Production of Protein A

Novel Protein A-producing Gram-positive bacterial strains and methods for their preparation are disclosed. Also disclosed are methods for producing Protein A using the novel Gram-positive strains.

BACKGROUND OF THE INVENTION 
Protein A is a cell wall component produced by nearly all strains of 
Staphylococcus aureus (see e.g. Forsgren, A., Infection and Immunity 2: 
672-673 [1970]); and Sjoquist, J. et al., Eur. J. Biochem. 30: 190-194 
[1972]). Protein A is useful in that it binds strongly and specifically to 
the Fc portion of immunoglobulin IgG from a variety of mammalian sources, 
including human (Kronvall, G. et al., J. Immunol. 103: 828-833 [1969]). 
Thus this protein has been used in diagnostic applications and has 
potential therapeutic value. 
In most S. aureus strains, at least 70% of the Protein A produced is 
covalently linked to the peptidoglycan of the cell wall (Sjoquist, J. et 
al., Eur. J. Biochem. 30: 190-194 [1972]. The site of attachment is the 
C-terminal region of the Protein A molecule (Sjodahl, J., Eur. J. Biochem. 
73: 343-351 [1977]). Some Protein A (15-30%) is generally excreted into 
the growth medium, and there are several circumstances under which the 
fraction of Protein A which is excreted can be increased. Some methicillin 
resistant strains of S. aureus excrete essentially all their Protein A 
(Lindmark, R. et al., Eur. J. Biochem. 74: 623-628 [1977]). Low levels of 
puromycin increase the amount of excreted Protein A, presumably by 
truncating the protein and thereby eliminating its C-terminal cell wall 
attachment site, and protoplasts excrete nearly all the Protein A which 
they synthesize (Movitz, J., Eur. J. Biochem. 68: 291-299 [1976]). 
Protein sequence information is available for Protein A from S. aureus 
strain Cowan I (Sjodahl, J., Eur. J. Biochem. 78: 471-490 [1977]). The 
Cowan I strain contains approximately 2.times.10.sup.5 molecules of 
Protein A per cell (Sjoquist, J. et al., Eur. J. Biochem. 30: 190-194 
[1972]). 
Protein A is synthesized in S. aureus only during exponential growth, and 
synthesis ceases as the culture approaches stationary phase (Movitz, J., 
Eur. J. Biochem. 48: 131-136 [1974]). The level of synthesis of Protein A 
in S. aureus is highly variable, and is strongly influenced by the growth 
conditions in some as yet poorly defined ways (Landwall, P., J. Applied 
Bact. 44: 151-158 [1978]). 
The Protein A gene from S. aureus strain Cowan I has been cloned in E. 
coli. Lofdahl, S., et al., Proc. Natl. Acad. Sci. USA, 80, 697-701 (1983). 
This gene is contained in a 2.15 kilobase insert bounded by EcoRV 
restriction sites. The gene has been inserted into a plasmid and cloned in 
E. coli, where low levels of expression have been achieved. The chimeric 
plasmid which contains the Protein A gene has been designated "pS." 
Currently, industrial production of Protein A is carried out using mutant 
strains of S. aureus. A major disadvantage of using S. aureus to produce 
Protein A is that all available production strains are human pathogens. 
Although many genetic engineering experiments have been conducted using 
Escherichia coli, that organism is not suitable for efficient production 
of Protein A, since it does not export protein outside the cell. 
Furthermore, E. coli possesses disadvantageous pathogenic properties as 
well, i.e., produces endotoxins. 
There thus remains a need for the production of Protein A by means which 
are both safe and efficient. 
SUMMARY OF THE INVENTION 
In accordance with the present invention, Protein A-producing Gram-positive 
bacteria are prepared by introduction into Gram-positive cells which do 
not normally produce Protein A, vectors containing the nucleotide sequence 
coding for Protein A and expression signals directing expression of the 
Protein A gene in the microorganism. Protein A can be produced by 
cultivating such cells in a nutrient medium under protein-producing 
conditions. 
DETAILED DESCRIPTION OF THE INVENTION 
A method of achieving high level production of Protein A in Gram-positive 
microorganisms without substantially inhibiting the growth of the host has 
been discovered. The method involves transformation of a Gram-positive 
microorganism by introduction therein of a vector containing the 
nucleotide sequence coding for Protein A. A Protein A gene may be obtained 
from Protein A-producing microorganisms, such as the above-mentioned 
strains of S. aureus. A preferred source of the gene is plasmid pS, 
which has been cloned in E. coli. See Lofdahl, S., et al. (supra). The 
gene may advantageously be excised from that clone by digestion with 
endonuclease EcoRV. 
The Protein A gene may contain its natural expression signals (i.e., 
transcriptional and translational initiation sequences), or those signals 
may be replaced by other expression signals recognizable by the 
Gram-positive host microorganism. Replacement of the natural expression 
signals with other recognizable Gram-positive expression signals may be 
accomplished using conventional methods of molecular biology. Such 
replacement involves cleavage of the natural expression signals from the 
Protein A sequence and fusion of the desired expression signals to the 
Protein A gene. 
Construction of the Protein A-producing strains of this invention involves 
inserting, by recombinant DNA techniques, the Protein A gene into a 
plasmid vector. Such a vector may be prepared in vitro and inserted 
directly into the Gram-positive bacterial host by transformation 
techniques. The vector is preferably cloned in another organism for 
amplification and purification prior to transformation of the ultimate 
Gram-positive host cells. The microorganism used for the intermediate 
cloning step may be an organism in which the vector will be maintained and 
express selectable phenotypical properties. E. coli is the preferred 
microorganism for the intermediate cloning step. 
When the vector is constructed in vitro and first cloned in E. coli, it 
advantageously contains a functional E. coli replicon as well as a 
phenotypic marker for E. coli. The vector also advantageously contains a 
phenotypic marker for the Gram-positive host microorganism. In one 
embodiment of the present invention, the vector also contains a functional 
replicon permitting autonomous replication in the Gram-positive bacteria 
selected. One or more copies of the Protein A gene may be inserted into 
this vector, and the vector then used to transform the appropriate 
Gram-positive microorganism. 
In preferred embodiments of this invention, the vector does not contain a 
replicon capable of functioning in the Gram-positive microorganism 
selected, but rather contains a DNA sequence homologous to a region of the 
chromosome of that Gram-positive microorganism. This construction permits 
linear integration of the vector into the host chromosome in the region of 
homology. The vector is again advantageously constructed in vitro and 
first cloned in E. coli as above; however, if desired, the gram-positive 
bacterial host may be transformed directly with the chimeric plasmid. Such 
a vector transforms the Gram-positive microorganism by recombination with 
the homologous region of Gram-positive host chromosome. An advantage of 
this method is that there is less likelihood of loss of the Protein A 
sequence from the host, due to negative selection favoring plasmid-free 
cells, and Protein-A producing strains prepared in this manner have been 
found to be genetically stable. 
The Gram-positive host microorganisms employed in this invention are 
advantageously selected from non-pathogenic strains which do not normally 
synthesize Protein A. Although the invention will be described in detail 
with regard to Bacillus subtilis, it is to be understood and will be 
appreciated by those skilled in the art that the invention is applicable 
to a variety of Gram-positive microorganisms. Particularly preferred host 
microorganisms are well known industrial strains of the genera, Bacillus 
and Streptomyces. Generally, it has been found that Protein A is produced 
at optimum levels during the exponential growth phase of the organisms, 
and production slows considerably thereafter. It has also been found that 
the period of Protein A synthesis can be extended using sporulation 
deficient (spo.sup.-) Gram-positive hosts. When spo.sup.- hosts are used, 
the resulting strains are generally genetically more stable, the level of 
Protein A is higher, and, because fewer proteases are produced by these 
cells, the Protein A product is more stable. 
Transformation of the Gram-positive microorganism may be accomplished by 
any suitable means. A particularly preferred transformation technique for 
these organisms is to remove the cell wall by lysozyme digestion, followed 
by transformation of the resulting protoplasts. Chang, S., et al. Molec. 
Gen. Genet., 168, 111-115 (1979). Alternatively, cells competent for 
transformation can be transformed by a modification of the method of 
Anagnostopoulos, C., et al., J. Bacteriol., 81, 741-746 (1961), as 
described in Example III below. 
The procedures used to clone the Protein A gene and construct Protein 
A-producing strains of B. subtilis described herein are, except where 
otherwise indicated, accomplished by using conventional techniques of 
molecular biology. Segments of DNA containing the sequence coding for 
Protein A are isolated. If the sequences contain the natural expression 
signals for Protein A, the segments may be inserted into an appropriate 
vector without further modification. If the sequences do not contain the 
natural expression signals, or it is desired to replace them, the existing 
expression signals (if present) may be enzymatically removed and a DNA 
sequence containing the desired expression signals may then be fused to 
the Protein A gene. The Protein A sequences attached to the desired 
expression signals may then be inserted into an appropriate vector. 
Vectors appropriate for transformation of B. subtilis are generally 
plasmids, and are advantageously constructed in E. coli. Such vectors 
contain a functional E. coli replicon, a phenotypic marker for E. coli, 
and a phenotypic marker for B. subtilis. The vector may also contain a B. 
subtilis replicon, but preferably it does not and instead contains a DNA 
sequence homologous to a region of the B. subtilis chromosome. Insertion 
of the homologous DNA sequence into the vector permits recombination of 
the vector with the B. subtilis chromosome, where it can be maintained at 
a copy number of one per genomic equivalent. 
One or more copies of the DNA sequences coding for Protein A and the 
desired expression signals are then inserted into the vector. The presence 
of the E. coli replicon and phenotypic marker in the vector permit its 
cloning and maintenance in E. coli, and allow for selection of clones 
containing the vector. 
When an intermediate cloning step in E. coli is employed, one or more E. 
coli colonies which carry the Protein A-containing plasmid are grown on 
suitable nutrient media, and the plasmids are isolated therefrom. Cells of 
B. subtilis (i.e. competent cells of protoplasts) are then transformed by 
introduction therein of the vector and successful transformants are 
selected by means of the B. subtilis phenotypic marker. Vectors containing 
a B. subtilis replicon are capable of reproducing in the host and 
producing Protein A when the cells are grown under protein-producing 
conditions. Alternatively, vectors not containing a B. subtilis replicon 
but instead containing a DNA sequence homologous with the host chromosome 
will recombine with the host chromosome and be replicated along with the 
host chromosome.

A further embodiment of the present invention involves preparation of 
vectors differing in their homologous chromosomal DNA sequences, but still 
containing one or more copies of the Protein A gene. Thus, vectors can 
contain sequences from different regions of the B. subtilis chromosome, or 
even from chromosomes of different species of Bacillus. This permits 
integration of the vectors into different parts of the host chromosome in 
the corresponding regions of homology resulting in transformants with more 
than one vector incorporated in the host chromosome. 
Transformed B. subtilis cells are grown in a nutrient medium under 
protein-producing conditions resulting in the production of Protein A by 
the cells and the secretion of Protein A into the medium. Protein A may 
then be purified from the medium after removing intact cells using 
conventional techniques. 
Those skilled in the art will recognize that, although the present 
disclosure describes cloning and expression of the entire Protein A gene, 
functional segments of that gene or fusions of the gene with other DNA 
segments can also be cloned and expressed in accordance with the teachings 
herein. Such segments and fusions are, therefore, intended to be within 
the scope of this invention. 
The invention is further illustrated by the following examples which are 
not intended to be limiting. For the DNA manipulation described in this 
and the following examples, the restriction endonucleases and other 
enzymes used were purchased from New England Biolabs, Inc., Bethesda 
Research Laboratories, Inc., Boehringer Mannheim GmbH, and were used in 
the conventional manner as recommended by the manufacturer, except as 
noted otherwise. 
EXAMPLE I 
Isolation of a DNA sequence containing the Protein A gene and Promoter 
Region 
Plasmid pSPAI (consisting of a 7.6 kilobase pair insert of DNA derived from 
S. aureus strain 8325-4 in E. coli vector pBR322) at a concentration of 
110 .mu.g/ml was digested with restriction endonuclease EcoRV at 256 
units/ml in a buffer ("EcoRV buffer") containing 150 mM NaCl, 6 mM 
Tris-HCl (pH 7.9), 6 mM MgCl.sub.2, 6 mM 2-mercaptoethanol for 1 hour at 
37.degree. C., then for an additional 30 min. with an additional 256 
units/ml EcoRV endonuclease. A small EcoRV fragment (2.15 kb) was isolated 
by agarose gel electrophoresis and electroelution, and found to obtain the 
Protein A gene and promoter region (see Examples II and IV). 
EXAMPLE II 
Insertion of the 2.15 kb pair fragment into Plasmid pGX251 
Plasmid pGX251 (containing an E. coli replicon derived from plasmid pBR322, 
a B. subtilis replicon derived from plasmid pC194, the gene for ampicillin 
resistance, the gene for chloramphenicol resistance and a unique EcoRV 
site) was linearized by restriction endonuclease digestion with EcoRV (640 
units/ml) at a concentration of 40 .mu.g/ml in EcoRV buffer for 1 hour at 
37.degree. C. Digestion was terminated by incubation for 8 minutes at 
65.degree. C. and was determined to be complete by agarose gel 
electrophoresis. The 2.15 kb EcoRV fragment from Example I and linearized 
pGX251 were ligated at a concentration of 200 .mu.g/ml EcoRV fragment, 100 
.mu.g/ml linearized pGX251, in a buffer ("ligation buffer") containing 50 
mM Tris-HCl (pH 7.8), 10 mM MgCl.sub.2, 2 mM dithiothreitol, 0.5 mM ATP, 
and 100 .mu.g/ml bovine serum albumin, and 4.times.10.sup.5 Units/ml T4 
DNA ligase at 5.degree. C. for 15 hours. 
Calcium-shocked E. coli strain SK2267 (F.sup.-, gal.sup.-, thi.sup.-, 
T.sub.1.sup.R, hsdR4, recA.sup.-, endA.sup.-, sbcB15) cells (0.2 ml), 
prepared as described by R. W. Davis, et al., "Advances Bacterial 
Genetics" Cold Spring Harbor Laboratory, N.Y. (1980) were transformed with 
the ligation mixture containing 0.2 .mu.g linearized pGX251 and 0.4 .mu.g 
of the 2.15 kb Eco RV fragment. Colonies were selected on standard L-broth 
plates containing 50 .mu.g/ml ampicillin. An ampicillin resistant 
transformant designated strain GX3311 produced approximately 1 
.mu.g/A.sub.600 unit of Protein A, determined by the method of Lofdahl, et 
al. (supra). The plasmid carried by this strain, designated pgX2901, 
consisted of a single copy of the 2.15 kb Eco RV fragment in pGX251. 
EXAMPLE III 
B. subtilis competent cell transformation by pGX251 containing Protein A 
gene and Protein A production therewith 
Competent cells of B. subtilis, strain BR151 (Lovett, P. S., et al., J. 
Bacteriol., 127, 817-828 (1976)) were transformed with 0.3 .mu.g/ml 
plasmid pGX251 containing the protein A gene. To prepare competent cells, 
B. subtilis strain BR151 was grown overnight at 37.degree. C. on tryptose 
blood agar base (Difco). Cells were resuspended in 10 ml SPI medium 
supplemented with 50 .mu.g/ml each of lysine, tryptophan, and methionine 
to give a reading of 50-70 on a Klett-Summerson colorimeter equipped with 
a green filter (Klett Mfg. Co., New York). SPI medium consists of 1.4% 
K.sub.2 HPO.sub.4, 0.6% KH.sub.2 PO.sub.4, 0.2% (NH.sub.4).sub.2 SO.sub.4, 
0.1% sodium citrate.multidot.2H.sub.2 O, 0.5% glucose, 0.1% yeast extract 
(Difco), 0.02% Bacto-Cusamino acids (Difco), and 0.02% MgSO.sub.4 
.multidot.7H.sub.2 O. The cultures were incubated at 37.degree. on a 
rotary shaker (200-250 rpm) for 3-41/2 hours until logarithmic growth 
ceased and the cells entered early stationary phase. The cells were then 
diluted 10 fold into the same medium supplemented with 0.5 mM CaCl.sub.2. 
Incubation was continued for 90 min. The cells were then centrifuged for 5 
min. at room temperature, and resuspended in 1/10 volume of spent medium. 
1 ml aliquots of the cell suspensions were frozen in liquid nitrogen and 
stored at -80.degree. C. for use. 
For transformation, the frozen competent cells were thawed quickly at 
37.degree. and diluted with an equal volume of SPII medium supplemented as 
above with amino acids. SPII is the same as SPI except that the 
concentration of MgSO.sub.4 is increased to 0.04% and 2 mM 
ethyleneglycol-bis-(.beta.-aminoethylether)-N,N,N',N'-tetraacetic acid 
(EGTA) is added. Cells (0.5 ml) are mixed with 0.1 to 5 .mu.g of DNA in 
13.times.100 mm glass tubes. The cell suspensions are rotated at 
37.degree. C. for 30 min. Penassay broth (Difco) (1-2 ml) is then added 
and incubation continued for 60 min. at 37.degree. C. Cells are then 
recovered by centrifugation, resuspended in 0-2 ml Penassay broth, and 
plated on LB agar plates containing 5-10 .mu.g/ml chloramphenicol. 
Successful transformants were selected at 5 .mu.g/ml chloramphenicol. The 
transformed B. subtilis strain was designated GX3308. This strain was 
shown to produce small quantitites of Protein A by the procedure of 
Lofdahl, et al. (supra), but lost the plasmid quickly upon culturing in a 
nutrient medium. 
EXAMPLE IV 
Insertion of 1 copy of the 2.15 kb fragment into Plasmid pGX284 
Plasmid pGX284 (containing an E. coli replicon derived from plasmid pBR322, 
the gene for ampicillin resistance, the gene for chloramphenicol 
resistance, a unique EcoRV site, and an undetermined B. subtilis 
chromosomal sequence) was linearized by endonuclease digestion with EcoRV 
at a concentration of 40 .mu.g/ml in EcoRV buffer for 1 hour at 37.degree. 
C. Digestion was terminated by incubation for 8 minutes at 65.degree. C. 
and was determined to be complete by agarose gel electrophoresis. The 2.15 
kb EcoRV fragment from Example I and linearized pGX284 were ligated at a 
concentratin of 200 .mu.g/ml EcoRV fragment, 100 .mu.g/ml linearized 
pGX284 under the conditions described in Example II. 
Calcium-shocked E. coli strain SK2267 cells were transformed with the 
ligation mixture containing 0.2 .mu.g linearized pGX284 and 0.4 .mu.g 2.15 
kb EcoRV fragment. Colonies were isolated on standard L-broth plates 
containing 50 .mu.g/ml ampicillin. An ampicillin resistant transformant 
designated strain GX3320 produced approximately 1 .mu.g/A.sub.600 unit of 
Protein A. The plasmid carried by this strain, designated pGX2907 was 
determined to consist of a single copy of the 2.15 kb EcoRV fragment 
inserted into pGX284. Transformed Strain GX3320 has been deposited with 
the American Type Culture Collection, Rockville, Md., USA and has been 
designated ATCC No. 39344. 
EXAMPLE V 
B. subtilis protoplast transformation by pGX284 containing single copy of 
Protein A gene and production of Protein A therewith 
Protoplasts derived from B. subtilis strain 1S53 (spo0A.DELTA.677) were 
transformed with 0.1 .mu.g/ml plasmid pGX2907 containing a single copy of 
the Protein A gene. Strain 1S53 was obtained from the Bacillus Genetic 
Stock Center, Ohio State University, Dept. of Microbiology, 484 West 12th 
Ave., Columbus, Ohio 43210 USA. Successful transformants were selected at 
5 .mu.g/ml chloramphenicol. A transformant (designated strain GX3305) was 
found to produce approximately 50 .mu.g/ml Protein A in the extracellular 
growth medium when grown in a medium containing (per liter) 33 g tryptone, 
20 g Yeast extract, 7.4 g NaCl, 12 ml 3M NaOH, 8 g Na.sub.2 HPO.sub.4, 4 g 
KH.sub.2 PO.sub.4 for 17 hours at 37.degree. C. GX3305 has been deposited 
with the American Type Culture Collection, Rockville, Md., U.S.A. and has 
been designated ATCC No. 39345. 
EXAMPLE VI 
Insertion of 2 tandem copies of the 2.15 kb fragment into Plasmid pGX284 
From the same tranformation described in Example IV, an ampicillin 
resistant transformant was isolated (designated strain GX3202-2) which was 
determined by restriction endonuclease digest anlaysis to carry a plasmid 
(designated pGX2907-2) in which two tandem copies of the 2.15 kb EcoRV 
fragment had been inserted into pGX284. 
EXAMPLE VII 
B. subtilis transformation by pGX284 containing two tandem copies of 
Protein A gene and production of Protein A therewith 
Competent cells of B. subtilis strain BR151 were transformed with 0.3 
.mu.g/ml plasmid pGX2907-2, containing two tandem copies of the Protein A 
gene. Successful transformants were selected at 10 .mu.g/ml 
chloramphenicol. One transformant designated strain GX3302-2 was grown in 
a standard fermenter (8L) containing 2.times.L Broth for 7 hours. The 
final yield of Protein A was 47 mg/l in the extracellular growth medium as 
determined by IgG binding activity by a competitive ELISA procedure as 
described by Lofdahl et al. (supra).