of 0.4% agar + lOOμg/ml thiostrepton. Transformants whic express the protein of interest were grown in trypticase so broth or ME1 + 5% HycaseSF (40g/liter glucose, 50g/lite HycaseSF, 50g/liter Hysoy, lg/liter yeast extract, lg/lite CaC04, and O.OOlg/liter CoC12 supplemented with 5μg/m thiostrepton. Cell-free supernatants used for the gpl2 binding assay and the gpl20 affinity chromatography wer harvested from cultures grown in ME1 + 5% Hycase. The V1V2 derivatives when expressed in S. lividans wer initially characterized by immunoblotting. Immunoblot Procedure - Cell free supernatants wer separated on a 15% polyacrylamide (30:0.8 acrylamide:bis) sodium dodecy] sulfate gel (Laemmli (1970) Nature 227: 680 685), then transferred to nitrocellulose (Towbin, et al. (1979) Proc. Natl Acad. Sci. USA 76: 4350-4354). Th nitrocellulose filter was processed to detect V1V2 (Brawne et al. (1985) Gene 4£: 191-201) using rabbit anti-seru prepared against denatured sCD4 protein. The bound antibod was detected with 125 I-protein A. The i munoreacting protein were visualized by autoradiography. The TKK-VIV2, TPAA-VIV2, TPAAA-VIV2, KA-VIV2 and 0gal*K derivatives were produced as a single, immunoreacting band. A doublet was produced from the KK-V1V2 derivative. One o the KK-V1V2 proteins co-migrated with V1V2 produced from CH cells. The other band, which migrated with a slower mobilit than the V1V2 reference protein, may be the result o incomplete processing of the LTI signal peptide or processin at an alternative cleavage site within the LTI signal peptide. Because of the heterogenous nature of the product expresse by the KK-V1V2 derivative no additional studies wer undertaken. The biological activity of these V1V2 derivatives was the determined by gpl20 binding. As judged by quantitative gpl20 im unoprecipitation (Arthos, et al., Cell 52:469-481 (1989), -39- the Streptomyces produced V1V2 proteins were fully active. Results of the immunopreciptation assay are shown in Table 2. ^ All of the VIV2 derivatives tested produce gpl20 binding of greater than 90%. V1V2 production of the TKK-V1V2, TPΛΛ-V1V2, TPΛAA-V1V2, and β-gal*KK derivatives was compared to that obtained from plasmid 12B1 which produces V1V2 at levels equal to or greater than 100 mg/1. V1V2 expression levels of the TPΛΛ-V1V2 and TPAAA-V1V2 derivatives were comparable to that obtained from 12B1; KA-V1V2 expression was 70% of that obtained from 12Bl; TKK-V1V2 expression was 30% of that from 12B1; βgal*KK expression was approximately 1 mg/1. Example 7 - N-terminal amino acid analysis of heterologous proteins produced bv plasmids PVIV2-2214. PVIV2-2253. PVIV2- 2254. pVIV2-2216 and PVIV2 gqal-2256. A. Purification of VIV2 from Streptomyces media for N- terminal sequencing by using GP-120 sepharose affinity column 1. Preparation of gpl20 Sepharose Purified gpl20 was immobilized on Sepharose CL-6B (Pharmacia) through amino group using active ester chemistry. Approximately 0.25 mg of gpl20 was coupled to 1ml of resin and binding capacity for sT4 was determined as 20 μg/ml. 2. Sample preparation from media. Fermentation media containing VIV2 constructs was diluted 5-fold in column eguilibration buffer (see below) , centrifuged for 15 minutes at 20,000 x g and filtered through 0.2μ Acrodisc low protein binding filter (Gelmen) . 3. Gpl20 affinity chromatography Gpl20 Sepharose column (1.6 cm x 6 cm) was equilibrated with 50mM Hepes pH 7.5, 150mM NaCl. The prepared media sample was applied to the column, washed with equilibration buffer until the absorbance reached to baseline, washed again with 50mM Hepes pH7.5, 500mM NaCl to remove any nonspecifically bound impurities. V1V2 was eluted with 0.1 M acetic acid. 4. Removal of peptide impurities from affinity purified V1V2 by using reverse phase HPLC -40- V1V2 eluted from affinity column was made to 0.05% TFA solution and applied to C3 RP-HPLC column (DuPont Pro 10/300, 4.6cm x 250 cm) equilibrated in 0.05% TFA. The column was eluted at lml/min for 60 minutes with linear gradient of o- 60% acetonitrile in 0.05% TFA. The V1V2 product was eluted as a single peak at 50 minutes. Pooled fractions were concentrated in Centricon 3 (Λmicon) and used for N-terminal seguencing and amino acid analysis. B. N-Terminal Sequence The N-terminal amino acid sequence was identified to determine if signal peptides processing had occurred at the predicted cleavage site. The V1V2 proteins were purified from culture supernatant using gpl20 sepharose affinity chromatography and reverse phase HPLC. The partially purified V1V2 preparations were further purified by polyacrylamide- SDS gel electrophoresis followed by electroelution into a polyvinylidene difluoride (PVDF) membrane in preparation for N-terminal amino acid sequencing (Matsudaria, J. Biol. Chem. 262:10035-10038 (1987)). The N-terminal amino acid sequence was determined using a gas-phase protein seguenator. This analysis showed that the V1V2 proteins TKK-V1V2, TPAAKK-V1V2, TPΛAAKK-V1V2 and KΛ-V1V2 fused to the carboxy terminus of the LTI signal peptide were correctly processed at the LTI signal peptide cleavage site. The N-terminal amino acid sequence is summarized in Table 2. The N-terminal end of the V1V2 peptide is LYS-LYS. The Bgal*KK-VlV2 derivative, however, was not processed at the natural signal sequence cleavage site. In this derivative, the amino acids preceeding LYS-LYS are derived from the coding sequence at the 3• end of the β-gal * signal peptide. Signal sequence cleavage for the R ( - 4)E/R(-3)D signal peptide mutation occured within the j Θgal signal sequence between positions -8 and -7. -41- Table 2 - Characteristics of the VIV2 derivatives gpl20 Derivative N-terminal Amino Acid Sequence Binding TKK-V1V2 Thr-LYS-LYS >90% TPAAKK-V1V2 Thr-Pro-Ala-Ala-LYS-LYS >90% TPAAAKK-V1V2 Thr-Pro-Ala-Ala-Ala-LYS-LYS >90% TPAAAKK-V1V2 LYS-LYS ND (1) KA-V1V2 LYS-Ala >90% Bgal*KK-VlV2 Ala-Ala-Val-GLu-Asp-Ala-Λla-LYS-LYS >90% (1) Not Determined. The above description and examples fully disclose the invention including preferred embodiments thereof. However, it is appreciated that the invention is not limited to the particular embodiments described above. Modifications of the methods described above that are obvious to those of ordinary skill in the art are intended to be within the scope of the following claims.

Claims: - 42 - Claims 1. A nucleic acid sequence comprising a nucleic acid sequence coding for the signal sequence of the Streptomyces lσnqisporus tyrosine inhibitor gene operatively linked with a propeptide sequence consisting essentially of an oligonucleotide coding for from one to about 6 amino acids, the sequence of said amino acids selected to result in the formation in Streptomyces of a heterologous protein having a homogeneous amino terminus after processing to remove the signal peptide formed on said heterologous protein during synthesis of said heterologous protein. 2. The nucleic acid sequence of claim 1 wherein said propeptide sequence causes processing of said heterologous protein at a position between the end of the nucleic acid sequence coding for said signal sequence and the beginning of said propeptide sequence. 3. The nucleic acid sequence of claim 2 wherein said propeptide codes for the amino acid threonine. 4. The nucleic acid sequence of claim 2 wherein said propeptide codes for the amino acid sequence thr-pro- ala-ala (SEQ ID NO:l). 5. The nucleic acid sequence of claim 2 wherein said propeptide codes for the amino acid sequence thr-pro- ala-ala-ala (SEQ ID NO:2) 6. The nucleic acid sequence of claim 1 wherein said nucleic acid sequence coding for the signal sequence of the Streptomyces longisporus tyrosine inhibitor gene comprises the sequence TGC GGA AGG ATG CAC ACA ATG CGG AAC ACC GCG CGC TGG GCA GCC ACC CTC GCC CTC ACG GCC ACC GCC GTC TGC GGA CCC CTC ACC GGA GCC GCG CTC GCC, or derivative thereof capable of acting as a signal peptide in Streptomyces. 7. The nucleic acid sequence of claim 6 wherein said propeptide sequence comprises the sequence ACC. 8. The nucleic acid sequence of claim 6 wherein said propeptide sequence comprises the sequence ACC CCG GCC GCT (SEQ ID NO:3) . 9. The nucleic acid sequence of claim 6 wherein said propeptide sequence comprises the sequence ACC CCG GCC GCT GCT (SEQ ID NO:4). 10. A nucleic acid sequence comprising a nucleic acid sequence coding for the signal sequence of the Streptomyces lonqjsporus tyrosine inhibitor gene operatively linked with a nucleic acid sequence coding for a polypeptide modified at the 3'end to code for lys-ala-. 11. A DNA vector for expressing heterologous proteins in Streptomyces which comprises a coding seguence for the heterologous protein operatively linked to a promoter and a nucleic acid sequence of claim 1. 12. The DNA vector of claim 11 wherein said coding sequence for the heterologous protein is a sequence coding for a HIV gpl20 binding region. 13. The DNA vector of claim 11 wherein said coding seguence for the heterologous protein is a sequence coding for a HIV gpl20 binding region joined to a portion of a human immunoglobulin constant region encoding a polypeptide lacking most or all of the CH3 domain. 14. A DNA vector for expressing heterologous proteins in Streptomyces which comprises a coding sequence - 44 - for the heterologous protein operatively linked to a promoter and the Streptomyces longispσrus trypsin inhibitor gene signal seguence, wherein said seguence coding for the heterologous protein is modified at its 3' end by adding bases coding for the amino acid sequence lys-ala, or by deleting bases coding for the two amino acids at the 3 * end and substituting for the deleted sequence a sequence coding for the the amino acid sequence lys-ala. _15. The DNA vector of claim 14 wherein said coding sequence for the heterologous protein is a sequence coding for a HIV gpl20 binding region and wherein the bases coding for the two amino acids at the 3 ' end have been deleted and a sequence coding for the amino acids lys-ala have been substituted for the deleted deguence. 16. The DNA vector of claim 14 wherein said coding sequence for the heterologous protein is a sequence coding for a HIV gpl20 binding region joined to a portion of a human immunoglobulin constant region encoding a polypeptide lacking most or all of the CH3 domain and wherein the bases coding for the two amino acids at the 3 1 end have been deleted and a sequence coding for the amino acids lys-ala have been substituted for the deleted sequence. 17. A method for using the DNA vector of claim 12 comprising the steps of introducing the vector of claim 12 into a Streptomyces host cell, and growing said host cell in a suitable culture medium. 18. A method for using the DNA vector of claim 14 comprising the steps of introducing the vector of claim 14 into a Streptomyces host cell, and growing said host cell in a suitable culture medium. 19. A Streptomyces cell transfected with the nucleic acid sequence of claim 1. - 45 - 20. A Streptomyces cell transfected with the nucleic acid seguence of claim 6. 21. A Streptomyces cell transfected with the DNA vector of claim 7. 22. A Streptomyces cell transfected with the DNA vector of claim 10.

Inventors: ARTHOS JAMES, BRAWNER MARY ELLEN, FORNWALD JAMES ALLAN, ARTHOS, JAMES, BRAWNER, MARY, ELLEN, FORNWALD, JAMES, ALLAN
Applicants: SMITHKLINE BEECHAM CORP, SMITHKLINE BEECHAM CORPORATION
Application year: 1991
Main classification: C07H15
IPC codes: C07K14, C07K16, C12N1, C12N15, C12P21, C12R1