Process for producing heterologous polypeptides

Processes for producing various heterologous polypeptides which when expressed are either incorrectly processed and hence asssociated with the surface of the host cell or are not processed to mature form. More specifically, processes for the production of heterologous non-human carbonyl hydrolases expressed either in host cells incapable of producing enzymatically active endoprotease or host cells deficient in enzymatically active extracellular endoprotease are disclosed. Such non-human carbonyl hydrolases generally are incapable of autoproteolytic maturation and become associated with the surface of expression hosts which are deficient in enzymatically active extracellular endoprotease. Processes for preparing non-human carbonyl hydrolase and heterologous polypeptides which are expressed as part of a fusion polypeptide are also disclosed, as well as non-human carbonyl hydrolases which are substantially free of the host cell membrane with which they are normally associated.

FIELD OF THE INVENTION 
The present invention relates to processes for producing various 
heterologous polypeptides which when expressed are either incorrectly 
processed and hence become associated with the surface of the host cell or 
are not processed to mature form. More specifically, the invention relates 
to processes for the production of heterologous non-human carbonyl 
hydrolases expressed either in host cells capable of producing 
enzymatically active endoprotease or in host cells deficient in 
enzymatically active extracellular endoprotease. Such non-human carbonyl 
hydrolases generally are incapable of autoproteolytic maturation and 
become associated with the surface of expression hosts which are deficient 
in enzymatically active extracellular endoprotease. 
The invention also relates to processes for preparing heterologous 
polypeptides which are expressed as part of a fusion polypeptide as well 
as non-human carbonyl hydrolases which are substantially free of the host 
cell membrane with which said hydrolases are normally associated. 
BACKGROUND OF THE INVENTION 
Various bacteria are known to secrete proteases at some stage in their life 
cycles. Bacillus species produce two major extracellular proteases, a 
neutral protease (a metalloprotease inhibited by EDTA) and an alkaline 
protease (or subtilisin, a serine endoprotease). Both generally are 
produced in greatest quantity after the exponential growth phase, when the 
culture enters stationary phase and begins the process of sporulation. The 
physiological role of these two proteases is not clear. They have been 
postulated to play a role in sporulation (J. Hoch, (1976) Adv. Genet. 18, 
69-98; P. Piggot, et al., (1976) Bact. Rev. 40, 908-962; and F. Priest, 
(1977) Bact. Rev. 41, 711-753), to be involved in the regulation of cell 
wall turnover (L. Jolliffe, et al., (1980) J. Bact. 141, 1199-1208), and 
to be scavenger enzymes (Priest, Id.). The regulation of expression of the 
protease genes is complex. They appear to be coordinately regulated in 
concert with sporulation, since mutants blocked in the early stages of 
sporulation exhibit reduced levels of both the alkaline and neutral 
protease. Additionally, a number of pleiotropic mutations exist which 
affect the level of expression of proteases and other secreted gene 
products, such as amylase and levansucrase (Priest, Id.). 
Subtilisin has found considerable utility in industrial and commercial 
applications (see U.S. Pat. No. 3,623,957 and J. Millet, (1970) J. Appl. 
Bact. 33, 207). For example, subtilisins and other proteases are commonly 
used in detergents to enable removal of protein-based stains. They also 
are used in food processing to accommodate the proteinaceous substances 
present in the food preparations to their desired impact on the 
composition. 
Classical mutagenesis of bacteria with agents such as radiation or 
chemicals has produced a plethora of mutant strains exhibiting different 
properties with respect to the growth phase at which protease excretion 
occurs as well as the timing and activity levels of excreted protease. 
These strains, however, do not approach the ultimate potential of the 
organisms because the mutagenic process is essentially random, with 
tedious selection and screening required to identify organisms which even 
approach the desired characteristics. Further, these mutants are capable 
of reversion to the parent or wild-type strain. In such event the 
desirable property is lost. The probability of reversion is unknown when 
dealing with random mutagenesis since the type and site of mutation is 
unknown or poorly characterized. This introduces considerable uncertainty 
into the industrial process which is based on the enzyme-synthesizing 
bacterium. Finally, classical mutagenesis frequently couples a desirable 
phenotype, e.g., low protease levels, with an undesirable character such 
as excessive premature cell lysis. 
Special problems exist with respect to the proteases which are excreted by 
Bacillus. For one thing, since at least two such proteases exist, 
screening for the loss of only one is difficult. Additionally, the large 
number of pleiotropic mutations affecting both sporulation and protease 
production make the isolation of true protease mutations difficult. 
Temperature sensitive mutants of the neutral protease gene have been 
obtained by conventional mutagenic techniques, and were used to map the 
position of the regulatory and structural gene in the Bacillus subtilis 
chromosome (H. Uehara, et al., (1979) J. Bact. 139, 583-590). 
Additionally, a presumed nonsense mutation of the alkaline protease gene 
has been reported (C. Roitsch, et al., (1983) J. Bact. 155, 145-152). 
Bacillus temperature sensitive mutants have been isolated that produce 
inactive serine protease or greatly reduced levels of serine protease. 
These mutants, however, are asporogenous and show a reversion frequency to 
the wild-type of about from 10.sup.-7 to 10.sup.-8 (F. Priest, Id. p. 
719). These mutants are unsatisfactory for the recombinant production of 
heterologous proteins because asporogenous mutants tend to lyse during 
earlier stages of their growth cycle in minimal medium when compared to 
sporogenic mutants, thereby prematurely releasing cellular contents 
(including intracellular proteases) into the culture supernatant. The 
possibility of reversion also is undesirable since wild-type revertants 
will contaminate the culture supernatant with excreted proteases. 
Bacillus sp. have been proposed for the expression of heterologous 
proteins, but the presence of excreted proteases and the potential 
resulting hydrolysis of the desired product has retarded the commercial 
acceptance of Bacillus as a host for the expression of heterologous 
proteins. Bacillus megaterium mutants have been disclosed that are capable 
of sporulation and which do not express a sporulation-associated protease 
during growth phases. However, the assay employed did not exclude the 
presence of other proteases, and the protease in question is expressed 
during the sporulation phase (C. Loshon, et al., (1982) J. Bact. 150, 
303-311). This, of course, is the point at which heterologous protein 
would have accumulated in the culture and be vulnerable. 
Accordingly, an object of U.S. Pat. application Ser. No. 614,615 (EPO 
Publication No. 0130756) is the construction of a Bacillus strain which is 
substantially free of extracellular neutral and alkaline protease during 
all phases of its growth cycle and which exhibits substantially normal 
sporulation characteristics. The need disclosed therein is for a 
non-revertible, otherwise normal protease deficient organism that can be 
transformed with high copy number plasmids for the expression of 
heterologous or homologous proteins. 
The present inventors have discovered that certain mutant subtilisins (made 
according to the methods disclosed in EPO Publication No. 0130756) were 
not completely secreted from Bacillus expression hosts which were rendered 
incapable of expressing and secreting enzymatically active neutral 
protease and subtilisin. These mutant subtilisins, containing mutations 
within the active site of subtilisin, were found to be incapable of 
autoproteolytic maturation and thus were bound to the Bacillus cell 
membrane making them far more difficult to isolate than if they were 
completely secreted into the culture medium. The inventors discovered that 
such mutants can be released from the surface of such Bacillus expression 
hosts by contacting the host cells with an enzymatically active 
subtilisin. 
Accordingly, an object of the invention herein is to provide processes for 
producing a heterologous non-human carbonyl hydrolase which is not 
secreted but which is bound to the surface of an expression host which 
does not produce extracellular enzymatically active subtilisin. 
In addition, an object of the invention is to provide processes for 
producing heterologous non-human carbonyl hydrolases which are released 
from the surface of a host cell by an enzymatically active subtilisin 
produced by the host cell. 
A further object of the present invention is to provide processes for 
preparing heterologous polypeptides from a fusion polypeptide which can be 
cleaved to produce a desired heterologous polypeptide. 
Still further an object of the present invention is to provide non-human 
carbonyl hydrolases which are normally membrane-associated and not 
released from the host cell expressing the hydrolase. Such hydrolases are 
substantially free of the host cell membrane with which they are normally 
associated. 
SUMMARY OF THE INVENTION 
The invention includes processes for isolating heterologous non-human 
carbonyl hydrolases which are bound to the surface of an expression host 
which does not secrete enzymatically active endoprotease. The process 
comprises expressing a prepro form of heterologous non-human carbonyl 
hydrolase in a host cell which permits the prepro hydrolase to be 
transported to the surface of the host cell but not its release in mature 
form. The heterologous carbonyl hydrolase is removed from the surface of 
the host cell either by the addition of enzymatically active exogenous 
subtilisin or by co-culturing such expression host cells with a second 
cell line which is capable of secreting enzymatically active subtilisin. 
The bound heterologous carbonyl hydrolase thereby is released into the 
culture medium from which it may be readily isolated. 
In addition, it includes processes for producing heterologous non-human 
carbonyl hydrolases from host cells which produce a different 
enzymatically active subtilisin which is capable of releasing the carbonyl 
hydrolase from the surface of the host cell. 
The invention also comprises processes for preparing a heterologous 
polypeptide expressed as part of a fusion polypeptide. First, a fusion 
polypeptide is expressed which has an amino-terminal first sequence and a 
carbonyl-terminal second sequence corresponding to the desired 
heterologous polypeptide. The fusion polypeptide is capable of being 
cleaved by subtilisin at a recognition site at the junction of the first 
and second sequences of the fusion polypeptide or within the first 
sequence of the fusion polypeptide. This fusion polypeptide is then 
contacted with a subtilisin which is capable of cleaving the fusion 
polypeptide at the recognition site. 
The invention also includes non-human carbonyl hydrolases which are 
normally membrane-associated and normally not released from the surface of 
the host cell expressing the hydrolase. Such carbonyl hydrolase are 
extracellular and are substantially free of the host cell membrane with 
which the hydrolase is normally associated.

DETAILED DESCRIPTION 
Carbonyl hydrolases are enzymes which hydrolyze compounds containing 
##STR1## 
bonds in which X is oxygen or nitrogen. They included naturally occurring 
carbonyl hydrolases and mutant carbonyl hydrolases. Naturally occurring 
carbonyl hydrolases principally include hydrolases, e.g. lipases and 
peptide hydrolases, e.g. subtilisins or metalloproteases. Peptide 
hydrolases include .alpha.-aminoacylpeptide hydrolase, peptidylaminoacid 
hydrolase, acylamino hydrolase, serine carboxy-peptidase, 
metallocarboxypeptidase, thiol proteinase, carboxylproteinase and 
metalloproteinase. Serine, metallo, thiol and acid proteases are included, 
as well as endo and exo-proteases. 
"Mutant carbonyl hydrolase" refers to a carbonyl hydrolase in which the DNA 
sequence encoding the naturally occurring carbonyl hydrolase is modified 
to produce a mutant DNA sequence which encodes the substitution, insertion 
or deletion of one or more amino acids in the carbonyl hydrolase amino 
acid sequence. Suitable modification methods are disclosed herein and in 
EPO Publication No. 0130756. 
Subtilisins are carbonyl hydrolases which generally act to cleave peptide 
bonds of proteins or peptides. As used herein, "subtilisin" means a 
naturally occurring subtilisin or a mutant subtilisin. A series of such 
naturally occurring proteases is known to be produced and often secreted 
by various bacterial species. Amino acid sequences of the members of this 
series are not entirely homologous, however, the proteins in this series 
exhibit the same or similar type of proteolytic activity. This class of 
serine proteases shares a common amino acid sequence defining the 
catalytic triad which distinguishes them from the chymotrypsin related 
class of serine proteases. The subtilisins and chymotrypsin related serine 
proteases both have a catalytic triad comprising aspartate, histidine and 
serine. In the subtilisin related proteases the relative order of these 
amino acids, reading from the amino to carboxy terminus is 
aspartate-histidine-serine. In the chymotrypsin related proteases the 
relative order, however, is histidine-aspartate-serine. Thus, subtilisin 
herein bears a functional definition--i.e., it refers to a serine protease 
directly or indirectly associated with or related to a bacterial source. 
"Mutant subtilisin" refers to a subtilisin in which the DNA sequence 
encoding the subtilisin is modified to produce a mutant DNA sequence which 
encodes the substitution, deletion, or insertion of one or more amino 
acids in the subtilisin amino acid sequence. Suitable methods to produce 
such modification include those disclosed herein and in EPO Publication 
No. 0130756. It is to be understood, however, that subtilisin mutants 
containing a change in the content or relative order of the catalytic 
triad, as defined above, are still mutant subtilisins. Thus, for example, 
a mutant subtilisin having an amino acid sequence which resembles a 
chymotrypsin related protease is a subtilisin mutant as herein defined. 
A subtilisin which is "enzymatically active" is one which is capable of 
cleaving either the prosequence normally associated with such a subtilisin 
or the prosequence of the heterologous non-human carbonyl hydrolases as 
hereinafter defined. 
Carbonyl hydrolases and their genes may be obtained from many procaryotic 
and eucaryotic organisms. Suitable examples of procaryotic organisms 
include gram negative organisms such as E. coli or pseudomonas and gram 
positive bacteria such as micrococcus or bacillus. Examples of eucaryotic 
organisms from which carbonyl hydrolase and their genes may be obtained 
include yeast such as S. cerevisiae, fungi such as Aspergillus sp., and 
non-human mammalian sources such as, for example, Bovine sp. from which 
the gene encoding the carbonyl hydrolase rennin can be obtained. A series 
of carbonyl hydrolases can be obtained from various related species which 
have amino acid sequences which are not entirely homologous between the 
member of that series but which nevertheless exhibit the same or similar 
type of biological activity. Thus, carbonyl hydrolase as used herein has a 
functional definition which refers to serine proteases which are 
associated, directly or indirectly, with procaryotic and non-human 
eucaryotic sources. 
The "non-human carbonyl hydrolases" of the present invention include the 
mature forms of carbonyl hydrolases as well as subtilisins which are 
derived from non-human sources. Such hydrolases, in addition, either lack 
endoprotease activity or are incapable of autoproteolytic maturation. 
Thus, the non-human carbonyl hydrolases are functionally defined as any 
heterologous non-human carbonyl hydrolase which either is incorrectly 
processed by a host cell expressing the same and thus not released from 
the surface of such host cell or which is produced by such host cells but 
not processed to the mature form of the carbonyl hydrolase. 
"Polypeptides" are polymers of .alpha.-amino acids which are covalently 
linked through peptide bonds. Polypeptides include low molecular weight 
polymers as well as high molecular weight polymers more commonly referred 
to as proteins. In addition, a polypeptide can be a phosphopolypeptide, 
glycopolypeptide or metallopolypeptide. Further, one or more polymer 
chains may be combined to form a polypeptide. 
"Heterologous" refers to non-human carbonyl hydrolases or other 
polypeptides which are not ordinarily produced by the host cell. Such 
heterologous polypeptides thus may comprise polypeptides which either do 
not have substantial amino acid sequence homology with those proteins 
produced by the host cell (e.g., protein from unrelated procaryotes or 
protein from eucaryotes such as yeast, fungi and other higher eucaryotes 
expressed in procaryotes) or may comprise polypeptides with substantial 
but incomplete homology to proteins produced by the host cell or the cell 
line from which the host cell is derived. For example, a mutant B. 
subtilis substilisin containing a single amino acid substitution as 
compared to wild-type B. subtilis subtilisin is heterologous whether 
expressed in a B. subtilis expressing wild-type subtilisin or a mutant B. 
subtilis strain which does not produce enzymatically active wild-type 
subtilisin. 
"Prosequence" refers to a sequence of amino acids bound to the N-terminal 
portion of the mature form of a carbonyl hydrolase or other polypeptide 
which when removed results in the appearance of the "mature" form of the 
carbonyl hydrolase. Many proteolytic enzymes are found in nature as 
translational proenzyme products and, in the absence of post-translational 
processing, are expressed in this fashion. The preferred prosequence is 
the putative prosequence of B. amyloliquifaciens subtilisin although other 
subtilisin prosequences may be used. The prosequence of B. 
amyloliquifaciens subtilis, was discovered by the inventors to be 
autolytically cleaved by naturally occuring subtilisin. This discovery was 
based on the observation that mutant subtilisins which are enzymatically 
inactive become associated with the surface of the expression host rather 
than being completely secreted into the expression host medium. 
A "signal sequence" or "presequence" refers to any sequence of amino acids 
bound to the N-terminal portion of a carbonyl hydrolase or other 
polypeptide or to the N-terminal portion of a prohydrolase or other 
propolypeptide which may participate in the secretion of the mature or pro 
forms of the hydrolase or other polypeptide. This definition of signal 
sequence is a functional one, meant to include all those amino acid 
sequences, encoded by the N-terminal portion of the subtilisin gene or 
other secretable carbonyl hydrolases, which participate in the 
effectuation of the secretion of subtilisin or other carbonyl hydrolases 
under native conditions. The present invention comprises the harnessing of 
said sequences to effect resultant partial or complete secretion of any 
heterologous carbonyl hydrolase or other polypeptide as defined herein. 
A "prepro" form of a heterologous carbonyl hydrolase consists of the mature 
form of the hydrolase having a prosequence operably linked to the 
amino-terminal first part of the hydrolase and a "pre" or "signal" 
sequence operably linked to the amino terminal part of the prosequence. 
The prepro forms of the heterologous non-human carbonyl hydrolases in one 
aspect of the invention, are not completely processed to the mature form 
of hydrolase and thus are not secreted from the host cell expressing the 
same. This is believed to result from the absence of or decrease in 
enzymatic activity of the mature form of the carbonyl hydrolase expressed. 
Such enzymatically inactive hydrolases typically are mutants containing 
the substitution, deletion or insertion of one or more amino acids of the 
wild type carbonyl hydrolase. This effect is observed for certain mutant 
subtilisins which are expressed in a prepro form in host cells which do 
not secrete enzymatically active endoprotease. As a result, the carbonyl 
hydrolase, presumably in its prepro form, is transported across the host 
cell membrane to become bound to the cell membrane or otherwise associated 
with the surface of the host cell (e.g., by association with the 
carbohydrate or glycoprotein of the cell membrane). Carbonyl hydrolases 
which are associated with the surface of an expression host, however, are 
not limited to mutant subtilisins but rather include all non-human 
carbonyl hydrolases which are associated with the surface of an expression 
host and which can be released therefrom by cleavage with subtilisin. 
A "fusion" polypeptide comprises at least two parts: an amino terminal 
first sequence and a carboxyl terminal second sequence comprising the 
amino acid sequence of a heterologous non-human carbonyl hydrolase or 
other heterologous polypeptide as defined herein. Examples of heterologous 
polypeptides include human growth hormone (hGH), tissue plasminogen 
activator (t-PA), and the subtilisins defined herein. The fusion 
polypeptide is capable of being cleaved by subtilisin or mutant subtilisin 
at a recognition site at the junction of the first and second sequences of 
the fusion polypeptide or at some point within the first sequence. The 
particular amino acid sequence comprising the first sequence of the fusion 
polypeptide is preferably the prosequence of B. amyloliquifaciens 
subtilisin although other subtilisin prosequences may be used. However, 
various other sequences which may be uniquely recognized and cleaved by 
mutant subtilisins are also included within the scope of this 
amino-terminal first sequence. The amino acid sequence used in the first 
sequence of the fusion polypeptide may thus be defined functionally as an 
amino acid sequence which when combined with a heterologous polypeptide to 
form a fusion polypeptide results in a fusion polypeptide which is capable 
of being cleaved by a subtilisin at the recognition site. Such cleavage 
produces either the mature heterologous polypeptide or non-human carbonyl 
hydrolase or the mature form of the hydrolase or polypeptide with one or 
more amino acids from the first sequence sequence attached to the amino 
terminus. 
"Expression vector" refers to a DNA construct containing a DNA sequence 
which is operably linked to a suitable control sequence capable of 
effecting the expression of said DNA in a suitable host. Such control 
sequences include a promoter to effect transcription, an optional operator 
sequence to control such transcription, a sequence encoding suitable mRNA 
ribosome binding sites, and sequences which control termination of 
transcription and translation. The vector may be a plasmid, a phage 
particle, or simply a potential genomic insert. Once transformed into a 
suitable host, the vector may replicate and function independently of the 
host genome, or may, in some instances, integrate into the genome itself. 
In the present specification, "plasmid" and "vector" are sometimes used 
interchangeably as the plasmid is the most commonly used form of vector at 
present. However, the invention is intended to include such other forms of 
expression vectors which serve equivalent functions and which are, or 
become, known in the art. 
The "host cells" used in one aspect of the present invention generally are 
procaryotic or eucaryotic hosts which preferably have been manipulated by 
the methods disclosed in EPO Publication No. 0130756 to render them 
incapable of secreting enzymatically active endoprotease. Host cells are 
transformed or transfected with vectors constructed using recombinant DNA 
techniques. Such vectors encode the heterologous non-human carbonyl 
hydrolases or heterologous polypeptides of the invention and are capable 
of replication and expression of such hydrolases or polypeptides. A 
preferred host cell for expressing such hydrolases is the Bacillus strain 
BG2036 which is deficient in enzymatically active neutral protease and 
alkaline protease (subtilisin). The construction of strain BG2036 is 
described in detail in EPO Publication No. 0130756 and further described 
by Yang, M. Y., et al. (1984) J. Bacteriol. 160, 15-21. Such host cells 
are distinguishible from those disclosed in PCT Publication No. 03949 
wherein enzymatically inactive mutants of intracellular proteases in E. 
coli are disclosed. In the case of host cells which produce an 
enzymatically active endoprotease to release the heterologous non-human 
carbonyl hydrolase from the surface of the host cell, B. subtilisin I-168 
and BG2044 each of which produce active alkaline protease are preferred. 
Stahl, M. L. and Ferrari, E. (1984) J. Bacteriol. 158, 411-418; Yang, M. 
Y., et al. (1984) J. Bacteriol. 160, 15-21. However, other host cells 
known to those skilled in the art may also be used in practicing the 
process of the present invention. 
"Operably linked" when describing the relationship between two DNA regions 
simply means that they are functionally related to each other. For 
example, a presequence is operably linked to a peptide if it functions as 
a signal sequence, participating in the secretion of the mature form of 
the protein most probably involving cleavage of the signal sequence. A 
promoter is operably linked to a coding sequence if it controls the 
transcription of the sequence; a ribosome binding site is operably linked 
to a coding sequence if it is positioned so as to permit translation. 
The genes encoding the carbonyl hydrolase may be obtained in accord with 
the general methods described in EPO Publication No. 0130756 published 
Jan. 9, 1985. As can be seen from the examples disclosed therein, the 
methods generally comprise synthesizing labelled probes having putative 
sequences encoding regions of the hydrolase of interest, preparing genomic 
libraries from organisms expressing the hydrolase, and screening the 
libraries for the gene of interest by hybridization to the probes. 
Positively hybridizing clones are then mapped and sequenced. 
The cloned carbonyl hydrolase is then used to transform a host cell in 
order to express the hydrolase. The hydrolase gene is then ligated into a 
high copy number plasmid. This plasmid replicates in hosts in the sense 
that it contains the well-known elements necessary for plasmid 
replication: a promoter operably linked to the gene in question (which may 
be supplied as the gene's own homologous promotor if it is recognized, 
i.e., transcribed, by the host), a transcription termination and 
polyadenylation region (necessary for stability of the mRNA transcribed by 
the host from the hydrolase gene in certain eucaryotic host cells) which 
is exogenous or is supplied by the endogenous terminator region of the 
hydrolase gene and, desirably, a selection gene such as an antibiotic 
resistance gene that enables continuous cultural maintenance of 
plasmid-infected host cells by growth in antibiotic-containing media. High 
copy number plasmids also contain an origin of replication for the host, 
thereby enabling large numbers of plasmids to be generated in the 
cytoplasm without chromosonal limitations. However, it is within the scope 
herein to integrate multiple copies of the hydrolase gene into host 
genome. This is facilitated by procaryotic and eucaryotic organisms which 
are particularly susceptible to homologous recombination. The resulting 
host cells are termed recombinant host cells. 
Once the carbonyl hydrolase gene has been cloned, a number of modifications 
are undertaken to enhance the use of the gene beyond synthesis of the wild 
type or precursor enzyme. A precursor enzyme is the enzyme prior to its 
modification as described in this application and in EPO Publication No. 
0130756. Usually the precursor is the enzyme as expressed by the organism 
which donated the DNA modified in accord herewith. The term "precursor" is 
to be understood as not implying that the product enzyme was the result of 
manipulation of the precursor enzyme per se but rather of the DNA encoding 
the precursor enzyme. 
In the first of these modifications, the gene may be deleted from a 
recombination positive (rec.sup.+) organism containing a homologous gene. 
This is accomplished by recombination of an in vitro deletion mutation of 
the cloned gene with the genome of the organism. Many strains of organisms 
such as E. coli and Bacillus are known to be capable of recombination. All 
that is needed is for regions of the residual DNA from the deletion mutant 
to recombine with homologous regions of the candidate host. The deletion 
may be within the coding region (leaving enzymatically inactive 
polypeptides) or include the entire coding region as long as homologous 
flanking regions (such as promoters or termination regions) exist in the 
host. Acceptability of the host for recombination deletion mutants is 
simply determined by screening for the deletion of the transformed 
phenotype. This is most readily accomplished in the case of carbonyl 
hydrolase by assaying host cultures for loss of the ability to cleave a 
chromogenic substrate otherwise hydrolyzed by the hydrolase. 
Transformed hosts containing the protease deletion mutants are useful for 
synthesis of products which are incompatible with proteolytic enzymes. 
These hosts by definition are incapable of secreting enzymatically active 
extracellular endoprotease encoded by the deleted protease genes described 
herein. In the case of Bacillus sp. these host cells also are 
substantially normally sporulating. In addition, the other growth 
characteristics of the transformants are substantially like the parental 
organism. Such organisms are useful in that it is expected they will 
exhibit comparatively less inactivation of heterologous proteins than the 
parents. Moreover, these hosts have growth characteristics which are 
superior to known protease-deficient organisms. However, the deletion of 
neutral protease and subtilisin as described in this application does not 
remove all of the proteolytic activity of Bacillus. It is believed that 
intracellular proteases which are not ordinarily excreted extracellularly 
"leak" or diffuse from the cells during late phases of the culture. These 
intracellular proteases may or may not be neutral protease or subtilisin 
as defined herein. Accordingly, the novel Bacillus strains herein are 
incapable of excreting the subtilisin and/or neutral protease enzymes 
which ordinarily are excreted extracellularly in the parent strains. 
"Incapable" means not revertible to the wild type. Reversion is a finite 
probability that exists with the heretofore known protease-deficient, 
naturally occurring strains since there is no assurance that the phenotype 
of such strains is not a function of a readily revertible mutation, e.g. a 
point mutation. This to be contrasted with the extremely large deletions 
provided herein. 
The deletion mutant-transformed host cells herein are free of genes 
encoding enzymatically active neutral protease or subtilisin, which genes 
are defined as those being substantially homologous with the genes set 
forth in FIGS. 1, 2 or 3 as well as the subtilisin sequences disclosed by 
Svendsen, et al. (1983) Carlberg Res Commun 48, 583-591, and Melourn, et 
al. FEBS, 183,195-200. "Homologous" genes contain coding regions capable 
of hybridizing under high stringency conditions with these sequences. 
The strains containing carbonyl hydrolase deletion mutants are useful in at 
least two principal processes. First, they are advantageous in the 
fermentative production of products ordinarily expressed by a host that 
are desirably uncontaminated with the protein encoded by the deletion 
gene. An example is fermentative synthesis of amylase, where contaminant 
proteases interfere in many industrial uses of amylase. These novel 
strains relieve the art from part of the burden of purifying such products 
free of contaminating carbonyl hydrolases. 
In a second process, protease deletion-mutant strains are useful in the 
synthesis of protein which is not otherwise encoded by the strain. These 
proteins will fall within one of two classes. The first class consists of 
proteins encoded by genes exhibiting no substantial pretransformation 
homology with those of the host cell or the organism from which the host 
cell was derived. These may be proteins from procaryotes as well as 
eucaryotic proteins from yeast or higher eucaryotic organisms. The novel 
strains herein serve as useful hosts for expressible vectors containing 
genes encoding such proteins because the probability for proteolytic 
degradation of the expressed, heterologous proteins is reduced. 
The second class, which is particularly relevant to the invention, consists 
of mutant host genes exhibiting substantial pretransformation homology 
with those of the host cell or organism from which the host cell is 
derived. These include mutations of non-human carbonyl hydrolases such as 
subtilisin and neutral protease. When so mutated, this group of 
polypeptides are heterologous to the host cell as defined herein. 
The following method was used to facilitate the construction and 
identification of such mutants as well as the host cells used in the 
present invention. First, the gene encoding the hydrolase is obtained and 
sequenced in whole or in part. Then the sequence is scanned for a point at 
which it is desired to make a mutation (deletion, insertion or 
substitution) of one or more amino acids in the expressed enzyme. The 
sequences flanking this point are evaluated for the presence of 
restriction sites for replacing a short segment of the gene with an 
oligonucleotide pool which when expressed will encode various mutants. 
Since unique restriction sites are generally not present at locations 
within a convenient distance from the selected point (from 10 to 15 
nucleotides), such sites are generated by substituting nucleotides in the 
gene in such a fashion that neither the reading frame nor the amino acids 
encoded are changed in the final construction. The task of locating 
suitable flanking regions and evaluating the needed changes to arrive at 
two unique restriction site sequences is made routine by the redundancy of 
the genetic code, a restriction enzyme map of the gene and the large 
number of different restriction enzymes. Note that if a fortuitous 
flanking unique restriction site is available, the above method need be 
used only in connection with the flanking region which does not contain a 
site. 
Mutation of the gene in order to change its sequence to conform to the 
desired sequence is accomplished by M13 primer extension in accord with 
generally known methods. Once the gene is cloned, it is digested with the 
unique restriction enzymes and a plurality of end termini-complementary 
oligonucleotide cassettes are ligated into the unique sites. The 
mutagenesis is enormously simplified by this method because all of the 
oligonucleotides can be synthesized so as to have the same restriction 
sites, and no synthetic linkers are necessary to create the restriction 
sites. The number of commercially available restriction enzymes having 
sites not present in the gene of interest is generally large. A suitable 
DNA sequence computer search program simplifies the task of finding 
potential 5' and 3' unique flanking sites. A primary constraint is that 
any mutation introduced in creation of the restriction site must be silent 
to the final construction amino acid coding sequence. For a candidate 
restriction site 5' to the target codon a sequence must exist in the gene 
which contains at least all the nucleotides but for one in the recognition 
sequence 5' to the cut of the candidate enzyme. For example, the blunt 
cutting enzyme SmaI (CCC/GGG) would be a 5' candidate if a nearby 5' 
sequence contained NCC, CNC, or CCN. Furthermore, if N needed to be 
altered to C this alteration must leave the amino acid coding sequence 
intact. In cases where a permanent silent mutation is necessary to 
introduce a restriction site one may want to avoid the introduction of a 
rarely used codon. A similar situation of SmaI would apply for 3' 
flanking sites except the sequence NGG, GNG, or GGN must exist. The 
criteria for locating candidate enzymes is most relaxed for blunt cutting 
enzymes and most stringent for 4 base overhang enzymes. In general many 
candidate sites are available. For the codon-221 target described herein a 
Bali site (TGG/CCA) would have been engineered in one base pair 5' from 
the KpnI site. A 3' EcoRV site (GAT/ATC) could have been employed 11 base 
pairs 5' to the PstI site. A cassette having termini ranging from a blunt 
end up to a four base-overhang will function without difficulty. In 
retrospect, this hypothetical EcoRV site would have significantly 
shortened the oligonucleotide cassette employed (9 and 13 base pairs) thus 
allowing greater purity and lower pool bias problems. Flanking sites 
should obviously be chosen which cannot themselves ligate so that ligation 
of the oligonucleotide cassette can be assured in a single orientation. 
It should be noted that the amino acid position numbers referred to herein 
are those assigned to B. amyloliquefaciens subtilisin as seen from FIG. 2. 
It should be understood that a deletion or insertion in the N-terminal 
direction from a given position will shift the relative amino acid 
positions so that a residue will not occupy its original or wild type 
numerical position. Also, allelic differences and the variation among 
various procaryotic and eucaryotic species will result in position shifts. 
For example, position 169 from B. amyloliquefaciens subtilisin may not be 
occupied by glycine in all other bacterial subtilisin. In such cases the 
new positions for glycine will be considered equivalent to and embraced 
within the designation glycine+169. The new position for glycine+169 is 
readily identified by scanning the subtilisin in question for a region 
homologous to glycine+169 in FIG. 2. 
The enzymes herein may be obtained as salts. It is clear that the 
ionization state of a protein will be dependent on the pH of the 
surrounding medium, if it is in solution, or of the solution from which it 
is prepared, if it is in solid form. Acidic proteins are commonly prepared 
as, for example, the ammonium, sodium, or potassium salts; basic proteins 
as the chlorides, sulfates, or phosphates. Accordingly, the present 
application includes both electrically neutral and salt forms of the 
designated carbonyl hydrolases, and the term carbonyl hydrolase referes to 
the organic structural backbone regardless of ionization state. 
The following disclosure is intended to serve as a representation of 
embodiments herein, and should not be construed as limiting the scope of 
this application. 
Glossary of Experimental Manipulations 
In order to simplify the Examples certain frequently occurring methods will 
be referenced by shorthand phrases. 
Plasmids are designated by a small p proceeded and/or followed by capital 
letters and/or numbers. The starting plasmids herein are commercially 
available, are available on an unrestricted basis, or can be constructed 
from such available plasmids in accord with published procedures. 
"Klenow treatment" refers to the process of filling a recessed 3' end of 
double stranded DNA with deoxyribonucleotides complementary to the 
nucleotides making up the protruding 5' end of the DNA strand. This 
process is usually used to fill in a recessed end resulting from a 
restriction enzyme cleavage of DNA. This creates a blunt or flush end, as 
may be required for further ligations. Treatment with Klenow is 
accomplished by reacting (generally for 15 minutes at 15.degree. C.) the 
appropriate complementary deoxyribonucleotides with the DNA to be filled 
in under the catalytic activity (usually 10 units) of the Klenow fragment 
of E. coli DNA polymerase I ("Klenow"). Klenow and the other reagents 
needed are commercially available. The procedure has been published 
extensively. See for example T. Maniatis, et al., (1982) Molecular 
Cloning, pp. 107-108. 
"Digestion" of DNA refers to catalytic cleavage of the DNA with an enzyme 
that acts only at certain locations in the DNA. Such enzymes are called 
restriction enzymes, and the sites for which each is specific is called a 
restriction site. "Partial" digestion refers to incomplete digestion by 
restriction enzyme, i.e., conditions are chosen that result in cleavage of 
some but not all of the sites for a given restriction endonuclease in a 
DNA substrate. The various restriction enzymes used herein are 
commercially available and their reaction conditions, cofactors and other 
requirements as established by the enzyme suppliers were used. Restriction 
enzymes commonly are designated by abbreviations composed of a capital 
letter followed by other letters and then, generally, a number 
representing the microorganism from which each restriction enzyme 
originally was obtained. In general, about 1 .mu.g of plasmid or DNA 
gragment is used with about 1 unit of enzyme in about 20 .mu.l of buffer 
solution. Appropriate buffers and substrate amounts for particular 
restriction enzymes are specified by the manufacturer. Incubation times of 
about 1 hour at 37.degree. C. are ordinarily used, but may vary in 
accordance with the supplier's instructions. After incubation, protein is 
removed by extraction with phenol and chloroform, and the digested nucleic 
acid is recovered from the aqueous fraction by precipitation with ethanol. 
Digestion with a restriction enzyme infrequently is followed with 
bacterial alkaline phosphatase hydrolysis of the terminal 5' phosphates to 
prevent the two restriction cleaved ends of a DNA fragment from 
"circularizing" or forming a closed loop that would impede insertion of 
another DNA fragment at the restriction site. Unless otherwise stated, 
digestion of plasmids is not followed by 5' terminal dephosphorylation. 
Procedures and reagents for dephosphorylation are conventional (T. 
Maniatis, et al., Id., pp. 133-134). 
"Recovery" or "isolation" of a given fragment of DNA from a restriction 
digest means separation of the digest on 6 percent polyacrylamide gel 
electrophoresis, identification of the fragment of interest by molecular 
weight (using DNA fragments of known molecular weight as markers), removal 
of the gel section containing the desired fragment, and separation of the 
gel from DNA. This procedure is known generally. For example, see R. Lawn, 
et al., (1981) Nucleic Acids Res. 9, 6103-6114, and D. Goeddel, et al., 
(1980) Nucleic Acids Res. 8, 4057. 
"Southern Analysis" is a method by which the presence of DNA sequences in a 
digest or DNA-containing composition is confirmed by hybridization to a 
known, labelled oligonucleotide or DNA fragment. For the purposes herein, 
Southern analysis shall mean separation of digests on 1 percent agarose 
and depurination as described by G. Wahl, et al., (1979) Proc. Nat. Acad. 
Sci. U.S.A. 76, 3683-3687, transfer to nitrocellulose by the method of 
Southern, E. (1975) J. Mol. Biol. 98, 503-517, and hybridization as 
described by T. Maniatis, et al., (1978) Cell 15, 687-701. 
"Transformation" means introducing DNA into an organism so that the DNA is 
replicable, either as an extrachromosomal element or chromosomal 
integrant. Unless otherwise stated, the method used herein for 
transformation of E. coli is the CaCl.sub.2 method of Mandel, et al., 
(1970) J. Mol. Biol. 53, 154, and for Bacillus, the method of 
Anagnostopolous, et al., (1961) J. Bacteriol. 81, 791-746. 
"Ligation" refers to the process of forming phosphodiester bonds between 
two double stranded nucleic acid fragments (T. Maniatis, et al., Id., p. 
146). Unless otherwise stated, ligation was accomplished using known 
buffers and conditions with 10 units of T4 DNA ligase ("ligase") per 0.5 
.mu.g of approximately equimolar amounts of the DNA fragments to be 
ligated. Plasmids from the transformants were prepared, analyzed by 
restriction mapping and/or sequenced by the method of Messing, et al., 
(1981) Nucleic Acids Res. 9, 309. 
"Preparation" of DNA from transformants means isolating plasmid DNA from 
microbial culture. Unless otherwise stated, the alkaline/SDS method of 
Maniatis, et al., Id., p. 90, was used. 
"Oligonucleotides" are short length single or double stranded 
polydeoxynucleotides which were chemically synthesized by the method of 
Crea, et al., (1980) Nucleic Acids Res. 8, 2331-2348 (except that 
mesitylene nitrotriazole was used as a condensing agent) and then purified 
on polyacrylamide gels. 
All literature citations are expressly incorporated by reference. 
EXAMPLE 1 
Site-specific mutagenesis of the B. amyloliquefaciens subtilisin gene at 
position 221; preparation of the gene for cassette insertion 
The construction of pS4 is described in detail in EPO Publication No. 
0130756. This plasmid is depicted in FIG. 4. pS4 contains 4.5 kb of 
sequence derived from pBS42 (solid line) and 4.4kb of sequence containing 
the B. amyloliquefaciens subtilisin gene and flanking sequences (dashed 
line). pBS42 was constructed as described in EPO Publication No. 0130756 
and Band, L. and Henner, D. J. (1984) DNA 3, 17-21. It was digested with 
BamHI and ligated with Sau3A partially digested chromosomal DNA from B. 
amyloliquefaciens (ATCC No. 23844) as described in EPO Publication No. 
0120756. pS4 was selected from this genomic library. 
pS4-5, a derivative of pS4 made according to Wells, et al. (1983) Nucleic 
Acids Res. 11, 7911-7924, was digested with EcoRI and BamHI, and the 1.5 
kb EcoRI-BamHI fragment recovered. This fragment was ligated into 
replicative form M-13 mp9 which had been digested with EcoRI and BamHI 
(Sanger, et al., (1980) J. Mol. Biol. 143, 161-178; Messing, et al., 
(1981) Nucleic Acids Res. 9, 304-321; Messing, J. and Vieira, J. (1982) 
Gene 19, 269-276). The M-13 mp9 phage ligations, designated M-13 mp9 SUBT, 
were used to transform E. coli strain JM101 (ATCC 33876) and single 
stranded phage DNA was prepared from a two mL overnight culture. An 
oligonucleotide primer was synthesized having the sequence 
5'-GTACAACGGTACCTCACGCACGCTGCAGGAGCGGCTGC-3'. 
This primer conforms to the sequence of the subtilisin gene fragment 
encoding amino acids 216-232 except that the 10 bp of codons for amino 
acids 222-225 were deleted, and the codons for amino acids 220, 227 and 
228 were mutated to introduce a KpnI site 5' to the Ser-221 codon and a 
PstI site 3' to the Ser-221 codon. See FIG. 5. Substituted nucleotides are 
denoted by asterisks, the underlined codons in line 2 represent the new 
restriction sites and the scored sequence in line 4 represents the 
inserted oligonucleotide. The primer (about 15 .mu.M) was labelled with 
[.sup.32 p] by incubation with [.gamma..sup.32 p]-ATP (10 .mu.L in 20 
.mu.L reaction)(Amersham 5000 Ci/mmol, 10218) and T.sub.4 polynucleotide 
kinase (10 units) followed by non-radioactive ATP (100 .mu.M) to allow 
complete phosphorylation of the mutagenesis primer. The kinase was 
inactivated by heating the phosphorylation mixture to 68.degree. C. for 15 
minutes. 
The primer was hybridized to M-13 mp9 SUBT as modified from Norris, et al., 
(1983) Nucleic Acids Res. 11, 5103-5112 by combining 5 .mu.L of the 
labelled mutagenesis primer (.about.3 .mu.M), .about.1 .mu.g M-13 mp9 SUBT 
template, 1 .mu.L of 1 .mu.M M-13 sequencing primer (17-mer), and 2.5 
.mu.L of buffer (0.3 M Tris pH 8, 40 mM MgCl.sub.2, 12 mM EDTA, 10 mM DTT, 
0.5 mg/ml BSA). The mixture was heated to 68.degree. C. for 10 minutes and 
cooled 10 minutes at room temperature. To the annealing mixture was added 
3.6 .mu.L of 0.25 mM dGTP, dCTP, dATP, and dTTP, 1.25 .mu.of 10 mM ATP, 1 
.mu.L ligase (4 units) and 1 .mu.L Klenow (5 units). The primer extension 
and ligation reaction (total volume 25 .mu.l ) proceeded 2 hours at 
14.degree. C. The Klenow and ligase were inactivated by heating to 
68.degree. C. for 20 minutes. The heated reaction mixture was digested 
with BamH1 and EcoRI and an aliquot of the digest was applied to a 6 
percent polyacrylamide gel and radioactive fragments were visualized by 
autoradiography. This showed the [.sup.32 p] mutagenesis primer had indeed 
been incorporated into the EcoRI-BamH1 fragment containing the now mutated 
subtilisin gene. 
The remainder of the digested reaction mixture was diluted to 200 .mu.L 
with 10 mM Tris, pH 8, containing 1 mM EDTA, extracted once with a 1:1 
(v:v) phenol/chloroform mixture, then once with chloroform, and the 
aqueous phase recovered. 15 .mu.L of 5 M ammonium acetate (pH 8) was added 
along with two volumes of ethanol to precipitate the DNA from the aqueous 
phase. The DNA was pelleted by centrifugation for five minutes in a 
microfuge and the supernatant was discarded. 300 .mu.L of 70 percent 
ethanol was added to wash the DNA pellet, the wash was discarded and the 
pellet lyophilized. 
pBS42 was digested with BamHl and EcoRI and purified on an acrylamide gel 
to recover the vector. 0.5 .mu.g of the digested vector, 0.1 .mu.g of the 
above primer mutated EcoRI-BamHI digested subtilisin genonic fragment, 50 
.mu.M ATP and 6 units ligase were dissolved in 20 .mu.l of ligation 
buffer. The ligation went overnight at 14.degree. C. The DNA was 
transformed into E. coli 294 rec.sup.+ (ATCC 31446) and the transformants 
grown in 4 ml of LB medium containing 12.5 .mu.g/ml chloramphenicol. 
Plasmid DNA was prepared from this culture and digested with KpnI, EcoRI 
and BamHI. Analysis of the restriction fragments showed 30-50 percent of 
the molecules contained the expected KpnI site programmed by the 
mutagenesis primer. It was hypothesized that the plasmid population not 
including the KpnI site resulted from M-13 replication before bacterial 
repair of the mutagenesis site, thus producing a heterogenous population 
of KpnI.sup.+ abd KpnI.sup.- plasmids in some of the transformants. In 
order to obtain a pure culture of the KpnI.sup.+ plasmid, the DNA was 
transformed a second time into E. coli to clone plasmids containing the 
new KpnI site. DNA was prepared from 16 such transformants and six were 
found to contain the expected KpnI site. 
Preparative amounts of DNA were made from one of these six transformants 
(designated p.DELTA.221) and restriction analysis confirmed the presence 
and location of the expected KpnI and PstI sites. 40 .mu.g of p.DELTA.221 
were digested in 300 .mu.L of KpnI buffer plus 30 .mu.L KpnI (300 units) 
for 1.5 h at 37.degree. C. The DNA was precipitated with ethanol, washed 
with 70 percent ethanol, and lyophilized. The DNA pellet was taken up in 
200 .mu.L HindIII buffer and digested with 20 .mu.L (500 units) PstI for 
1.5 h at 37.degree. C. The aqueous phase was extracted with 
phenol/CHCl.sub.3 and the DNA precipitated with ethanol. The DNA was 
dissolved in water and purified by polyacrylamide gel electrophoresis. 
Following electroelution of the vector band (120 v for 2 h at 0.degree. C. 
in 0.1.times. TBE (Maniatis, et al., Id.)) the DNA was purified by 
phenol/CHCl.sub.3 extraction, ethanol precipitation and ethanol washing. 
Although p.DELTA.221 could be digested to completion (&gt;98 percent) by 
either KnpI or PstI separately, exhaustive double digestion was incomplete 
(&lt;&lt;50 percent). This may have resulted from the fact that these sites were 
so close (10 bp) that digestion by KnpI allowed "breathing" of the DNA in 
the vicinity of the PstI site, i.e., strand separation or fraying. Since 
PstI will only cleave double stranded DNA, strand separation could inhibit 
subsequent PstI digestion. 
EXAMPLE 2 
Ligation of oligonucleotide casette Ser221.fwdarw.Ala into the subtilisin 
gene 
10 .mu.M of the complementary oligonucleotides (under and overscored in 
FIG. 5), which were not 5' phosphorylated were annealed in 20 .mu.l ligase 
buffer by heating for five minutes at 68.degree. C. and then cooling for 
15 minutes at room temperature. 1 .mu.M of the annealed oligonucleotide 
encoding the substitution of alanine for serine 221, .about.0.2 .mu.g KpnI 
and PstI digested p.DELTA.221 obtained in Example 1, 0.5 mM ATP, ligase 
buffer and 6 units T.sub.4 DNA ligase in 20 .mu.L total volume was reacted 
overnight at 14.degree. C. to ligate the Ser(221).fwdarw.Ala cassette in 
the vector. A large excess of cassette (.about.300.times. over the 
p.DELTA.221 ends) was used in the ligation to help prevent intramolecular 
KpnI-KpnI ligation. The reaction was diluted by adding 25 .mu.L of 10 mM 
Tris pH 8 containing 1 mM EDTA. The mixture was reannealed to avoid 
possible cassette concatemer formation by heating to 69.degree. C. fo five 
minutes and cooling for 15 minutes at room temperature. The ligation 
mixture was transformed into E. coli 294 rec.sup.+ cells. A small aliquot 
from the transformation mixture was plated to determine the number of 
independent transformants. The large number of transformants indicated a 
high probability of multiple mutagenesis. The rest of the transformants 
(.about.200-400 transformants) were cultured in 4 ml of LB medium plus 
12.5 .mu.g chloramphenicol/ml. DNA was prepared from each transformant. 
This DNA was digested with KpnI. Approximately 0.1 .mu.g was used to 
retransform E. coli rec.sup.+ and the mixture was plated to isolate 
individual colonies. Since ligation of the Ser221.fwdarw.Ala cassette into 
the gene and bacterial repair upon transformation destroyed the KpnI and 
PstI sites, only p.DELTA.221 was cut when the transformant DNA was 
digested with KpnI. This linearized plasmid thus would have a much lower 
transformation efficiency than the circular plasmid containing the 
Ser221.fwdarw.Ala cassette. Individual transformants were grown in culture 
and DNA was prepared for direct plasmid sequencing. A synthetic 
oligonucleotide primer having the sequence 5'-GAGCTTGATGTCATGGC-3' was 
used to prime the dideoxy sequencing reaction. The sequence obtained 
corresponded to that expected for the substitution of alanine for serine 
at residue 221. This plasmid, pAla+221, encoded the mutant subtilisin 
designated A221 or Ser221.fwdarw.Ala. 
EXAMPLE 3 
Site-Directed Mutagenesis of B. amyloliquefaciens subtilisin gene at 
position 32 
Three hydrogen bonds have been proposed that may help stabilize the 
transition state of the subtilisinsubstrate complex. (For example, see 
Kossiakoff, A. A. (1985) in Biological Macromolecules and Assemblies, Vol. 
III, in press.) One of these hydrogen bonds is between the aspartic acid 
at residue 32 and the positivity charged histidine at position 64. 
Kossiakoff, A. A. and Spencer, S. A. (1981) Biochem. 20, 6462-6474. The 
second involves a hydrogen bond between the scissile amide nitrogen of the 
substrate and the proton of histidine 64. A third set of hydrogen bonds 
forms between the enzyme and the oxyanion that is produced from the 
carbonyl oxygen of the substrate. Crystallographic studies of subtilisin 
show that two hydrogen bonds are formed with the substrate oxyanion: one 
hydrogen bond being from the catalytic serine at residue 221 while the 
other is from asparagine at residue 155. Robertus, J. D., Kraut, J., 
Alden, R. A. and Birktoft, J. J. (1972) Biochem. 11, 4293-4303; Matthews, 
D. A., Alden, R. A., Birktoft, J. J., Freer, S. T. and Kraut, J. (1975) J. 
Biol. Chem. 250, 7120-7126; and Poulos, T. L., Alden, R. A., Freer, S. T., 
Birktoft, J. J. and Kraut, J. (1976) J. Biol. Chem. 250, 1097-1103. In 
this example site-directed mutagenesis of Asp32.fwdarw.Asn was undertaken 
to determine the effect of such substitution on catalytic activity. 
pS4-5 was digested with EcoRI and BamHI, and the 1.5 kb EcoRI-BamHI 
fragment recovered. This fragment was ligated into replicative form M-13 
mpll which had been digested with EcoRI and BamHI. This vector was 
designated M13mpll SUBT and single-stranded phage DNA was prepared from 
it. Sanger, et al., (1980) J. Mol. Biol. 143, 161-178; Messing, et al., 
(1981) Nucleic Acids Res. 9, 304-321; Messing, J. and Vieira, J. (1982) 
Gene 19, 269-276. An oligonucleotide primer was synthesized having the 
sequence: 
5'-CGGTTATCAACAGCGGTAT-3'. 
This primer includes the sequence of the subtilisin gene fragment encoding 
amino acids 30-34 except that the codon for amino acid 32 was changed from 
GAC (aspartic acid) to AAC (asparagine). The single stranded M13mpll SUBT 
DNA was primed with the 5'-phosphorylated M13 sequencing primer and the 
mutagenesis primer, as previously described. Adelman, J. P., Hayflick, J. 
S., Vasser, M. and Seeburg, P. H. (1983) DNA 2, 183-193. Mutant phage were 
identified by hybridization with the .sup.32 P-mutagenic primer using a 
tetramethyl ammonium chloride washing procedure. Wood, W. I., Gitschier, 
J., Lasky, L. A. and Lawn, R. M. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 
1585-1588. All mutations were confirmed by M13 dideoxy sequencing. Sanger, 
F., Coulson, A. R., Barrell, B. G., Smith, A. J. H. and Roe, B. A. (1980) 
J. Mol. Biol. 143, 161-178. The mutagenized 1.5 kb EcoRI-BamHI fragment 
was sub-cloned into the E. coli-B. subtilis shuttle plasmid, pBS42. This 
plasmid was designated pAsn+32 and was used to transform E. coli 
294rec.sup.+. Mandel, M., et al. (1970) J. Mol. Biol. 53, 159-162. The 
mutant subtilisin encoded by this plasmid was designated N32 or 
Asp+32.fwdarw.Asn. To ensure that no second site mutation(s) had occurred, 
the region of DNA that was sequenced was replaced with wild type sequence 
containing Asp+32. This reconstruction restored the wild type protease 
phenotype. 
EXAMPLE 4 
Site-Directed mutagenesis of the B. amyloliquefaciens subtilisin gene by 
deletion of carboxyl terminal residues containing Ser221 
The procedure of Example 3 was followed in substantial detail. In this 
example, a primer having the sequence 
5'-AAGGCACTTCCGGGAGCTCAACCCGGGTAAATACCCT-3' 
directed a 7 bp deletion and a frame-shift starting at codon 163 giving 
rise to the plasmid p.DELTA.66. This frame shift causes premature chain 
termination 21 codons downstream. The mutant subtilisin encoded by this 
plasmid was designated .DELTA.166. 
Plasmid p.DELTA.66 was transformed into E. coli strain 294 rec.sup.+ as 
described by Mandel, M. and Higs, A. (1970) J. Mol. Biol. 53, 159-162. 
Plasmid DNA prepared from transformed E. coli 294 rec.sup.+ was used to 
transform various B. subtilis hosts as described hereinafter. 
EXAMPLE 5 
Site directed mutagenesis of B. amyloliquefaciens subtilisin gene by 
deletion of amino acid residues in the signal sequence 
The procedure of Example 3 again was followed except that a primer having 
the sequence 
5'-GAGAGGCAAAAAGCTTTTTGCTTTAGC-3' 
was used. This primer directed an in-frame deletion of codons -102 to -98 
in the subtilisin signal sequence. This mutant DNA sequence when subcloned 
into pBS42 was designated p.DELTA.(-102). The mutant subtilisin was 
designated .DELTA.(-102). 
The plasmids and polypeptides encoded by pS4-5 and the mutated plasmids of 
Examples 3, 4 and 5 are shown in FIG. 6. 
EXAMPLE 6 
Site-Directed mutagenesis of B. amyloliquefaciens subtilisin gene at 
position 48 
The procedure of Example 3 again was followed in substantial detail to 
produce a mutant subtilisin containing the substitution of arginine for 
the alanine at position 48. In this example, mutagenesis of 
Ala48.fwdarw.Arg was directed by a 26-mer oligonucleotide having the 
sequence: 
5'-GTAGCAGGCGGACGCTCCATGGTTCC-3' 
The primer included the sequence of the subtilisin gene fragment encoding 
amino acids 44 through 52 except that the codon normally encoding alanine 
was substituted with the codon CGC encoding arginine; the serine codon at 
49(AGC) was also converted to TCC to introduce a convenient NcoI site. The 
plasmid containing this mutation after subcloning into pBS42 was 
designated pAla48.fwdarw.Arg and was used to transform E. coli 294 
rec.sup.+. The mutant subtilisin encoded by this plasmid was designated 
R48 or Ala48.fwdarw.Arg and was used to transform various species of B. 
subtilis. 
EXAMPLE 7 
Cell fractions and Western Blot analysis 
Stationary phase cultures of various strains of B. subtilis transformed 
with pBS42, pAsn+32 or p.DELTA.166 in LB medium containing 1.25 .mu.g/ml 
chloramphenicol (1.25 ml) were treated with lmM PMSF to inactivate 
subtilisin activity. Samples were centrifuged at 11,000.times.g for 10 
minutes and a medium fraction of 0.6 ml was mixed with 0.6 ml 20 percent 
trichloroacetic acid. The suspension was incubated at 4.degree. C. for 30 
minutes and the precipitated protein was recovered by centrifugation for 
10 minutes at 19000 rpm in a Sorvall SS34 rotor (37,000.times.g). After 
washing with 0.6 ml acetone followed by centrifugation, the pellet was 
dried. The sample was disassociated in NaDodSO.sub.4 sample buffer (4 
percent glycerol, 2 percent NaDodSO.sub.4 and 10mM Na phosphate, pH 6.8) 
at 95.degree. C. for 3 minutes. A modified cell fractionation procedure 
was employed Kaback, H. R. (1971) Methods in Enzymology, ed. Jakoby, W. 
B., Acad. Press, N.Y., Vol. 22, pp. 99-120. The cell pellet was washed 
with 100 .mu.l of 10 mM Tris-HCl, pH 7.4, treated with PMSF as before and 
centrifuged. The cells were resuspended in 100 .mu.l of 30 mM Tris-HCl, pH 
8.0 and treated with 20 .mu.g/ml T4 lysozyme at 37.degree. C. for 20 
minutes. After centrifugation at 40,000.times.g for 20 minutes, the 
supernatant (i.e., cytosol fraction) was saved. The pellet (i.e., crude 
membrane fraction) was resuspended in 100 .mu.l of 50 mM Na phosphate, pH 
6.6 and treated with 5 mM EDTA. The crude membrane fraction was treated 
with 10 .mu.g/ml DNAse, 10 mM MgCl.sub.2, and incubated at 37.degree. C. 
for 10 minutes. The membranes were recovered by centrifugation at 
40,000.times.g for 20 minutes, washed once in 100 .mu.l of 50 mM sodium 
phosphate buffer and disassociated as described above. 
The samples were electrophoresed on 12.5 percent polyacrylamide gels (0.75 
mm.times.15 cm.times.15 cm) as described by Laemmli (Laemmli, U.K. (1970) 
Nature 227, 680-685; Laemmli, U. K. and Favre, M. (1973) J. Mol. Biol. 80, 
575-599) except that 10 percent glycerol was added to the separating gel 
to enable simultaneous casting and polymerization with the stacking gel. 
For greater resolution, particular samples were electrophoresed in 10 
percent polyacrylamide gels (0.4 mm.times.50 cm). The polyacrylamide gels 
were transferred to nitrocellulose treated with 10 percent acetic acid, 
neutralized, and probed with .sup.125 I labelled antibodies to B. 
amyloliquefaciens subtilisin. Towbin, H., Stdaehelin, T. and Gordon, J. 
(1979) Proc. Natl. Acad. Sci. U.S.A. 76, 4350-4354. 
EXAMPLE 8 
Preparation of antibodies and radioimmunoassay 
B. amyloliquefaciens subtilisin was purified (Philipp, M. and Bender, M. L. 
(1983) Mol. Cell. Biochem. 51, 5-32) and digested with 100 mg/ml CNBr (5 
mg/ml protein) in 88 percent formic acid. After incubation at room 
temperature for 2 hours, the reaction mixture was lyophilized and 
resuspended in 0.1 M Tris-HCl, pH 8.0 containing 0.05 percent Triton 
X-100. This was mixed with an equal volume of complete Freund's adjuvant 
and distributed intracutaneously (1 mg/animal) over several sites on New 
Zealand white-female rabbits. Booster injections were given in the ear 
vein (200 .mu.g/animal) on days 21 and 42. Bleeds were collected weekly 
following each boost. 
Subtilisin antigens in the membrane, media, and cytosol fractions were 
quantitated by radioimmunoassay. The membrane fractions were treated with 
5M urea prior to analysis in order to solubilize the antigen. The urea 
extract was diluted 1:20 with assay buffer (10 mM Na phosphate, pH 7.4, 
0.9 percent NaCl, 0.5 percent bovine serum albumin and 1 mM PMSF) just 
before analysis. Affinity purified anti-subtilisin immunoglobulin fraction 
(Eveleigh, J. W. and Levy, D. E. (1977) J. Solid-Phase Biochem. 2, 45-78) 
was coated at 10 .mu.g/ml onto the 96 well microtiter plate for 2 hours at 
room temperature. After washing with assay buffer, samples and standards 
were incubated in the wells as before. After washing, 100,000 cpm of 
.sup.125 I-antibody was introduced and the plate incubated again for 2 
hours. After washing, the plate was counted. 
EXAMPLE 9 
Expression and secretion of wild type B. amyloliquefaciens subtilisin 
The wild type B. amyloliquefaciens subtilisin gene was introduced on the 
plasmid, pS4-5 into B. subtilis strain BG2036. The construction of this 
strain is described in detail in EPO Publication 0130756. In this strain 
both the endogenous alkaline protease (apr) (i.e., subtilisin) and neutral 
protease (npr) genes have been deleted (Yang, M. Y., Ferrari, E. and 
Henner, D. J. (1984) J. Bacteriol. 160, 15-21). FIG. 7 shows the Western 
blot analysis of media (a) and membrane (b) fractions sampled as a 
function time of culture growth. Numbers 1-5 refer to time points of 4.8, 
6.5, 7.8, 10.8 and 24 h, respectively. ST denotes 0.1 .mu.g of a standard 
mature B. amyloliquefaciens subtilisin (P27). The band appearing in the 
media below P27 is a degradation product of P27. A sample containing 0.3 
ml equivalents of culture for medium samples or 0.2 ml equivalents of 
culture for membrane samples was loaded onto the gels shown. 
This Western analysis shows that at both early and late stages of cell 
growth only the 27,000 dalton mature form of subtilisin (P27) was detected 
in media fractions. In contrast, analyses of membrane fractions shows the 
presence of a 42,000 dalton precursor (P42). Initially, the appearance of 
P42 in the membrane was coincident with the appearance of P27 in the 
medium. Subsequently, P42 was seen to disappear as P27 accumulated. 
FIG. 8 shows the time course of cell growth (+), secretion of mature 
subtilisin (P27) into the medium (x), and accumulation of subtilisin 
precursor (P42) into cell membrane fractions (.DELTA.). Cell growth was 
measured by absorbance at 550nm while subtilisin was quantified by 
radioimmunoassay. Values were normalized to their maxima (cell growth was 
A.sub.550 =4; mature subtilisin (P27) was 36 .mu.g/ml; and the precursor 
(P42) was 0.06 .mu.g/ml). The accumulation of P42 measured by this RIA 
peaked at approximately 6.5 h of cell growth which was just prior to the 
expected onset of sporulation. As the cells sporulated, as measured by the 
drop in absorbance at 550 nm, P27 continued to accumulate in the media at 
the expense of P42 in the membrane. Thus, wild type subtilisin can be 
detected in both the membrane of the expression host and media supporting 
the same shortly after the media is innoculated. 
EXAMPLE 10 
Non-secretion of certain mutant subtilisins 
B. subtilis BG2036 was transformed separately with pAsn+32 and p.DELTA.66 
and grown for 20 hours. Cells were fractionated into membrane and media 
components, and fractions were loaded onto gels as previously described in 
Example 7. The results are shown in FIG. 9. ST denotes a gel lane 
containing 0.1 .mu.g of subtilisin. The media membrane fractions in tracks 
1 to 4 were derived from pS4-5, pAsn+32, p.DELTA.66 and the vector 
control, pBS42, respectively. 
Mature subtilisin (P27) was not observed in the medium for pAsn+32. 
Initially, the precursor of Asn+32, P42, appeared in the membrane with 
normal synthesis kinetics (data not shown). However, unlike the wild-type 
precursor, it continued to accumulate reaching a plateau late in 
stationary phase. The Asn+32 precursor migrated roughly 1,000 daltons 
smaller in size than the Asp+32 precursor. This is presumably due to 
charge and not a size difference as similar differences have been observed 
between basic and acidic substitutions in other subtilisin positions. 
In p.DELTA.166, the region of the gene coding for the carboxy-terminus of 
the protein was deleted thereby removing the catalytic serine+221 which is 
required for subtilisin activity. As can be seen in FIG. 9, subcellular 
fractions of stationary phase cells expressing p.DELTA.66 showed only a 
31,000 dalton membrane bound antigen (P31). The difference in 
electrophoretic migration observed for P31 and P42 was consistent with the 
length of the p.DELTA.66 deletion. Immunologically reactive mutant 
subtilisin was not observed in the media fraction. 
EXAMPLE 11 
Membrane bound P42 contains the subtilisin signal sequence 
To determine whether the membrane bound precursor, P42, contained the 
signal sequence, a deletion of five residues, -102 through -98, was 
constructed in the subtilisin gene (p.DELTA.-102). FIG. 10 is the high 
resolution (50 cm) Western blot analysis of membrane bound precursors from 
wild type (pS4-5) and the signal peptide deletion mutant p.DELTA.-102. 
Lane 1 contains 0.1 .mu.g of mature subtilisin; lanes 2 and 4 contain 
membrane fractions from a p.DELTA.-102 culture of B. subtilis, BG2036; 
lane 3 contains membranes from a wild type subtilisin (pS4-5) culture of 
B. subtilis, BG2036. This Western analysis revealed an alteration in the 
electrophoretic mobility of the translated precursor for p.DELTA.-102 
consistent with a five amino acid deletion, indicating that the membrane 
precursor P42 from pAsn+32 probably also contains the subtilisin signal 
sequence. 
EXAMPLE 12 
Maturation of membrane bound mutant subtilisin 
In contrast to the wild-type subtilisin, the maturation of the Asn+32 
mutant was blocked when produced in B. subtilis BG2036 which lacked both 
subtilisin (apr) and neutral protease (npr) genes. To test whether 
enzymatically active subtilisin or neutral protease was necessary for 
processing, pAsn+32 was expressed in various B. subtilis hosts. FIG. 11 
shows the Western analysis of medium fractions (lanes a and A) and 
membrane fractions (lanes b and B) from B. subtilis cultures containing 
either the pAsn+32 plasmid (lanes A and B) or the vector, pBS42, minus the 
subtilisin gene (lanes a and b). These plasmids were expressed in the 
following B. subtilis strains which have been described by Stahl, M. L. 
and Ferrari, E. (1984) J. Bacteriol. 158, 411-418 and Yang, M. Y., et al. 
(1984) J. Bacteriol 160, 15-21: B. subtilis I-168 (apr.sup.+ npr.sup.+), 
B. subtilis BG2044 (apr.sup.+ npr.sup.-), B. subtilis BG2019 (apr.sup.- 
npr.sup.+), and B. subtilis BG2036 (apr.sup.- npr.sup.+). Cultures were 
grown and fractionated as previously described. As can be seen, processing 
of the membrane bound precursor to the mature Asn+32 enzyme was only 
observed in apr(+) hosts. Maturation of Asn+32 subtilisin was independent 
of an intact npr gene as shown by the continued presence of P42 in the 
apr.sup.- npr.sup.+ strain, BG2019. It should be noted that antibodies 
elicited by the B. amyloliquefaciens subtilisin do not cross-react with 
the B. subtilis subtilisin. Thus, a P27 band was not seen with the vector 
control, pBS42, in the apr.sup.+ hosts. 
EXAMPLE 13 
In vitro removal of a membrane bound mutant subtilisin 
The P42 precursor of Asn+32 subtilisin derived from the apr.sup.- npr.sup.- 
host, BG2036, can also be matured in vitro by addition of subtilisin to B. 
subtilis BG2036 transformed with pAsn+32. Cell pellets were treated with 
exogenous subtilisin in vitro prior to analysis. Cells from 12.5 ml of a 
stationary phase (16 hour) culture were resuspended in 1.0 ml of 
osmotically supported Tris buffer (30 mM Tris-HCl, 25 percent (w/v) 
sucrose, pH 8.0) and treated with 1 .mu.g/ml B. licheniformis subtilisin 
(Sigma). After overnight incubation at room temperature with stirring, the 
cells and medium were separated by centrifugation and fractionated as 
described above. 
FIG. 12 shows the Western analysis of B. subtilis BG2036 cells containing 
pAsn+32 (lanes labeled b) or vector pBS42 (lanes labeled a) plasmids. 
Medium and membrane fractions are shown for samples taken immediately 
after cultruing (labeled 0) or after overnight incubation in the presence 
(labeled +sbt) or absence (labeled -sbt) of 1 .mu.g/ml B. licheniformis 
subtilisin. As can be seen, the majority of the P42 material was released 
from the membrane and converted to P27. The B. licheniformis enzyme was 
used because B. amyloliquefaciens subtilisin antibodies do not cross-react 
with it. In contrast, cells incubated in the absence of protease showed 
neither accumulation of P27 nor loss of P42. Cells containing only the 
vector plasmid, pBS42, did not show any detectable P27 in the presence of 
added B. licheniformis subtilisin. These studies suggest that P42 and P27 
have a precursor-product relationship. 
EXAMPLE 14 
Removal of membrane bound mutant subtilisin by co-culturing 
In addition to the in vitro method, it is also possible to mature an 
inactive subtilisin by co-culturing a strain possessing the inactive 
protein with a strain carrying a plasmid copy of an active endoprotease. 
For clarity and detection purposes, the active gene chosen was B. 
amyloliquifaciens subtilisin with the substitution Ala48.fwdarw.Arg 
prepared as described in Example 6. This latter enzyme migrates much 
faster on SDS gels than the normal wild type or the inactive mutant 
Ser221.fwdarw.Ala221 used in this experiment. Plasmids pAla+221 and 
pArg+48 were separately transformed into the BG2036 host which lacks both 
alkaline and neutral protease. 
Strains were precultured to a cell density of .about.0.5 A550/ml (log 
phase) and then fresh flasks were innoculated (1:100) such that equal cell 
densities were introduced into each flask. Samples were innoculated as 
ratios of R48 (Arg+48) cells to A221 (Ala+221) cells as follows: (1) 1:0, 
(2) 0:1, (3) 0.5:0.5, (4) 0.25:0.75, (5) 0.1:0.9, and a final flask (6) of 
the vector control pBS42. Cultures were then grown to stationary phase, 
and the cells removed by centrifugation at 11,000.times. g for 10 minutes 
in a Sorvall SS34 rotor. 600 .mu.l samples of the media were treated with 
an equal volume of 20% TCA and the pellets collected by centrifugation at 
19000 rpm in a Sorvall SS34 rotor (4.degree. C.). After washing with 600 
.mu.l of acetone and centrifugation as before, the pellets were dried in a 
SpeedVac and then dissociated in SDS sample buffer (25 .mu.l percent 
glycerol, 2 percent SDS, 10 mM Na phosphate pH 6.8) at 95.degree. C. for 3 
minutes. Samples were electrophoesed on 50 cm, 12.5 percent gels as 
described. 
FIG. 13 depicts the electrophoretic pattern of media samples as detected by 
Coumassie Blue staining. As shown therein, the band corresponding to A221 
subtilisin is clearly resolved from the R48 subtilisin. Even in the 
presence of low amounts of R48 cells, the production of A221 is 
significantly enhanced over the control A221 containing cells grown in the 
absence of active enzyme. Thus, active enzyme secreted by co-cultured 
cells is capable of maturing inactive subtilisin expressed by another 
strain which is incapable of secreting enzymatically active endoprotease. 
EXAMPLE 15 
Binding of a mutant subtilisin (Ser221.fwdarw.Ala) to subtilisin inhibitor 
To test the binding of the catalytically inactive mutant of wild type 
subtilisin, A221, to a subtilisin inhibitor, turkey ovomucoid third domain 
(TOM3) was utilized. Upon incubation with TOM3, the wild type enzyme binds 
the inhibitor leading to a quantitative decrease in activity. 
Wild type subtilisin (1.07.times.10-5 M) was incubated with TOM3 
(0.09.times.10-5 M) in the presence or absence of A221 enzyme 
(1.67.times.10-5 M). Activity was measured on the 
succinyl-Ala-Ala-Pro-Phe-pNa substrate. Results were as follows: 
TABLE 1 
______________________________________ 
Activity (units) 
Solution total bound to TOM3 
______________________________________ 
WT .896 0 
WT + TOM3 .143 .753 
WT + TOM3 + A221 .430 .466 
______________________________________ 
As shown in Table 1, the competitive binding of A221 to TOM3 inhibitor 
causes a 38 percent reduction in the binding of the wild type enzyme to 
the inhibitor. Therefore, although the A221 mutant is catalytically 
inactive, it is still capable of binding a subtilisin inhibitor. 
EXAMPLE 16 
Construction of a thermolabile mutant subtilisin 
A thermolabile mutant subtilisin was prepared containing the substitution 
of methionine at position 222 with aspartic acid (Met222.fwdarw.Asp or 
D222) as described in EPO Publication No. 0130756. 
This particular mutant is thermolabile as demonstrated by the approximate 
90% decrease in its enzymatic activity on skim milk plates at 45.degree. 
C. versus 37.degree. C. when compared to wild type subtilisin or other 
mutants of subtilisin at position 222. 
EXAMPLE 17 
Thermal inactivation of enzymatically active endoprotease after maturation 
of subtilisin Ser221.fwdarw.Ala 
In Example 14, the Ser221.fwdarw.Ala mutant was matured by coculturing with 
Ala48.fwdarw.Arg. In this example, the experiment was repeated, this time 
using the thermolabile, Met222.fwdarw.D222 mutant subtilisin of Example 16 
as the active enzyme. In this example B. subtilis BG2036, transformed 
separately with pD222 and pA221, were co-cultured at a ratio of 
l(D222):9(A221). A control coculture of D222 transformed B. subtilis 
BG2036 and untransformed BG2036 at the same ratio was similarly treated. 
At the conclusion of growth the media was treated at 50.degree. C. for up 
to 15 minutes. As shown in FIG. 14, essentially all of the activity of the 
D222 subtilisin in the coculturing experiment was eliminated after 15 
minutes while only 10% of the enzyme detectable on SDS gels was lost. 
Under these conditions, which are similar to those in Example 14 (except 
that the D222 subtilisin co-migrates with the A221 subtilisin), only 10% 
of the material would be expected to be the D222 enzyme. Thus, the 
Ser221.fwdarw.Ala mutant was produced by co-culturing with the 
thermolabile Met222.fwdarw.D222 mutant and the active enzyme removed by a 
simple heat step. 
EXAMPLE 18 
Cleavage of prosubtilisin hGH fusion polypeptide with subtilisin to 
generate mature hGH 
p22 is an expression plasmid designed to secrete human growth hormone 
(hGH) in Bacillus subtilis. The plasmid replicates in E. coli as an extra 
chromosomal element but can only integrate into the Bacillus subtilis 
chromosome at the Trp region. The integrated plasmid can be amplified to 
several copies per cell by altering the concentration of chloramphenical 
in the media. Albertini, A. M. and Galizzi, A. (1985) J. Bact., 162(2), 
1203. Transcription of the hGH gene is under the control of the Pac 
promoter (Yansura, D. G. and Henner, D. J. (1984) Proc. Natl. Acad. Sci. 
U.S.A., 81, 439) and secretion of hGH is facilitated by the Bacillus 
amyloliquifaciens amylase signal sequence and 32 amino acids of mature 
amylase. Cleavage of the secreted fusion protein is made possible by 
inserting the Bacillus amyloliquifaciens subtilisin pro sequence between 
the partial mature amylase sequence and the hGH gene. Several plasmids 
were constructed as precursors to the final plasmid 22. 
The plasmid p22 was constructed by a three way ligation. The vector was 
pJH101Trp2 in which the 375 basepair EcoRI-BamHI fragment had been 
removed. The second piece was a 650 basepair EcoRI-PstI fragment from 
p90 which contained the Pac Promoter and ribosome binding site, amylase 
signal sequence, 32 codons of mature amylase, codons 45 to 107 of 
preprosubtilisin, and the first 45 codons of hGH. The third fragment is a 
1075 basepair PstI-BamHI fragment from PHGH207. It contains codons 46 to 
191 of hGH (Goeddel, D. V., et al. (1979) Nature, 281(5732), 544), 
followed by the last 180 basepairs of E. coli lipoprotein gene (Nakamura, 
K., et al. (1980) J. Biol. Chem, 255, 210), 100 basepairs 3' of the 
lipoprotein gene encoding its transcriptional terminator, and finally 346 
basepairs of pBR322 between the Hind III and BamHI sites (Bolivar, F., et 
al. (1977) Gene, 2, 95). 
The three fragments, 300 ng of pJH101Trp2 vector and 50 ng of the other two 
pieces, were ligated in 30 .mu.l with T4DNA ligase. After 3 hours, 
competent E. coli D1210 (ATCC 31449) (Goeddel, D. V., et al. (1979) Proc. 
Natl. Acad. Sci. U.S.A., 76, 106-110), were transformed with the mixture 
and plated on LB ampicillin (30 .mu.g/ml) plates. The vectors containing 
the three fragments of p22 were constructed as follows. 
pJH101Trp2 
pJH101Trp2 was constructed by inserting a 375 basepair Sau3A fragment 
coding for amino acids 160-276 of the Bacillus subtilis Trp E gene 
(Henner, D. J., et al. (1984) Gene, 34, 169) into the BamHI site of 
pJH101. Ferrari, F. A., et al. J. Bact., 154(3), 1513. JH101 was digested 
with BamHI, treated with Bacterial Alkaline Phosphatase, and then ligated 
to the Trp Sau3A fragment with T4 DNA ligase. Competent E. coli strain 294 
rec.sup.+ was then transformed with the ligation mixture and plated on 
ampicillin (30 .mu.g/ml) LB plates. Colonies were screened by plasmid 
isolation (Birnboim, H. C. and Doly, J. (1979) Nucl. Acids Res, 2, 1513) 
and restriction analysis. One plasmid containing the orientation in which 
the BamHI site was recreated closest to the EcoRI site was designated 
pJH101Trp2. 
p2 
p2 fuses the Pac promoter and ribosome binding site to the amylase 
signal sequence. A three piece ligation was used to create the plasmid. 
The first piece was the vector pJH101Trp2 in which the 375 basepair 
EcoRI-BamHI fragment had been removed. 
The second piece was an EcoRI blunt fragment which contains the Pac 
promoter and ribosome binding site and the first two codons of the 
Bacillus licheniformis penicillinase gene. This fragment was created by 
the "Primer Repair" reaction. Goeddel, D. V., et al. (1980) Nucl. Acids 
Res., 8, 4057. The primer sequence was TTCATCAAAA and the 175 basepair 
fragment upon which the primer sat was isolated from an RsaI digest of a 
pBSA105 subclone. Yansura, D. G. amd Henner, D. H. (1983) Biology and 
Biotechnology of the Bacilli, eds. Ganesan A. T. and Hoch, J. A. Academic 
Press, New York. The reaction was subsequently digested with EcoRI to 
generate the 140 basepair fragment. 
The third piece was a 215 basepair blunt BamHI fragment which contains 
codons 2 through 63 of preamylase (Palva, I., et al. (1981) Gene 15, 43) 
followed by the following double stranded DNA linker. 
5'-TCTAGAATTCATGGCAGAAATAACAAG AGATCTTAAGTACCGTCTTTATTGTTCCTAG-5' 
The ligation was accomplished by mixing 300 ng pJH101Trp2 vector, and 50 ng 
of the other two fragments in 30 .mu.l in the presence of T3DNA ligase. 
After 3 hours competent E. coli D1210 (ATCC 31449) were transformed with 
the mixture and plated on ampicillin (30 .mu.g/ml) LB plates. 
pPS11 
The plasmid pPS11 was-constructed by ligating four fragments. The first was 
the vector pJH101Trp2 in which the 375 basepair EcoRI BamHI fragment had 
been removed. The second was a 375 basepair piece obtained by digesting 
pPS4-5 (Wells, J. A., et al. (1983) Nucl. Acids Res., 11(22), 7911) with 
EcoRI and AvaI. This piece contains the subtilisin promoter, signal 
sequence, and 93 codons of preprosubtilisin. Wells, J. A., et al. (1983) 
Nucl. Acids Res,, 11(22), 7911. The third fragment was a 46 basepair blunt 
AvaI piece which was generated by digesting pPS4-5 with HinpI followed by 
treatment with DNA Polymerase Klenow, and then second cutting with AvaI. 
This fragment codes for amino acids 94 to 108 of preprosubtilisin. The 
final fragment was a 937 basepair blunt BamHI piece generated by digesting 
plasmid pHGH207 (Kleid, D. G., et al. EPO Publication No. 0154133 
published Sep. 11, 1985) with EcoRI followed by filling in with DNA 
Polymerase Klenow and second cutting with BamHI. 
D72 
The plasmid p72 required four pieces for construction. The first was the 
vector pJH10Trp2 in which the 375 basepair EcoRI-BamHI fragment had been 
removed. The second was a 187 basepair EcoRI-HgiAI fragment containing the 
Pac promoter and ribosome binding site and the first 17 codons of the 
amylase signal sequence. This was obtained by the appropriate digestion of 
a p2 subclone. The third piece was a 139 basepair HgiAI-EcoRV fragment 
containing codons 17 to 32 of preamylase. Palva, I., et al. (1981) Gene 
15, 43. The last was a 1129 basepair DraI-BamHI pPsll fragment containing 
codons 45 to 108 of preprosubtilisin followed by an EcoRI linker and the 
hGH gene. Goeddel, D. V., et al. (1979) Nature, 281(5732) , 544. 
The ligation mixture included 300 ng of pJH101Trp2 vector, 200 ng of the 
DraI-BamHI fragment, and 50 ng of the final two pieces in 30 .mu.l in the 
presence of T4DNA ligase. After 3 hours competent E. coli D1210 was 
transformed with this mixture and then plated on LB ampicillin plates 30 
.mu.g.ml). 
p90 
Plasmid p90 was constructed from four pieces. The first was the vector 
pBR322 (Bolivar, F., et al. (1977) Gene, 2, 95) in which the 748 basepair 
EcoRIPstI section had been removed. The second was a 496 basepair 
EcoRI-Sau3A fragment generated by digesting p72 completely with EcoRI 
and partially with Sau3A. This fragment contains the Pac promoter and 
ribosome binding site, amylase signal sequence, 32 codons of mature 
amylase and codons 45 to 100 of preprosubtilisin. The third piece was a 
synthetic DNA duplex with the following sequence. 
5'-GATC ACG TAG CAC ATG CGT AC TGC ATC GTG TAC GCA TG-5' 
The fourth piece was a 135 basepair blunt PstI fragment containing the 
first 45 codons of hGH. (Goeddel, D. V., et al., Nature (1979) 281 (5732), 
544.) This fragment was generated by the primer repair reaction. The 
primer used had the sequence 
TTC CCA ACT ATA CCA CTA TCT CGTCT ATT and the fragment upon which the 
primer sat was a 941 basepair XbaI-BamHI fragment from pHGH207. The primer 
repair reaction was digested with PstI to generate the 135 basepair 
fragment. 
The four fragments, 300 ng pBR322, 30 ng. EcoRI-Sau3A fragments, 1 .mu.g 
synthetic DNA duplex, and 5 ng blunt PstI fragment, were ligated in 30 
.mu.l with T4 DNA ligase. After 4 hours competent E. coli D1210 were 
transformed with the mixture and then plated on tetracycline LB plates 
(.mu.g/ml). 
Transformation and Amplification 
The plasmid p22 was used to transform competent Bacillus subtilis strain 
I-168(TrpC2). Gryczan, T. J., et al. (1978) J. Bacteriol., 134, 318. The 
transformation mixture was plated on LB plates containing 12.5 .mu.g/ml 
chloramphenicol. 12 colonies were picked and grown with shaking at 
37.degree. C. in 2 YT broth containing 12.5 .mu.g/ml chloramphenicol until 
4 hours after the start of stationary phase. All cultures were then 
centrifuged to remove cells, and the supernatant assayed for hGH by RIA. 
The cells from 5 ml of the above cultures which showed the highest level of 
hGH in the media, were suspended in 300 .mu.l 50 mM glucose, 10 mM EDTA, 
25 mM Tris pH 8 containing 4 mg/ml lysozyme. After 30 minutes at 
37.degree., 100 .mu.l of 10 mM Tris 1 mM EDTA were added along with 1 
.mu.l 10% SDS. The mixture was then phenol extracted, chloroform 
extracted, and then 1 ml ethanol added. The cellular DNA was removed, 
dried, and taken up in 50 .sup..mu. l 10 mM Tris pH 8, 1 mM EDTA. 
5 .mu.l of the above cellular DNA was used to transform competent Bacillus 
subtilis BG84 (spoOA-). EPO Publication No. 0130756; Wells, J. A., et al. 
(1983) Nucl. Acids Res., 11(22), 7911. The transformation mixture was 
plated on LB plates containing 25 .mu.g/ml chloramphenicol. 
Cleavage of Fusion Polypeptide with Subtilisin 
B. subtilis BG84 containing the plasmid PA 422 was grown in a 10 liter 
fermenter with yeast extracts and protein hydrolysates to early stationary 
phase. After removing the cells from the fermentation broth by continuous 
centrifugation, the clear culture supernatant containing the secreted 
fusion protein was loaded onto a coupled anti-hGH antibody sepharose GMB 
column. The fusion protein was eluted from the column into fractions. The 
fractions containing hGH as measured by hGH RIA activity were pooled for 
subtilisin cleavage. The reaction mixture for cleavage of the fusion 
polypeptide contained about 2 .mu.g of antibody purified fusion 
polypeptide and 16 ng of subtilisin BPN (Albertini, A.M. et al. (1985) J. 
Bacteriol. 162(2), 1203) in 10 mM Tris pH 8.6. The reaction temperature 
was 25.degree. C. Time points at 1 min. intervals were taken and the 
reaction was stopped by 15% TCA. The TCA precipitates were washed twice by 
acetone and finally dissolved in SDS buffer for SDS polyacrylamide gel 
electrophoresis. The proteins on the gel were transferred onto 
nitrocellulose paper for immunoblot analysis with anti-hGH antibody. Lanes 
1-5 (top) in FIG. 15 represent processing incubation times 0, 0.5, 1, 2 
and 4 min., respectively. To the right is indicated the mobility of 
molecular weight standards of 30,000; 20,000; and 17,000 daltons. To the 
left is indicated the position of the unprocessed prosubtilisin-hGH fusion 
(1, the heavy band at 29-30 kd) and the position of mature hGH (2, the 
lighter band, at 22 kd). The results shown in FIG. 15 indicate that the 
fusion protein at 29-30 kd was cleaved by subtilisin with the subsequent 
appearance of a protein at 22 kd (mature hGH). 
Having described the preferred embodiments of the present invention, it 
will appear to those ordinarily skilled in the art that various 
modifications may be made to the disclosed embodiments, and that such 
modifications are intended to be within the scope of the present 
invention.