Adenovirus promoter system

The adenovirus major late promoter is employed as a promoter for expression in a yeast host. Constructions are provided for expression in yeast with the adenovirus major late promoter and a coding segment.

BACKGROUND OF THE INVENTION 
The present invention relates to a development of recombinant expression 
vectors useful for the expression of DNA coding segments in yeasts, fungi 
and mammalian cells, and to the development of shuttle expression vectors, 
capable of expressing a DNA coding segment in both yeasts and mammalian 
cells. The vector system herein described employs an adenovirus promoter 
to control expression of the inserted DNA coding segment. The described 
promoter system was not previously known to be functional in non-mammalian 
cells. 
The adenoviruses constitute a class of viruses infective to animals, 
including humans, and known to transform infected mammalian cells in 
culture with low frequency. The adenoviruses have been extensively studied 
and are well-characterized. The DNA nucleotide sequences are known for 
specific strains. (For general background, see Tooze, J., Molecular 
Biology of Tumor Viruses, Part II: DNA Tumor Viruses, Cold Spring Harbor 
Laboratory, Cold Spring Harbor, New York (1980). Genetically, the 
functions of the virus are classified as "early" or "late," depending on 
whether they are expressed before or after the onset of viral DNA 
synthesis in the infection cycle. Early functions are expressed under 
control of several early promoters. In contrast, the majority of late 
functions are expressed under the control of a single, very active, 
promoter known as the adenovirus major late promoter (herein AML 
promoter). The nucleotide sequence of the AML promoter has been published, 
Tooze, supra. Comparison with the known nucleotide sequences of active 
yeast promoters reveals few points of similarity between the AML promoter 
and yeast promoters, other than certain short segments which appear to be 
characteristic of almost all eukaryotic promoters. By contrast, many 
points of structural similarity are known, between the yeast promoters for 
alcohol dehydrogenase (ADH) and glyceraldehyde phosphate dehydrogenase 
(GAPDH). [See Dobson, M. J., et al., Nucl. Acids Res. 10, 2625 (1982)]. 
A promoter is defined herein as a DNA segment capable of functioning to 
initiate transcription of an adjoining DNA segment. Transcription is the 
synthesis of RNA (herein termed messenger RNA, or mRNA), complementary to 
one strand of the DNA adjoining the promoter region. In eukaryotes, mRNA 
synthesis is catalyzed by an enzyme termed RNA polymerase II. The minimum 
essential elements of promoter function are two: to provide a starting 
point for the initiation of transcription and to provide a binding site 
for RNA polymerase II near the start site permitting selection of the 
proper strand of DNA as a template for mRNA synthesis. In addition, a 
eukaryotic promoter functions to regulate the relative efficiency of 
transcription of coding segments under its control. An active promoter is 
one which induces the synthesis of relatively large amounts of mRNA 
complementary a strand of the adjacent DNA coding segment. 
The structural correlates of promoter function have not been clearly 
established. A promoter segment can be identified in nature as a region 
lying adjacent to a given structural gene at its 5' end. (References to 
the 5' and 3' ends of a gene will be understood to indicate the 
corresponding respective ends of mRNA transcribed therefrom, and these, in 
turn, will be understood to correlate with the NH.sub.2 -- and --COOH 
termini of the encoded protein, respectively. Mutations in the 5' 
untranslated region, adjacent to a coding segment of DNA, and extending 
from 200 to 400 nucleotides from the start codon thereof, display a 
variety of functional defects in transcription, ranging from reduced rate 
or efficiency of transcription, to total cessation of transcription. Where 
several genes are transcribed together in a single transcription unit, 
such mutations can result in the simultaneous loss or reduction in amount 
of several gene products. Such mutations define the promoter region for 
the structural gene or genes they affect. Comparisons of nucleotide 
sequences of promoters of various genes from various species have revealed 
only a few short regions of nucleotide sequenced similarity in common 
between them. Most notable of these is the "TATA box," a segment of about 
five to ten nucleotides located generally about 70 to 230 nucleotides 
upstream from the start of a coding segment, having a sequence generally 
resembling TATAA. For a review of structural comparisons, see Rosenberg, 
M. et al., Ann. Res. Genetics, 13, 319 (1979). The TATA box is believed to 
function in the initiation of transcription. Other examples of short 
regions of sequence similarity having similar locations in a number of 
promoters, include segments with such whimsical descriptors as "CAAT BOX," 
and "CACA BOX,". However, many promoters lack one or more of these 
features and their function is not established. 
Comparative studies of the effects of promoter mutations lying distal to 
the start of the coding segment have been undertaken, e.g., by McKnight, 
S., et al., Science 217, (1982). Such studies have shown, in general, that 
certain regions lying upstream from the TATA box appear to be involved in 
the binding and orientation of the RNA polymerase to the DNA segment to be 
transcribed. Structural variations in this portion of the promoter 
presumably affect the efficiency of transcription and are known to vary 
substantially from one species to another. 
The structures of over a dozen yeast promoters have been determined and the 
structures were compared by Dobson, M. J. et al., Nucleic Acids Research 
10, 2625 (1982). The yeast promoters have many points of similarity not 
shared by non-yeast promoters, such as the adenovirus major late promoter 
(supra, Tooze et al.), and herpes virus thymidine kinase gene (Kiss, G., 
et al., J. Bact. 149, 542, 1982; Kiss, G., et al., J. Bact. 150, 465 
(1982). Further, it has been shown (Kiss, G. et al., supra) that the 
thymidine kinase promoter of herpes virus does not function in yeast. 
The present invention stems from the surprising discovery that the major 
late promoter of adenovirus is functional and highly active in yeast. That 
discovery has made it possible to construct, for the first time, 
expression vectors for the expression of DNA coding segments in yeast, 
controlled by the AML promoter, and for methods of synthesizing proteins 
in yeast cells transformed by such vectors. 
The construction of vectors suitable for the expression of a DNA coding 
segment in yeast has been described, see, e.g., Ammerer, G. et al., 
Recombinant DNA, Proc. 3rd Cleveland Symp. Macromolecules (Walton, A. G., 
ed.), p. 185, Elsevier, Amsterdam (1981). Shuttle vectors, capable of 
replication either in a bacterial strain such as Escherichia coli and in 
yeast have been described. However, such vectors have relied upon the use 
of known yeast promoters for expression in yeast. Previously described 
shuttle vectors have been limited to one of the alternative hosts for 
expression. For example, shuttle vectors having yeast promoters are 
limited to expression of the DNA coding sequence in yeast only. A 
disadvantage of such vectors is that an extensive region of yeast 
homology, the promoter region, provides an opportunity for genetic 
recombination between the vector and the yeast chromosome, possibly 
resulting in integration of the vector. Consequently, the copy number per 
cell of sequences represented by the vector is one per chromosome. The 
reduction in copy number makes it impossible to achieve the highest levels 
of expression. 
Expression vectors for yeast, containing the AML promoter, provide distinct 
advantages over vectors previously available in the art. In addition to 
promoting a high level of expression of any DNA coding sequence under AML 
promoter influence, such vectors, according to the present invention, lack 
the regions of DNA homology between vector and chromosome provided by 
prior art vectors employing a yeast promoter. In fact, vectors lacking any 
homology with yeast chromosomal DNA can be constructed, using a 
replication origin provided by yeast two micron circle plasmid DNA. 
Furthermore, the discovery of a promoter functional in both yeast and 
mammalian cells makes possible the construction of shuttle vectors capable 
of expressing a DNA coding sequence in either host. Therefore, the present 
invention makes possible, for the first time, the construction of true 
expression shuttle vectors. 
SUMMARY OF THE INVENTION 
The invention is based upon the surprising discovery that the major late 
promoter of adenovirus (hereinafter the AML promoter) functions in yeast 
to provide high efficiency transcription and translation of a DNA segment 
coding for a protein. This discovery makes possible the construction of 
expression vectors, such as plasmids, containing a desired DNA coding 
segment, which vector is able to replicate in yeast, providing multiple 
copies per cell, and which coding segment is expressed in yeast cells 
transformed by the vector. The combination of AML promoter and DNA coding 
segment may be placed within a vector capable of replicating in both yeast 
and mammalian cells, to provide an expression shuttle vector between yeast 
and mammalian cells. Expression of the DNA coding segment in such a vector 
takes place in either host cell. Alternatively, the AML promoter-DNA 
coding segment unit may be transferred to a vector capable of replication 
in mammalian cells, thereby providing for expression of the coding segment 
in the mammalian cell. It is even feasible to construct triple shuttle 
vectors, capable of replication in a bacterial host, a yeast, or a 
mammalian host, capable of expression in both the yeast and mammalian 
hosts. 
It follows from the discovery that the AML promoter functions in yeast that 
there are many other organisms similar to yeast in which the AML promoter 
can be shown to function. Such organisms include, by way of example, and 
without limitation, other members of the genus Saccharomyces, members of 
the genus Aspergillus, and members of the genus Neurospora. The screening 
of organism strains suitable for use with the AML promoter can be 
accomplished according to the teachings herein, without undue 
experimentation. In addition, it will be understood that any of the many 
strains of adenovirus, whether infective to humans or other animals, may 
be employed as the source for the AML promoter sequence. It is deemed 
preferable to employ a virus strain that is non-oncogenic. 
The principles for construction of a vector having proper orientation and 
juxtaposition of the promoter sequence and coding segment with respect to 
each other are matters well known to those skilled in the art. For 
example, such minimal conditions for operativeness as the existence of a 
start codon in the DNA coding segment, at its beginning, and location of 
the promoter segment proximal to the 5' end of the coding segment, are 
matters deemed within the scope of knowledge of those skilled in the art. 
The DNA coding segment may be any DNA segment from which, as a minimum, an 
RNA transcript is to be made. In many instances, translation of the RNA 
transcript will be desired, and in such circumstances, it will be 
understood that the coding segment will include nucleotide sequences 
providing the necessary signals to provide RNA processing and translation. 
The coding segment may comprise cDNA, genomic DNA and synthetic DNA.

DETAILED DESCRIPTION OF THE INVENTION 
Construction of a yeast vector comprising the AML promoter of adenovirus 2 
is described in detail to provide an example of the invention. The use of 
a yeast vector comprising the AML promoter for the expression of hepatitis 
B surface antigen coding segment under the influence of the AML promoter 
is demonstrated. Two promoter constructions are shown, one having a 
somewhat shorter nucleotide sequence than the other. Nucleotide sequences 
at the 5' end of the promoter sequence (in the same orientation as the 
coding segment), tend to be less significant for promoter function than 
sequences closer to the translational start point. However, for maximum 
promoter activity, the AML promoter sequence should include approximately 
200-300 nucleotides, counting from the initiation point of translation. 
EXAMPLE 1 
Construction of a Yeast Vector Comprising the AML Promoter 
The SmaF fragment of adenovirus 2, which contains a region previously 
identified as the major late promoter, was isolated and purified as 
described by Weil et al. Cell, 18, 469 (1979). The fragment was cloned in 
the vector in pBR313, and amplifed in amount by multiple cycles of growth 
in an E. coli host. (The techniques used herein of restriction 
endonuclease digestion, ligation, transformation, gel electrophoresis, 
isolation and purification of plasmid DNA were standard techniques known 
in the art unless otherwise specified. Detailed descriptions of such 
techniques are found, for example, in Methods of Enzymology, Vol 68, R. 
Wu, Ed., 1980. (The AML promoter segment was reisolated by SmaI digestion 
of the plasmid into which it was initially cloned, and further digested 
with Pvu II. Decameric, HindIII linkers (commercially available from 
Collaborative Research, Inc., Waltham, Mass.) were attached to the ends by 
a DNA ligase-catalyzed reaction. The material was then digested with 
HindIII endonuclease and a fragment of about 1150 base pairs was 
identified and isolated by gel electrophoresis. That fragment was digested 
with Alu I endonuclease, generating a single blunt end, resulting from Alu 
I endonuclease cleavage. An oligonucleotide BamHI linker was attached by 
blunt end ligation. The end not affected by the Alu I digestion was 
similarly unaffected by the ligation, because the previous HindIII 
endonuclease digestion did not generate a blunt end to which the BamHI 
oligonucleotide linker could be joined. After BamHI endonuclease 
digestion, the resulting fragment, now approximately 440 base pairs 
length, was isolated by gel electrophoresis and inserted into pBR327 
previously digested with BamHI and HindIII endonucleases. Transformants 
were screened for the existence of a fragment of about 440 base pairs 
produced by combined digestion with BamHI and HindIII endonucleases. 
Positive clones were confirmed by nucleotide sequence analysis, using the 
technique of Maxam, A., et al., Proc. Nat. Acad. Sci. USA 74, 560 (1977). 
The sequence analysis confirmed that the 440 base pair fragment contained 
the AML promoter. Orientation of the promoter sequence was such that the 
transcription initiation site was nearest the HindIII end of the fragment 
and farthest from the BamHI end of the fragment. 
The DNA coding segment used to demonstrate expression was a fragment 
containing a nucleotide sequence coding for the hepatitis B surface 
antigen. The coding segment was previously described in copending 
application Ser. No. 402,330, obtained by combined digestion with HindIII 
and EcoRI endonucleases of the plasmid pHBS-5 described therein. 
The plasmid pHBS-5 was digested with EcoR1 endonuclease generating a 
fragment of approximately 850 base pairs including the S-protein coding 
region and flanking 3'-and 5'-untranslated regions, terminated by EcoR1 
linker oligonucleotide segments. The HBV-DNA segment was reisolated by 
preparative gel electrophoresis, electroeluted and divided into samples 
which were digested with the exonuclease Bal-31 for varying times from 0.5 
to 30 minutes at 37.degree. C. The extent of exonuclease digestion was 
characterized qualitatively by digesting a portion of each sample with 
XbaI endonuclease. The surface antigen coding region contains an XbaI site 
beginning 92 base pairs from the first base of the start codon. Therefore, 
samples in which Bal-31 digestion had proceeded beyond the Xba1 site would 
yield only one fragment upon gel electrophoresis after XbaI endonuclease 
incubation while samples with fewer bases removed would yield two classes 
of fragment: a homogenous large fragment and a heterogeneously sized small 
fragment. Samples yielding only one XbaI fragment were discarded. Samples 
yielding two size classes of fragments were blunt-ended by incubation with 
DNA polymerase I (Klenow fragment, see Klenow, H., et al., Proc. Nat. 
Acad. Sci. 65, 168 (1970) in the presence of all four deoxynucleotide 
triphosphates. Linker oligonucleotides containing the HindIII recognition 
site were added by blunt-end ligation using T4 DNA ligase. HindIII 
specific cohesive ends were generated by digestion with HindIII 
endonuclease. The mixture of fragments was then digested with XbaI 
endonuclease and fractionated by gel electrophoresis. Fractions of 
approximately 90-110 nucleotides length were isolated from the gel and 
joined by ligation to pHBS-5 previously cut with HindIII and XbaI 
endonucleases. E. coli transformants were screened for tetracycline 
sensitivity. Clones were further screened for the presence of plasmid DNA 
yielding HindIII-XbaI fragments of about 90-110 nucleotides length. The 
largest of these (about 110 nucleotides) was chosen. The HindIII specific 
end of the fragment was located proximal to the start codon of the surface 
antigen coding segment, on the 5' side thereof. Consequently, the AML 
promoter fragment and surface antigen coding segment could be joined in 
correct orientation to one another by virtue of the complementarity of 
their respective HindIII-specific ends. 
A mixture of the approximately 440 base pair AML promoter fragments and the 
surface antigen coding segment was treated with DNA ligase under reaction 
conditions favorable to the DNA joining reaction. The ligation mixture was 
then digested with a mixture of BamHI and EcoR1 endonucleases, to separate 
concatamers arising from self-ligation at the BamHI and EcoR1 sites. The 
mixture was then fractionated by gel electrophoresis, and a fragment of 
about 1300 base pairs was isolated as the main reaction product. 
The 1300 base pair composite fragment, designated the AML-HBsAg gene, was 
inserted into the plasmid vector pHBS16, previously described in copending 
application Ser. No. 402,330. The plasmid, and a microorganism transformed 
thereby, were placed on deposit with the ATCC on Aug. 4, 1981, and have 
accession Numbers 40,043 and 20,619 for the plasmid and microorganism, 
respectively. The plasmid pHBS16 was digested with EcoR1 and BamHI 
endonucleases and the largest fragment resulting therefrom was reisolated 
by gel electrophoresis. The large pHBS16 fragment was joined with the 
AML-HBsAg gene, using DNA ligase, and the resulting plasmid was used to 
transform E. coli HB101. The transformants were screened to identify those 
yielding the correctly sized fragments following digestion with EcoR1, 
BamHI and HindIII endonucleases. The resulting plasmid was designated 
pAH1. A schematic diagram of the construction of pAH1 is shown in FIG. 1. 
A second plasmid was constructed by modification of pAH1. The modification 
consisted of deleting a segment of about 150 nucleotides length, well 
upstream from the TATA box at the 5' end of the promoter fragment. Plasmid 
pAH1 was then digested with BamHI and XhoI endonucleases. The resuling 
single-stranded ends were filled in, using the Klenow fragment of DNA 
polymerase. The reaction mixture was diluted to minimize the likelihood of 
intermolecular ligation, and treated with DNA ligase to regenerate closed 
loop DNA by blunt end ligation. The nucleotide sequences of the BamHI and 
XhoI, cleaved and blunt-ended as described, are such that, when joined 
together, they regenerate a complete XhoI site. 
The resulting plasmid was designated pAH2. Its route of synthesis and 
structure are shown schematically in FIG. 2. 
A third vector was constructed, as a control, by deleting the AML promoter 
region from plasmid pAH1. If expression of the surface antigen in pAH1 
was, in fact, controlled by the AML promoter, deletion of the promoter 
should result in loss of expression. 
The plasmid pAH1 was digested with BamHI endonuclease, and then partially 
digested with HindIII endonuclease. Since digestion of both HindIII sites 
of pAH1 would result in excision of the surface antigen coding region, a 
partial digestion was necessary, the desired result being a single 
cleavage at the HindIII site joining the surface antigen coding region and 
the AML promoter. After partial digestion, the unpaired ends were filled 
in with the Klenow fragment of DNA polymerase. The reaction mixture was 
diluted to enhance the probability of intramolecular ligation, and closed 
loop DNA was regenerated by blunt end ligation. Plasmids initially cleaved 
at the desired HindIII site resulted in a larger closed loop DNA than 
those cleaved at the undesired site, or at both sites. After identifying 
the correctly cleaved plasmid by gel electrophoresis, these were further 
screened for a subset containing a new, second HindIII site. The existence 
of a subset having a new HindIII site was discovered to have occurred as a 
result of partial exonuclease activity of the DNA polymerase preparation. 
When two base pairs are removed from the BamHI end, in the blunt end 
reaction using the DNA polymerase Klenow fragment, subsequent blunt end 
ligation regenerates a HindIII site near the 5' end of the surface antigen 
coding segment. A schematic of the construction and structure of the 
resulting plasmid, pAH3, is shown in FIG. 3. 
A fourth vector was constructed, to incorporate a terminator region 
adjacent to the 3' end of the coding segment. For this purpose, the AML 
promoter fragment was derived and purified from pAH1 digested with HindIII 
endonuclease and SphI endonuclease. The hepatitis B surface antigen coding 
segment was a TacI-HpaI fragment of hepatitis B virus DNA, obtained as 
described by Valenzuela, P., et al., Nature 298, 347 (1982). The surface 
coding antigen fragment was then modified by the addition of HindIII 
linkers at each end, by blunt end ligation. The ADH terminator region of 
the yeast ADH gene was a HindIII-SphI fragment prepared as described in 
copending application Ser. No. 402,330, supra. The specificity of the 
resulting ends made it possible to join the promoter, coding region and 
terminator to form a single composite, DNA segment herein termed the 
surface antigen cassette. After the three fragments were joined by DNA 
ligase, the reaction product was treated with SphI endonuclease to destroy 
dimers and concatamers incorrectly joined at the SphI sites. After gel 
electrophoresis to isolate and purify the cassette DNA, the cassette was 
joined with plasmid pHBS56 digested with endonuclease. The plasmid pHBS56 
has been described in copending application Ser. No. 402,330, supra, and, 
together with a yeast strain transformed thereby, has been placed on 
deposit with ATCC on July 7, 1982, accession numbers 40,047 and 20,648, 
for the plasmid and yeast strain, respectively. The cassette DNA was 
joined to the cut plasmid by DNA ligase and the resulting plasmid, termed 
pAH56, was used to transform yeasts, after screening for clones having the 
predicted restriction site pattern. Details of construction and structure 
of pAH56 are shown schematically in FIG. 4. The orientation of the 
cassette shown in the diagram is arbitrary; however, since the cassette is 
self-contained with respect to the elements necessary for expression of 
the coding region contained therein, it makes no difference, so far as 
function is concerned, which orientation was isolated, since both are 
functional. 
The structures of 5' untranslated region in pAH1 and pAH56 are shown in 
Tables 1 and 2, respectively, based on nucleotide sequence analysis. The 
sequence also shows the 5' end of the surface antigen coding segment, up 
to the XbaI site. The sequence for pAH2 is identical to the sequences 
shown, except that the first approximately 150 nucleotides of the 
sequence, up to the XhoI site, are deleted in pAH2. The sequence differs 
at two points from the published sequence for the AML promoter, at 
position 204, where the published sequence shows an AT pair instead of the 
GC pair, found in the present study, and at position 339, where the 
published sequence shows a GC pair, and the present results indicate an AT 
pair. The TATA box begins at about position 381, and has the sequence 
TATAAAA. The cap site, where transcription is initiated in mammalian 
cells, is the AT pair at position 412. The start codon of the coding 
sequence is the ATG sequence, which commences at position 464. In pAH56, 
the start codon is the ATG triplet commencing at position 481. 
EXAMPLE 2 
Expression in Yeast Mediated by the AML Promoter Yeast cells transformed 
with either pAH56, pAH1 or pAH3, were grown in 100 ml cultures at 
30.degree. C. in YEPD medium. The host cell strain for pAH56 was S. 
cerevisiae 2150-2-3, and for pAH1 and pAH3, the host cell strain was S. 
cerevisiae AB35-14D, as previously described in copending application Ser. 
No. 402,330. The total protein concentration was determined by 
spectrophotometric measurement of coomassie blue dye binding. The surface 
antigen concentration was determined by radioimmunoassay, using a 
commercially available assay kit (Abbott Laboratories, North Chicago, 
Ill.). The results are shown in Table 3. The pAH56 vector produced about 
ten times the surface antigen expressed under the influence of the pAH1 
vector. However, no radioimmunoassay-detectable surface antigen was 
produced by pAH3, the vector lacking the AML promoter. For comparison, the 
amount of antigen produced with the AML promoter, in vector pAH1, was 
about 80% of the amount produced by the corresponding vector having a 
yeast ADH promoter. Therefore, it can be seen that the AML promoter is 
highly active in yeast, and that the expression observed with pAH1 and 
pAH56 is due to the presence of the AML promoter in the vector, and not 
due to some other, outlying promoter. 
While the invention has been described by reference to specific operating 
examples, it is intended that the scope of the invention shall include 
such alternative embodiments and variations as lie within the grasp of 
those ordinarily skilled in the art, or combined with expedients known in 
the art. 
TABLE 1 
__________________________________________________________________________ 
1 
##STR1## 
##STR2## 
61 
##STR3## 
AAGAATGGAGACCAAAGG TACTCGGCCACAGGTGC GAGCCACTGCTTTTT CCGACAGGCA 
121 
##STR4## 
##STR5## 
181 
##STR6## 
##STR7## 
241 
##STR8## 
##STR9## 
301 
##STR10## 
##STR11## 
361 
##STR12## 
##STR13## 
421 
##STR14## 
##STR15## 
481 
##STR16## 
##STR17## 
541 
##STR18## 
##STR19## 
__________________________________________________________________________ 
TABLE 2 
__________________________________________________________________________ 
1 
##STR20## 
##STR21## 
61 
##STR22## 
AAGAATGGAGACCAAAGG TACTCGGCCACAGGTGC GAGCCACTGCTTTTT CCGACAGGCA 
121 
##STR23## 
##STR24## 
181 
##STR25## 
##STR26## 
241 
##STR27## 
##STR28## 
301 
##STR29## 
##STR30## 
361 
##STR31## 
##STR32## 
421 
##STR33## 
##STR34## 
481 
##STR35## 
##STR36## 
541 
##STR37## 
##STR38## 
__________________________________________________________________________ 
TABLE 3 
______________________________________ 
Expression of Surface Antigen in Yeast 
One hundred milliliter cultures of the genetically engineered yeast, 
i.e., containing the 56-type plasmid, were grown in leucine 
deficient medium, at 30.degree. C. A cell free lysate was prepared by 
agitation with glass beads. The protein concentration was 
determined by the coomassie dye binding method. The surface 
antigen concentration was determined with the Abbott 
radioimmunoassay kit. 
Protein sAntigen 
Concentration 
Concentration 
Specific (.mu.g Ag) 
Expt. # 
(mg/ml) (.mu.g/ml) Activity (mgPtn) 
______________________________________ 
1 25 10.2 0.41 
2 38 12.5 0.38 
3 17 5.7 0.33 
4 17 5.8 0.34 
Average 
24 8.6 0.37 
______________________________________