Expression vector containing PL6M promoter and TAT32 ribosome binding site and host cells transformed therewith

Disclosed are expression vectors useful as vectors in recombinant methods to facilitate expression of exogenous genes in E. coli. Specifically, the disclosed expression vector has the following elements in operable linkage: the PL6m promoter, the TAT32 ribosome binding site and a gene encoding a heterologous polypeptide, Also disclosed are E. coli host cells transformed with this expression vector.

FIELD OF THE INVENTION 
This invention relates to expression of recombinant DNA sequences. More 
particularly, it relates to expression of DNA sequences encoding soluble 
CD4 (sCD4) polypeptides in microbial hosts. 
BACKGROUND OF THE INVENTION 
CD4, a normal membrane component of the T4 lymphocyte, binds gp120, an 
envelope glycoprotein of the human immunodeficiency virus (HIV). This RNA 
virus, which is responsible for acquired immune deficiency syndrome (AIDS) 
in humans, uses CD4 as its receptor for infection (Klatzmann, D., et al. 
(1984) "Selective tropism of lymphadenopathy associated virus (LAY) for 
helper-inducer T lymphocytes." Science 225:59-63). CD4 has 4 extracellular 
domains (Maddon, P., et al. (1985) "The isolation and nucleotide sequence 
of a cDNA encoding the T cell surface protein T4: A new member of the 
immunoglobulin gene family." Cell 42:93-104). A soluble molecule including 
some or all of these domains is referred to as sCD4. The two N-terminal 
domains of CD4 appear to be the most important for gp120 binding and 
proteins which incorporate this gp120 binding capability have been 
proposed as potential therapeutics for AIDS because they may target the 
protein to the virus, to HIV-infected cells, or to other species that 
might have exposed gp120 (Hussey, R., et al. (1988) "A soluble CD4 protein 
selectively inhibits HIV replication and syncitium formation." Nature 
331:768-81; Deen, K., et al. (1988) "A soluble form of CD4 (T4) protein 
inhibits AIDS virus infection." Nature 331:82-84; Traunecker, A., et al. 
(1988) "Soluble CD4 molecules neutralize human immunodeficiency virus type 
1." Nature 331:84-86; Berger, E., et al. (1988) "A soluble recombinant 
polypeptide comprising the amino-terminal half of the extracellular region 
of the CD4 molecule contains an active binding site for human 
immunodeficiency virus." Proc. Natl. Acad. Sci. U.S.A. 85:2357-2361). The 
determinants for high affinity binding of gp120 are in domain 1, residues 
1-109 of CD4 (Arthos, J., et al. (1989) "Identification of the residues in 
human CD4 critical for the binding of HIV." Cell 57:469481). 
sCD4-PE40 is such a potential therapeutic agent for the treatment of AIDS. 
(Chaudhary, V., et al. (1988) "Selective Killing of HIV-Infected Cells by 
Recombinant Human CD4-Pseudomonas Exotoxin Hybrid Protein." Nature 
335:369-372). This hybrid protein consists of an N-terminal methionine 
(amino acid 1) followed by the first two domains of CD4 (178 amino acids), 
several linker amino acids, and the last two domains of Pseudomonas 
exotoxin A (amino acids 253-613 of the toxin). The resulting protein 
contains 545 amino acids and has a calculated molecular weight of 
approximately 59,200 daltons. Amino acids 2-110 in sCD4-PE40 (Chaudhary, 
supra) correspond to residues 3-111 in the cDNA sequence of Maddon, supra, 
except that residue 3 of the Maddon sequence should be lysine, and 
residues 1-109 (domain 1) of Arthos, supra. The gene for sCD4-PE40 has the 
sequence reported by Chaudhary, supra except that the codons that 
correspond to the N-terminal portion of the protein have been modified as 
described for sCD4-183 in PCT Application No. PCT/US90/01367, and codon 
179, corresponding to Ala, is GCT rather than GCG. 
Upon expression of sCD4-PE40 in E. coli, we have found a major contaminant 
which is immunologically-related to sCD4-PE40 and has a molecular mass of 
approximately 50,000 daltons. This protein has the N-terminal sequence 
Met-Leu-Val-Phe-Gly-Thr-Ala- which corresponds to the C-terminal 449 
residues of sCD4-PE40, i.e., beginning with Leu.sup.97 (preceded by a 
methionine). The 50,000 dalton protein results from internal initiation 
within domain 1 of sCD4; a UUG codon down-stream of potential 
Shine-Dalgarno sequences is read as an initiation codon by f-Met-tRNA. 
Since the contaminant is closely related to the full length sCD4-PE40 
product, it has similar biochemical properties. Accordingly, it 
co-purifies with the desired product and may interfere with the oxidation 
and folding of sCD4-PE40 to its biologically active conformation. 
Among the potential causes investigated for the impurity is internal 
initiation. A gene including the above-described region of domain 1 with 
the potential for internal initiation may generate an impurity with an 
N-terminal Met-Leu-Val-Phe-Gly-Thr-Ala- sequence. Such internal initiation 
could result from translating a sCD4-containing gene in many prokaraytic 
organisms, including but not limited to E. coli. Since proteins including 
sCD4 components are potential human drugs, it is desirable to eliminate 
the cause of the contaminating protein. 
Four sequence-related features appear to positively favor translation 
initiation in prokaryotes. First, the preferred initiation codon is AUG. 
GUG and UUG can function as initiation codons although at only about 10 
and 1 percent of the frequency of AUG, respectively. (Hershey, J. (1987) 
Protein Synthesis. In "Escherichia coli and Salmonella typhimurium: 
Cellular and Molecular Biology". F. C. Neidhardt, et al., eds. (American 
Society for Microbiology: Washington, DC) p.613-641.) These codons are 
recognized by f-Met-tRNA as the site where amino acid polymerization is to 
begin (Gold, L. (1988) "Posttranscriptional Regulatory Mechanisms in E. 
coli." Ann. Rev. Biochem. 57:199-233.) 
The second feature that favors prokaryotic initiation is the Shine-Dalgarno 
sequence, e.g. 5'-UAAGGAGGUGA-3', a sequence in the mRNA which is 
complementary to the 3' terminal sequence of 16s rRNA, such that base 
pairs can be formed to stabilize the initiation complex. (Shine, J., and 
Dalgarno, L. (1974) "The 3' terminal sequence of E. coli 16s ribosomal 
RNA: Complementarity to nonsense triplets and ribosome binding sites." 
Proc. Natl. Acad. Sci USA 71:1342-1346; Steitz, J., and Jakes, K. (1975) 
"How ribosomes select initiator regions in mRNA: Base pair formation 
between the 3' terminus of 16s rRNA and the mRNA during initiation of 
protein synthesis in E. coli." Proc. Natl. Acad. Sci. USA 72:4734-4738.) A 
variety of sequences which retain complementarity to the 16s RNA can 
function in this role. Shine-Dalgarno-like sequences, usually include GGAG 
or GAGG, and typically are located about 5-13 bases upstream of the 
initiation codon for most effective initiation. (Gold, L., supra.) 
The third feature is a region which facilitates ribosome binding and 
initiation. A preferred pattern of nucleotides spanning at least -20 to 
+13 bases about the initiation codon of many E. coli genes has been 
detected by in vitro analysis of ribosome protected sequences (Steitz, J., 
supra) and by statistical analysis (Stormo, G., et al. (1982) 
"Characterization of translational initiation sites in E. coli." Nucleic 
Acids Res. 10:2971-2996; Schneider, T., et al. (1986) "Information Content 
of Binding Sites on Nucleotide Sequences." J. Mol. Biol. 188:415-431). 
Additionally, translation reinitiation can occur if a translational start 
signal overlaps (Oppenheim, D., and Yanofsky, C. (1980) "Translational 
Coupling During the Expression of the Tryptophan Operon of Escherichia 
Coli." Genetics 95:785-795) or follows one of the translational stop 
signals. (Steitz, J. (1979) "Genetic signals and nucleotide sequences in 
messenger RNA." In "Biological Regulation and Development. 1. Gene 
Expression.") Such reinitiation does not require a Shine-Dalgarno sequence 
and differs from the intragenic initiation discussed herein. 
The fourth feature is the absence of significant mRNA secondary structure 
in the initiation codon region that might block the necessary annealing 
events with the 16s RNA or the initiator tRNA (Gold, L. (1988), supra). 
The presence of potential translation initiation points can be identified 
in several ways. First, the sequencing of the N-terminus of immunoreactive 
peptides should yield methionine for peptides resulting from initiation 
although in some cases, methionine aminopeptidase can remove methionine 
leaving the adjacent residue in the sequence at the N-terminus (Waller, J. 
(1963) "The NH.sub.2 -terminal residue of the proteins from cell-free 
extracts of E. coli." J. Mol. Biol. 7:483-496; Ben-Bassat, A., et al. 
(1987) "Processing of the initiation methionine from proteins: Properties 
of the E. coli methionine aminopeptidase and its gene structure." J. 
Bacteriol. 169:751-757). In that case, one must rely on the gene sequence 
to determine if the terminal amino acid was encoded with an adjacent codon 
capable of initiating translation. Codons which direct the insertion of 
the N-terminal Met can be AUG, GUG or UUG (Gold, L., supra). Secondly, one 
can analyze the gene for sequences approximating a good initiation region. 
Many of these sequences are not functional. (Stormo, G., and Schneider, 
T., supra). Translation initiation points can be found through 
"footprinting" or "toeprinting" experiments in which regions of the mRNA 
to which ribosomes bind either are protected from nuclease digestion or 
block the elongation of a primed, reverse-transcribed DNA copy. (Gold, L., 
supra.) 
Intragenic ribosome initiation sites have been identified in a number of 
genes. Following expression in E. coli of poliovirus 3C protease, 
initiation at the AUG of codon 27 gave rise to significant levels of an 
unstable internal initiation product (Hanecak, R., et al. (1984) 
"Expression of a cloned gene segment of poliovirus in E. coli: Evidence 
for autocatalytic production of the viral proteinase." Cell 37:1063-1073; 
Ivanoff, L., et al. (1986) "Expression and site-specific mutagenesis of 
the poliovirus 3C protease in E. coli." Proc. Natl. Acad. Sci. USA 
83:5392-5396). Furthermore, expression of xylanase in E. coli was 
accompanied by the production of a species apparently initiating at GUG, 
codon 471. (Grepinet, O., et al. (1988) "Nucleotide sequence and deletion 
analysis of the xylanase gene (xynZ) of Clostridium thermocellum." J. 
Bacteriol. 170:4582-4588.) Translation initiation within the porcine 
parvovirus structural protein B occurs at internal initiation sites, with 
at least two of these internal initiation peptides produced at higher 
levels than the full length recombinant protein. (Hailing, S., and Smith, 
S. (1985) "Expression in E. coli of multiple products from a chimaeric 
gene fusion: Evidence for the presence of procaryotic translational 
control regions within eucaryotic genes." Bio/Technology 3:715-720.) 
Finally, expression of a simian rotavirus glycoprotein in E. coli 
generated an apparent product of internal initiation at a level similar to 
that of the full length molecule. (Arias, C., et al. (1986) "Synthesis of 
the outer-capsid glycoprotein of the simian rotavirus SAl1 in E. coli." 
Gene 47:211-219.) It has been proposed that commercial production can be 
facilitated by removing internal initiation sites through mutagenesis 
(Hailing, S., supra). 
Once the cause of the impurity has been determined to be internal 
initiation, a method of eliminating the internal initiation needs to be 
developed. 
INFORMATION DISCLOSURE 
As discussed above, several intragenic ribosome initiation sites have been 
identified. Initiation at the AUG of codon 27 gave rise to significant 
levels of an unstable internal initiation product following poliovirus 3C 
protease expression in E. coli. See, e.g., Hanecak, R., et al. (1984) 
"Expression of a cloned gene segment of poliovirus in E. coli: Evidence 
for autocatalytic production of the viral proteinase." Cell 37:1063-1073 
and Ivanoff, L., et al. (1986) "Expression and site-specific mutagenesis 
of the poliovirus 3C protease in E. coli." Proc. Natl. Acad. Sci. USA 
83:5392-5396. E. coli expression of xylanase was accompanied by the 
production of a species initiating at GUG. See, e.g., Grepinet, O., et al. 
(1988) "Nucleotide sequence and deletion analysis of the xylanase gene 
(xynZ) of Clostridium thermocellum." J. Bacteriol. 170:4582-4588. 
Translation initiation within the porcine parvovirus structural protein B 
occurs at internal initiation sites. See, e.g., Halling, S. and Smith, S. 
(1985) "Expression in E. coli of multiple products from a chimaeric gene 
fusion: Evidence for the presence of procaryotic translational control 
regions within eucaryotic genes." Bio/Technology 3:715-720. Halling and 
Smith suggest that mutagenesis could remove internal initiation sites. 
Simian retrovirus glycoprotein expression in E. coli also generated a 
product of internal initiation. See, e.g., Arias, C., et al. (1986) 
"Synthesis of the outer-capsid glycoprotein of the simian rotavirus SAl1 
in E. coli." Gene 47:211-219 (1986). None of these references mention CD4 
proteins. 
SUMMARY OF THE INVENTION 
The present invention provides DNA sequences that eliminate internal 
translation initiation and do not change the amino acid sequence from 
genes containing portions of sCD4. More specifically, the modified 
sequence comprises: 
##STR1## 
This sequence may be modified by various codon substitutions, deletions, 
additions or replacements. All such allelic variations and modifications 
resulting in a sCD4 protein in which internal translation initiation has 
been eliminated are included within the scope of this invention. 
The present invention further provides recombinant DNA molecules which do 
not support internal initiation of sCD4. The present invention also 
provides host cells transformed with these recombinant DNA molecules. 
The present invention also provides methods of eliminating internal 
initiation of sCD4 which comprises substituting base sequences for the 
Shine-Dalgarno-like sequences that precede the codon of amino acid 96 of 
sCD4 and/or modifying codon 96 or other codons which can be recognized for 
translation initiation. 
The term "Shine-Dalgarno-like sequences" as used herein means sequences 
with complementarity to the 3' end of 16s rRNA and which could be used as 
a ribosome binding site. These can include but are not limited to GGAG, 
GAGG, AGGAGGT, GGAGG, and AAGGAGG. 
The term "sCD4" refers to any protein or hybrid molecule that includes 
sequences related to those in T cell CD4 and is capable of binding to 
gp120, the external subunit of the HIV envelope glycoprotein. Such 
molecules are exemplified in Chaudhary, V., supra; Klatzmann, D., supra; 
Smith, D., supra; Fisher, R., supra; Hussey, R., supra; Deen, K., supra; 
Traunecker, A., supra; Berger, E., supra; Capon, D., et al. (1988) 
"Designing CD4 immunoadhesions for AIDS therapy." Nature, 337:525-531; and 
Till, M., et al. (1988) "HIV-infected cells are killed by rCD4-ricin A 
chain." Science 242: 1166-1168; European Patent Application No. 0,331,356. 
The term "hybrid molecule" refers to a molecule that contains functional 
components derived from two independent molecule species. The independent 
molecule species can be used in whole or in part to produce the "hybrid 
molecule". 
The term "host cell" as used herein means any procaryotic cell capable of 
being transformed with the modified DNA sequence encoding the first domain 
of an sCD4 molecule wherein internal initiation expression has been 
eliminated without altering the amino acid sequence of the sCD4, including 
but not limited to E. coli. 
DETAILED DESCRIPTION OF THE INVENTION 
The present invention is based upon the discovery that proteins made from 
genes that include the CD4 sequence in its cDNA form can make additional 
polypeptides because of an intragenic nucleotide sequence which favors 
translation initiation. The invention is thus directed to a novel method 
for preventing such initiation, particularly comprising a modified 
sequence which minimizes the potential for internal initiation. 
sCD4-PE40 is a four domain hybrid protein. It consists of N-terminal 
methionine, the first two domains of CD4 (178 amino acids), several linker 
amino acids, and the last two domains of Pseudomonas exotoxin A (amino 
acids 253-613 of the toxin). The resulting protein contains 545 amino 
acids and has a calculated molecular weight of approximately 59,200 
daltons. See Chaudhary, V., supra. 
A variety of lower molecular weight species cross-reacting with antibodies 
to sCD4 have been found by Western Blot analysis in a variety of E. coli 
stains producing sCD4-PE40. The major contaminant has a molecular weight 
of approximately 50,000 daltons and represented 5-20% of the level of 
sCD4PE40 in isolated inclusion bodies. Although such a species could 
result from errors in biosynthesis, e.g., frameshifting or termination, it 
seemed more likely to represent a proteolytic fragment. To test this 
hypothesis, it was necessary to identify the putative clip site in order 
to develop approaches for eliminating the protease(s) responsible for the 
proteolysis. 
The impurity protein was characterized by N-terminal sequence analysis 
following isolation by electroblotting from SDS-PAGE or Reversed-Phase 
HPLC. The amino acid sequence of the impurity lacked the first 96 residues 
of sCD4-PE40; it began with N-terminal methionine and continued with the 
sequence starting at residue 97. The apparent molecular weight of 50,000 
daltons observed on SDS-PAGE was in good agreement with the calculated 
molecular weight of 48,375 daltons for such a fragment of sCD4-PE40 
comprising residues 96-545. In view of our original hypothesis, the 
presence of N-terminal methionine on the protein was surprising in that 
there are no known mechanisms for generating the identified sequence from 
the intact protein by proteolysis. 
Protein synthesis in E. coli is initiated with N-formyl methionine. The 
N-terminal methionine is usually deformylated as the nascent peptide chain 
is elongated. Furthermore, depending on the adjacent amino acids, a 
methionyl amino peptidase often removes the N-terminal methionine. The 
cleavage is inhibited by the adjacent lysine in sCD4-183 and sCD4-PE40. 
The presence of methionine at the N-terminus of the impurity and the 
observed composition indicated that the impurity was not a proteolytic 
fragment but resulted from internal initiation at amino acid-96. 
For initiation of protein synthesis, an initiation codon (usually AUG) is 
required. The presence of a Shine-Dalgarno-like sequence enhances the 
efficiency of initiation. To generate the observed impurity, an initiation 
codon must be present at a position corresponding to amino acid 96. The 
codon corresponding to Leu.sup.96 is UUG. One of six codons specifying 
leucine, UUG is rarely found in the mRNA of highly expressed E. coli genes 
and the corresponding tRNA is found in low abundance. UUG is read, 
although infrequently (at &lt;1% of normal initiation), by the f-Met-tRNA as 
an initiation codon. For this unusual initiation to occur, an upstream 
ribosome binding site is required. Inspection of the sequence encoding the 
impurity revealed three good Shine-Dalgarno-like sequences only five, 
eight and twenty nucleotides upstream of the UUG. Thus, internal 
translation was a reasonable explanation for the presence of a subsequence 
of sCD4-PE40 beginning with Met-Leu.sup.97. 
A modified sCD4-PE40 can be constructed in which the leucine codon has been 
changed from UUG to CUG and the GGAGG sequences have been changed to 
remove these Shine-Dalgarno-like sequences. These changes eliminate 
expression of the internal initiation product but do not alter the amino 
acid sequence of the full length sCD4-PE40 protein. Other similar 
alterations in sequences in this area will be readily apparent to those 
skilled in the art.

The present invention is exemplified in more detail in the examples below. 
EXAMPLE 1 
In this example, we set forth the construction of cells and their induction 
to express sCD4-PE40 in E. coli. 
The UC12656 strain of E. coli is used as the host for sCD4-PE40 expression. 
This strain is derived from NRRL B-18303. The derivation of the NRRL 
B-18303 strain is described in International Application No. PCT/US88/0038 
which is incorporated herein by reference. The UC12656 strain is made in 
three steps which employ techniques well known to those skilled in the 
art. First, NRRL B-18303 is crossed with an Hfr strain to replace the 
rpoH112 allele with rpoH.sup.+. In addition this cross removes a Tn10 
adjacent to the rpoH locus, and introduces the rpsL100 allele. Second, the 
NRRL B-18303 culture is resistant to lambda owing to an alteration in its 
lamB gene; a lamB.sup.+ allele is transduced into the strain. Finally, a 
cryptic lambda lysogen from the strain TAP106 (obtained from Dr. Donald 
Court, NCI-Frederick Cancer Institute, Frederick, Md. 21701; Chen, S., et 
al. (1990) "Expression and characterization of RNaseIII and Era proteins: 
Products of the rnc operon of Escherichia coli." J. Biol. Chem 
265:2888-1895) is P1 transduced into the strain to create UC12657. The 
lambda cryptic from TAP106 contains the following genetic configuration: 
(int-ral).tangle-solidup., N::Kan, cI857, (cro-bioA).tangle-solidup.. 
A vector used to express the sCD4-PE40 protein is pUC1456. The vector is 
derived from pBR322 (available from Pharmacia LKB Biotechnologies, 
Piscataway, N.J. 08854) by cloning into the EcoRI and HindIII restriction 
sites a fragment containing the lambda P.sub.L promoter, the TAT32 
ribosome binding site, and the sCD4-PE40 gene. The P.sub.L promoter is 
taken from the pJL-6 vector (Lautenberger, J., et al. (1983) "High-level 
expression in Escherichia coli of the carboxyl-terminal sequence of the 
avian myelocytomatosis virus (MC29) v-myc protein." Gene:75-84; the vector 
can be obtained from Dr. Donald Court). The promoter, ribosome binding 
site and the sCD4-PE40 gene are constructed and cloned using techniques 
that are well known to those skilled in the art The P.sub.L promoter is 
modified by introducing an XbaI restriction site shortly after the +1 
nucleotide of the promoter. The modified promoter is designated P.sub.L6m. 
The TAT32 ribosome binding site is derived from synthetic oligonucleotides 
that contained a sequence derived from the ribosome binding site of the 
bacteriophage T4 gene 32 (Gorski, K., et al. (1985) "The stability of 
bacteriophage T4 gene 32 mRNA: A 5' leader sequence that can stabilize 
mRNA transcripts." Cell 43:461-469). The sCD4-PE40 gene is obtained from 
Chaudhary, V., supra, and modified by making changes in codon usage for 
several N-terminal codons. The codons that correspond to the N-terminal 
portion of the protein are modified as described for sCD4 in PCT 
Application No. PCT/US90/01367. 
The pUC1456 vector is transformed into competent cells of UC12656. The 
culture is developed from one of the transformed colonies and is 
designated UC12575. 
UC12575 cells are grown at 30.degree. C. and induced by heat shifting to 
40.degree. C. This results in the formation of intracellular aggregates 
(inclusion bodies) containing sCD4-PE40. 
The vector pUC1456 and transformed culture UC12575 of Example 1 were 
deposited at The Agricultural Research Culture Collection (NRRL), Northern 
Regional Research Center, 1815 North University Street, Peoria, Ill. 
61604, under the Accession No. NRRL B-18667 on Jun. 27, 1990, in 
accordance with the requirements of the Budapest Treaty on the 
International Recognition of the Deposit of Microorganisms for the 
Purposes of Patent Procedure. 
EXAMPLE 2 
This example describes the isolation and characterization of the impurity 
that contaminates preparations of sCD4-PE40. 
Samples containing sCD4-PE40 in inclusion body form are analyzed by 
SDS-polyacrylamide gel electrophoresis(SDS-PAGE), electroblotting on PVDF 
membranes, Western blotting, and sequencing according to methods readily 
apparent to those skilled in the art. In particular, the solids from cells 
or inclusion body preparations are collected by centrifugation. SDS-PAGE 
is performed essentially as described by Laemmli (Laemmli, U. (1970) 
"Cleavage of structural proteins during the assembly of the head of 
bacteriophage T4." Nature 227:680-685), except that samples are heated at 
100.degree. C. for 5 minutes in ethanolamine sample buffer (10 g SDS; 45 
ml water, 20 ml 1M ethanolamine, pH 10; 25 ml glycerol; 10 ml 0.05% (w/v) 
Bromophenol Blue) for five minutes before application to gels. Following 
electrophoresis, these gels are rinsed immediately, arranged in a blotting 
sandwich containing polyvinylidine difluoride (PVDF) membranes (Immobilon, 
pore size 0.45 .mu.m), and blotted electrophoretically. This protein 
transfer to the PVDF membrane is by the discontinuous semi-dry method of 
Hirano, H. (1989) "Microsequence analysis of winged bean seed proteins 
electroblotted from two-dimensional gel." J. Protein Chem. 8(1):115-130. 
The blots are either visualized with anti-sera or with Coomassie Blue 
R250. 
SDS-PAGE analysis of cells expressing sCD4-PE40 reveals a major band at 
about 60,000 daltons corresponding to the recombinant product as well as a 
variety of other bands. When the inclusion bodies are separated from 
soluble proteins, the sCD4-PE40 is enriched, increasing from 10-20% of the 
total protein to 50-80%. The major impurity band, which has an apparent 
weight of about 50,000 daltons, is also greatly enriched. By densitometric 
scanning, this band is 5-20% of the sCD4-PE40 band. 
Western analysis of the PVDF blots is conducted to determine if the 
observed bands are related to sCD4-PE40. Immunodetection is accomplished 
with rabbit anti-sera to sCD4-183, to sCD4-PE40, and to Lys-PE40 (Domains 
2 and 3 of Pseudomonas exotoxin A). Many immunoreactive bands are 
observed, including the major impurity. The band with an apparent weight 
of 50,000 daltons is immunoreactive with each antibody tested, indicating 
that it contains domains of both sCD4 and PE40. 
For N-terminal sequence analysis, the PVDF membranes containing the blotted 
protein are stained with Coomassie Blue R250 for 2 minutes, destained with 
an aqueous solution of 50% methanol and 10% acetic acid for 3 minutes, 
rinsed with Milli-Q water and air dried. The 50 Kd protein band is excised 
from the dried blot, cut into approximately 2.times.4 mm pieces, and 
loaded into the upper block of a sequencer cartridge above a 
Polybrene-loaded, precycled filter. N-terminal sequence analysis is 
performed on an Applied Biosystems (ABI) 470A sequencer equipped with an 
on-line ABI 120A PTH analyzer. 
Following SDS-PAGE of sCD4-PE40 from inclusion bodies, the 50,000 dalton 
impurity located with Coomassie Blue R-250 on a PVDF membrane was 
sequenced through residues on the ABI 470A. Two sequences were apparent, 
with the minor species presumably representing cross-contamination by the 
neighboring 60,000 dalton band since it had the N-terminus of sCD4-PE40 
(e.g., Met.sup.1 -Lys.sup.2 -Lys.sup.3 -Val.sup.4 -Val.sup.5 -Leu.sup.6 
-Gly.sup.7). The most abundant sequence begins with 
Met-Leu-Val-Phe-Gly-Leu-Thr-Ala, corresponding to N-terminal methionine 
followed by Leu.sup.97 through Leu.sup.110 of sCD4-PE40. 
EXAMPLE 3 
In this example, we set forth the design and construction of a synthetic 
DNA fragment from oligonucleotides to eliminate a Shine-Dalgarno-like 
sequence in sCD4-PE40. 
The DNA sequence between the BclI and EcoNI restriction sites of the cDNA 
encoding sCD4-PE40 is shown below. These restriction sites are unique for 
the CD4-PE40 gene. The BclI-EcoNI fragment encompasses codons 71 (ATC) 
through 111 (CAG) of the sCD4-PE40 sequence, which corresponds to codons 
72-112 in the cDNA sequence determined by Maddon, supra. 
##STR2## 
In the sequence preceding the codon for amino acid 96 there are three 
regions that have strong homology to the so-called Shine-Dalgarno sequence 
(e.g. a sequence complementary to the 3' end of the 16s rRNA) and could be 
used as ribosome binding sites. These sequences are indicated below: 
##STR3## 
To disrupt these Shine-Dalgarno-like sequences the following base 
substitutions can be made without altering the amino acid sequence of the 
encoded protein. 
##STR4## 
In addition the TTG initiation codon can be changed from TTG to CTG. The 
CTG codon is not a known initiation codon. Additional codon changes have 
been made in the DNA sequence to optimize the codon usage in this region. 
The use of codon optimization is known to those skilled in the art. These 
changes are shown above the native sequence. 
##STR5## 
The codon optimization changes in combination with the codon change for 
removal of the three ribosome binding sites and the TTG initiation site 
are shown as a composite below. The codon changes are indicated above the 
native sequence. 
##STR6## 
Four oligonucleotides are synthesized as described in International 
Application No. PCT/US88/00328 which is incorporated herein by reference. 
These oligonucleotides, when hybridized and ligated, will form a synthetic 
fragment that contains the codon changes presented above and can be cloned 
into the unique BclI/EcoNI sites of the CD4-PE40 gene thus replacing the 
native sequence that contains the intragenic ribosome binding site. The 
sequences of the four oligonucleotides are: 
##STR7## 
The crude oligonucleotides are purified by cutting out the product band on 
a 20% acrylamide gel and desalting over a Waters Sep-Pak column as 
described in PCT Application No. PCT/US88/00328. The oligonucleotides form 
strands of the synthetic fragment as indicated below. 
##STR8## 
The procedures used can found in Current Protocols in Molecular Biology 
(edited by Ausubel, F., et al., and published by John Wiley and Sons). 
Oligonucleotides 2 and 4 are kinased using .sup.32 P gamma labeled ATP. 
Oligonucleotides 1 and 4, and 2 and 3 are hybridized to each other. The 
two set of hybridized oligonucleotides 1/4 and 2/3 are ligased and run on 
a 12% acrylamide gel. The synthetic oligonucleotide derived DNA fragment 
is visualized by autoradiograph, cut from the gel and isolated. The 
sequence of the synthetic DNA fragment is as follows: 
##STR9## 
EXAMPLE 4 
In this example we set forth the cloning of the BclI/EcoNI fragment into 
the CD4-PE40 gene. 
A detailed description of the cloning methodologies employed herein can be 
found in Current Protocols in Molecular Biology (supra). These techniques 
and the pBR322 vector use in the clonings described are well known to 
those skilled in the art. 
The pUC1456 vector described in Example 1 contains BclI and EcoNI 
restriction sites in the sCD4-PE40 gene and a second EcoNI restriction 
site resident in the pBR322 sequence downstream of the HindIII restriction 
site. The pUC1456 vector is transformed into the E. coli strain CGSC 6580 
which had been lysogenized with the bacteriophage lambda. This strain 
carries the dam13::Tn9 allele (the strain can be obtained from Dr. Barbara 
Bachmann, Coli Genetic Stock Center, Department of Biology, 255 OML, Yale 
University, P.O. Box 6666, New Haven, Conn. 06511-7444). The dam13::Tn9 
allele prevents methylation of the adenine in the sequence GATC. 
Methylation of this site prevents the BclI restriction enzyme from cutting 
the DNA. The use of dam deficient host to permit the BclI enzyme to cut is 
well known to those skilled in the art. Vector DNA is isolated and 
digested with BclI and EcoNI restriction endonucleases. This digestion 
produces a large vector fragment, a 1837 bp fragment and a 115 bp 
fragment. The vector fragment is isolated from an agarose gel, and is 
ligated to the synthetic oligonucleotide derived fragment and transformed 
into competent cells of UC12656. The juncture formed between the EcoNI 
site of the oligonucleotide derived fragment of Example 3 and the EcoNI 
site in the vector generates a PstI restriction site which can be used to 
identify candidates with the oligonucleotide fragment inserted. One of the 
candidates identified by restriction analysis is selected and the presence 
of the oligonucleotide fragment insert is confirmed by DNA sequence 
analysis. The vector is designated pUC1470. 
In order to reconstruct the sCD4-PE40 gene an additional vector pUC1469 is 
constructed. The pBR322 vector contains EcoRI, ClaI, HindIII, EcoNI and 
NdeI restriction sites. Each one of these sites are unique in the vector. 
The CD4-PE40 gene can be cloned as a ClaI/HindIII fragment into the 
corresponding ClaI/HindIII restriction site in the pBR322 vector. However, 
in such a vector the EcoNI site in the CD4-PE40 gene would not be unique. 
To prevent this the pBR322 vector is cut with the HindIII and NdeI 
restriction enzymes. The "sticky" ends are filled with PolA Klenow 
fragment in the presence of dNTPs. The DNA is run on an agarose gel and 
the fragment containing the ampicillin resistance gene and origin of 
replication is isolated, ligated and transformed into competent cells of 
MC1061. Candidates are analyzed by restriction digestion, and a clone with 
the deletion identified. The ligation of the HindIII and NdeI restriction 
sites regenerates a HindIII site. This vector is designated pUC1468. The 
ClaI/HindIII CD4-PE40 fragment is isolated from pUC1456 and is cloned into 
the pUC1468 vector at the corresponding ClaI/HindIII sites. The resultant 
vector is designated pUC1469. 
To regenerate the sCD4-PE40 gene, the pUC1470 vector is digested with EcoNI 
and EcoRI restriction enzymes and a fragment of approximately 600 bp 
containing the P.sub.L6m promoter, the TAT32 ribosome-binding site and the 
5' portion of the sCD4-PE40 gene with the modifications of the internal 
ribosome-binding sites is isolated. The pUC1469 vector is digested with 
EcoRI and EcoNI restriction enzymes to generate a vector fragment and a 
fragment of approximately 600 bp. The vector fragment is isolated. The two 
isolated fragments are ligated and transformed into UC12656. The DNA 
sequence derived from the oligonucleotide fragment contains an XmnI site 
that can be used for characterizing clones. A candidate with the correct 
restriction analysis is identified. The sequence modified by the cloning 
of the oligonucleotide fragment is sequenced for confirmation. The 
resultant vector is designated pUC1467. This vector is transformed into 
UC12656, and the resultant culture, designated UC12657, is capable of high 
level expression of the unmodified sCD4-PE40 protein from the modified 
gene. The vector pUC1467 and transformed culture UC12657 of Example 4 were 
deposited at The Agricultural Research Culture Collection (NRRL), Northern 
Regional Research Center, 1815 North University Street, Peoria, Ill. 
61604, under the Accession No. NRRL B-18676 on Jul. 13, 1990, in 
accordance with the requirements of the Budapest Treaty on the 
International Recognition of the Deposit of Microorganisms for the 
Purposes of Patent Procedure. 
EXAMPLE 5 
In this example, it is shown that the use of a modified gene, such as 
described in Example 4, eliminates the production of the 50 kilodalton 
fragment which was described in Example 2. 
Strain UC12657 containing pUC1467 is grown and induced as described for 
strain UC12575 in Example 1. The cells are analyzed for sCD4-PE40 using 
SDS-PAGE and Western blotting as described in Example 2. An examination of 
the gel reveals the 50 kilodalton, immunoreactive species apparent in the 
UC12575 culture is not detected in the induced UC12657, indicating that 
internal initiation has been eliminated.