NO892683L - PROCEDURE FOR THE PREPARATION OF ANIMAL LYSOSOM C THROUGH PICHIA PASTORIS SECRETARY AND COMPOSITION THEREOF. - Google Patents

Description

The present invention generally relates to a microbiological method for the production of an animal lysozyme c using recombinant DNA technology and more particularly the present invention relates to the production of an animal lysozyme c by cultivating Pichia pastoris yeast cells which contain a gene which, in such cells , are capable of expressing the pre-form of the lysozyme in such cells, the DNAs used to transform such cells to express the pre-form of the lysozyme, and cultures and subcultures of the transformed cells.

Lysozymes are basic enzymes that directly exhibit anti-bacterial action, as a result of their ability to lyse bacterial cells, and indirectly, as a result of their ability to provide a stimulating effect on the phagocytic activity of polymorphonuclear leukocytes and marcophages (Jollés et al., Mol. and Cell. Biochem. 63, 165 (1984)). Lysozymes are found in a variety of tissues and secretions in humans, other vertebrates and invertebrates, as well as in plants, bacteria and phage. In carrying out their primary function of protecting organisms against bacterial infections, lysozymes, which are also known as 1,4-|3-N-acetylmuramidases, cleave the glycosidic bond between C-1 of N-acetylmuramic acid and C-4 of N- the acetylglucosamine in the bacterial peptidoglycan, a polysaccharide of amino sugars linked to short cross-linked peptides which are a component of bacterial cell walls. Gram-negative bacteria have cell walls that contain mono- or bi-layers of peptidoglycan while gram-positive bacteria have cell walls that contain highly complex multi-layers of peptidoglycan. As a result of the above cutting, all such bacteria will lighten and consequently die. Some lysozymes also exhibit a more or less pronounced chitinase activity, corresponding to a random hydrolysis of 1,4-p-N-acetylglucosamine bonds in chitin, and consequently have the additional capacity of extensive protection of organisms against a number of chitin-covering pathogens. A small esterase activity of lysozymes has also been reported.

Due to their bacteriolytic activity, lysozymes are used, alone or in combination with other components, such as lactotransferrin, which inhibit the growth of certain microorganisms by means of chelating iron, complement, antibodies, vitamins, other enzymes and various antibiotics, such as tetracycline and bacitracin, as antimicrobial agents, as food preservatives, such as cheese, sausage and marine products, as ripening agents for cheese and for various other uses. As lysozymes also have the ability to indirectly stimulate the formation of antibodies against a number of antigens, such enzymes can also be used to increase resistance to infections.

c- or "chicken"-type lysozymes contain 129-130 amino acids in their mature, secreted forms. 40 of these 129-130 have been found to be invariant among different species. Two of the many carboxyl groups in lysozyme c (corresponding to Glu-3 5 and Asp-52 in the lysozyme amino acid sequence in chicken egg whites) which participate in the enzyme's catalytic activity and which are essential for lysozyme activity, appear in approx. the same positions in all c-type lysozymes. A third carboxyl group (corresponding to Asp-101 in lysozyme from chicken egg white), which is involved in a substrate-binding interaction, occurs in most c-type lysozymes. The eight half-cysteine residues in all c-type lysozymes are invariant. The disulphide bridges formed by the cysteines play an important role in the formation and maintenance of the enzymes' secondary and tertiary structures. These three-dimensional structures, and processes of folding to form these structures upon translation of mRNA appear to be very similar for all lysozymes c. The complete primary structures are known for the mature lysozymes c obtained from the following sources: (1) hen, quail, turkey, Italian, duck, pheasant and chicken egg whites; (2) human milk and urine; (3) moths; (4) baboon, rat, and bovine stomachs; and (5) T2 and T4 subjects. DNA sequences encoding mature lysozyme c from human milk are known. See EP patent application with publication numbers 0181 634, 0 208 472 and 0 222 366.

Only three complete amino acid sequences in mature lysozymes of the other major type, the g or "goose" type, have been determined. These include lysozyme g sequences from Emden goose, black swan and ostrich egg white.

Although lysozymes of both types c and g have acidic amino acid residues as part of their active sites, a number of differences exist between the two types. g-type lysozymes contain approximately 185 amino acids in their mature form, exhibit low activity on N-acetylglucosamine polymers, and do not immunologically cross-link with c-type lysozymes. G-type lysozymes have an unusually high incidence of paired amino acids (i.e. the same amino acids occurring in neighboring positions in the sequence) in the molecules, and all four half-cysteine residues in the g-type molecules are located in the N-terminal half of the chain . c-type lysozymes are likewise active on peptide-substituted or unsubstituted peptidoglycan, and are also active on chitin oligosaccharides. g-type lysozymes, which have activity towards the linear peptidoglycan similar to that of the c-type enzymes, do not act on chitin oligosaccharides. Furthermore, unlike c-type lysozymes, which are capable of both hydrolysis and transglycosylation, g-type enzymes act only as hydrolases.

The occurrence of other distinct lysozyme types, which differ from types c and g on the basis of structural, catalytic and immunological criteria, has also been reported. A number of mammalian species that have a foregut fermentation and that use lysozyme in their digestive systems are known. Domesticated cattle of the species Bos taurus and other ruminant mammals of the species Artiodactyla (i.e. ruminants) have developed a symbiotic relationship with microbes that live in the intestine (foregut) and break down cellulose and other forage constituents. Such mammals have an unusually high level of lysozyme in the stomach lining. As a result of this form of decomposition, a number of microbes will enter the fundus area (front part) of the fourth stomach, where lysozymes are secreted from the mucosal layer, and will be broken down by the lysozymes, thus causing the ruminants to use the nutritional content of the microbes . Metabolic equilibrium studies that have been carried out suggest that the ruminant uses lysed bacteria as a carbon, nitrogen and phosphorus source for energy and growth.

The three forms of lysozyme present in the fourth stomach of domestic cattle make up about 10% of the total protein that can be secreted from the mucosa of the fourth stomach. These three, non-allelic lysozymes, which are of type c (and designated cl, c2 and c3) are antigenically and in amino acid composition closely related to each other.

Jolles et al., J. Biol. Chem. 259, 11617 (1984), determined the complete 129 amino acid sequence of a mature bovine lysozyme c2, which is the most abundant of the three lysozymes found in the bovine stomach, to be as follows:

The three lysozymes c present in the abomasum of domestic cattle differ, in certain respects, from other lysozymes c in that (1) the optimum pH for enzymatic activity of lysozymes c present in the bovine abomasum is about 5, instead of 7 for other lysozymes c; and (2) the lysozymes c present in the bovine abomasum are more stable in acidic environments, such as in the abomasum, and are more resistant to proteolytic enzymes, such as pepsins, found in the abomasum, than other lysozymes c .

The production of heterologous proteins by secretion into the culture medium mediated by signal sequences has been described for a number of organisms, including various Aspergillus species, Saccharomyces cerevisiae, and various types of mammalian cells. In these species, it has been shown that both native (ie intra-generic) and signal sequences of mammalian origin are capable of directing secretion of certain heterologous proteins into the culture media. However, each of these hosting systems has different advantages and disadvantages.

Aspergillus strains excrete e.g. large amounts of endogenous proteins into the culture media, thus significantly increasing the complexity and expense of purifying a desired heterologous protein product. The productivity of heterologous protein production by secretion from S. cerevisiae turns out to be severely limited for those proteins that can be secreted at all. A significant disadvantage in connection with mammalian cell hosts is the difficulty of, and the large expenses associated with, maintaining such host systems and cultivating such hosts on a large scale.

Certain polypeptides expressed in, and secreted from, heterologous hosts exhibit no, or reduced, degree of biological activity compared to their naturally occurring counterparts. This reduced biological activity may be the result of a number of factors, including the inability of the polypeptide to pass into and through the host's secretion apparatus without activity-reducing degradation or cleavage and to fold correctly and form disulfide bridges during the secretion process.

Yeast provides certain advantages over other host systems with respect to large-scale production of heterologous proteins in biologically active form. Yeast can generally be grown to a higher cell density than bacteria. A particularly preferred yeast is P. pastoris. The methylotropic yeast, Pichia pastoris, is known in the art. Such yeast has been found to be particularly advantageous for use as a host system for large-scale production of the heterologous proteins which it is able to secrete in significant quantities in biologically active form in its culture medium. P. pastoris can easily be adapted to continuous fermentation treatment in an industrial scale, whereby the yeast grows to a high cell density in a defined and cheap fermentation medium. With P. pastoris, production volumes can usually be scaled up from shake flask cultures to large fermentation cultures. Simple culture media that are cheap and free of undetermined constituents, which may be potential sources of pyrogens and toxins, can be used for this yeast. Moreover, since many critical functions in yeast, such as oxidative phosphorylation, are carried out in organelles, such functions are not directly exposed, as they are in prokaryotic hosts, to the possible deleterious effects of the production of polypeptides foreign to the host cells. Since yeasts are also eukaryotes, their intercellular environment tends to be more suitable than that of prokaryotes, for the correct folding of eukaryotic proteins. In addition, cultivation of yeast, particularly P. pastoris, is easier than cultivation of most other host systems. Contamination of P. pastoris cultures grown on methanol can be more easily prevented than contamination of cultures of other host types, since the reliability and safety of the heterologous polypeptide products can thus be increased.

In cases where a yeast secretes a desired protein into the culture medium, the separation of the protein from cellular components and therefore purification of the protein is simplified.

Although P. pastoris is a particularly advantageous host system for the production of heterologous proteins which it accidentally secretes into the medium, it cannot be predicted that a particular protein type, e.g. lysozyme c, will be secreted by the yeast in a biologically active form and, if this type is secreted at all, how effective the secretion will be. However, when a particular protein is found to be efficiently secreted from the yeast, it is likely that other proteins of similar sequence and three-dimensional structure will also be secreted.

Even for the yeast S. cerevisiae, which has been studied to a significantly greater extent than P. pastoris, secretion systems for proteins remain poorly understood. Secretion of a mature protein in biologically active form into the medium requires, upon expression, that the mature protein has a "signal sequence"

(also referred to as "signal peptide") attached to its amino-terminal end. This signal sequence has a little understood role when it comes to introducing a protein into the cell's secretion apparatus and is cut during translation to give the mature protein.

SUMMARY OF THE INVENTION

The present invention provides a new, surprising and unexpectedly efficient method for the production of large quantities of a biologically active animal lysozyme c which is easily purified by culturing P. pastoris cells containing a gene capable of expressing the pre-form of the animal lysozyme c (ie the animal lysozyme c with its naturally occurring signal sequence attached to the N-terminal end of the mature protein) in such cells, under conditions such that the gene is transcribed in the cells.

In accordance with the present invention, large amounts of an animal lysozyme c are efficiently secreted from P. pastoris cells into the media. Moreover, production levels are maintained when transformed P. pastoris cultures are scaled up from shake flask cultures to large fermentation cultures. Virtually all animal lysozyme c that is secreted into and recovered from the media is correctly processed at the junction of the signal sequence and the mature protein, has disulfide bridges formed correctly to give the correctly folded protein, and is biologically active. Furthermore, the secreted mature protein has essentially the same immunological properties and specific activity as the naturally occurring animal lysozyme c. In addition, the secreted animal lysozyme c comprises at least 50% of the protein present in the media and proteins different from animal lysozyme c are present in small amounts compared to the animal lysozyme c.

The present invention also includes DNA1 er, for transforming P. pastoris cells to express an animal pre-lysozyme c, and cultures of P. pastoris cells which have been transformed with such DNAs.

P. pastoris cells which have been transformed with the DNAs according to the invention are cultured to carry out the method according to the invention for the production of an animal lysozyme c.

Finally, the present invention involves the discovery of the signal peptide, of bovine lysozyme c2 and of bovine pre-lysozyme c2, and the invention comprises DNAs with sequences that code for these polypeptides.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a restriction map of the generalized Pichia pastoris expression vector pA0804. Important functional features and restriction sites for the plasmid are indicated and are described in the examples. Figure 2 provides a restriction map for bovine pre-lysozyme c2 expression plasmid pSL12A and illustrates construction of the plasmid from plasmid pA0804 and the EcoRI site-terminated bovine pre-lysozyme c2 coding segment of plasmid pBLI6C (derived from clone XBL3). Important functional features and restriction sites for pSL12A are indicated in the figure and are described in the examples that follow. Figure 3 is a restriction map of bovine pre-lysozyme c2 expression plasmid pBL11. Important functional features and restriction sites for the plasmid are indicated and are described in the examples that follow.

In the figures, restriction enzyme sites are indicated in parentheses, sites where a fragment, on one end thereof formed with one of the indicated enzymes, was joined to another fragment, on one end thereof formed with the other of the indicated enzymes, and, as a result of the association of fragments in the site, sites for both of the indicated enzymes were eliminated.

DETAILED DESCRIPTION OF THE INVENTION

In one aspect, the present invention comprises a method for producing an animal lysozyme c

extensive cultivation of P. pastoris cells, which have a gene capable of expression of the corresponding pre-lysozyme c in P. pastoris, said cultivation being carried out under conditions such that the gene is transcribed in the cells.

The invention further relates to a DNA which comprises (1) a promoter segment of a first P. pastoris gene, said segment comprising the promoter and the transcription initiation site of said first gene, and a terminator segment of another P. pastoris gene, wherein said terminator segment comprises polyadenylation signal coding segments and polyadenylation site coding segments and the transcription termination signal of said second gene, wherein said first and second genes are the same or different and said terminator segment is oriented, with respect to the direction of transcription from the promoter of said first gene in said promoter segment, operative for termination of transcription from said promoter on said transcription terminator of said second gene in said terminator segment; and (2) a DNA segment encoding an animal pre-lysozyme c which is oriented and positioned, between said promoter and terminator segments, operative for transcription, from said promoter of said first gene of the animal pre-lysozyme c encoding DNA- segment and polyadenylation signal coding segments and polyadenylation site coding segments of said second gene in said terminator segment.

In another aspect, the invention comprises a DNA, which is able to transform P. pastoris cells to express an animal pre-lysozyme c and which has the properties of a DNA according to the invention described in the preceding paragraph, and which in additionally comprising a gene for providing a selectable marker to cells containing the DNA.

In a further aspect, the invention comprises a culture of P. pastoris cells transformed with a DNA according to the invention.

In a further aspect, the invention comprises a polypeptide having the sequence of bovine pre-lysozyme c2, a polypeptide having the sequence of the signal peptide of bovine pre-lysozyme c2, and a DNA segment comprising a segment encoding bovine pre-lysozyme c2 or the signal peptide thereof.

The present invention provides a new, surprising and unexpectedly effective method for producing large quantities of a biologically active animal lysozyme c which can be easily purified.

The various terms used in the present application are defined generally as follows: (1) The term "culture" means a propagation of cells in a medium leading to their growth, and all subcultures thereof. (2) The term "subculture" means a culture of cells grown from cells of another culture (stock culture), or any subculture of the stock culture, without regard to the number of subcultures carried out between the subculture of interest and the stock culture. (3) The term "animal pre-lysozyme c" means the pre-form of the animal lysozyme c protein, which consists of the naturally occurring signal peptide of lysozyme c fused to the amino-terminal end of the mature lysozyme c. Pre-bovine lysozyme c2 prepared in accordance with the present invention has the 147 amino acid sequence as indicated in Example 1, comprising the signal peptide of 18 amino acids and the mature protein of 129 amino acids. Pre-human lysozyme c produced in accordance with the invention has the 148 amino acid sequence as described in example 12, comprising the signal peptide with 18 amino acids and the mature protein with 130 amino acids. (4) The amino acids occurring in the various amino acid sequences appearing herein can be identified in accordance with the following three- or one-letter abbreviations: (5) The nucleotides occurring in the various nucleotide sequences appearing herein have their common designations of a letter, which is routinely used in the prior art. (6) The term "animal lysozyme c" means a lysozyme c from an organism of the animal kingdom and from either the group Aves or the group Mammalia (birds and mammals).

Methods for transforming Pichia pastoris with DNAs, including vectors comprising genes for expression of heterologous proteins, are known in the art. Likewise, methods are known for culturing P. pastoris cells, which have a gene for a heterologous protein, for expression of the heterologous protein from such a gene. Furthermore, there are known methods for isolating heterologous proteins from the medium of such cultures of P. pastoris, which are secreted from the cells into the medium. These known methods can be used for the production of cultures of P. pastoris according to the invention and for carrying out the method according to the invention with such cultures for the production of an animal lysozyme c. Some of these methods are described in more detail in the examples that follow.

In a DNA according to the present invention comprising a selectable marker gene, any selectable marker gene can be used which acts in P. pastoris cells to allow the cells transformed with the DNA according to the invention to be distinguished from cells which have not been transformed thus. Among the types of selectable marker genes, the selectable marker gene on a DNA according to the invention can provide a dominant selectable marker or a marker that complements an auxotropic mutation in cells to be transformed. A gene that can provide a dominant selectable marker in P. pastoris cells is the well-known neomycin resistance gene from bacterial transposon Tn5 which provides resistance to the antibiotic G418. Among the genes that provide complementation for euxotropic mutations are the P. pastoris HIS4 gene (for transformation of His4~ strains of P. pastoris),

The S. serevisiae HIS4 gene (for transformation of the His4~ strain of P. pastoris), the P. pastoris ARG4 (arginosuccinate lyase) gene (for transformation of the Arg4~ strains of P. pastoris), and the S. cerevisiae ARG4 gene (for transformation of Arg4~ strains of P. pastoris).

In the promoter segment of a DNA according to the invention, comprising DNAs for transformation of P. pastoris to form an animal lysozyme c (referred to herein as

"transformation DNAs according to the invention"), the promoter of any P. pastoris gene can be used to drive the transcription of the animal pre-lysozyme c-encoding DNA segment of the DNA<1> according to the invention. Preferably, the promoter driving the transcription of the animal pre-lysozyme c coding segment will be the promoter of a P. pastoris gene whose transcription is carefully regulated by means of factors that can be easily varied in P. pastoris cultures, e.g. the carbon source. Among such P. pastoris genes, the preferred gene is the important alcohol oxidase gene (A0X1 gene), whose promoter is essentially completely inactive unless methanol is present in the culture medium, but which is highly active, yielding large amounts of the gene product when methanol is present. In the promoter segment of a DNA according to the invention, the transcription initiation signal and the segment between the promoter and the transcription initiation signal will preferably be from the same P. pastoris gene as the promoter.

The "terminator segment" of a DNA according to the invention (including transformation DNAs) has a subsegment which codes for a polyadenylation signal and polyadenylation site in the transcript, from the promoter of the DNA according to the invention, whose transcript comprises the transcript of the animal pre-lysozyme c-coded DNA segment of the DNA according to the invention, and a subsegment which provides a transcription termination signal for transcription from said promoter. The entire "terminator segment" of a transformation DNA according to the invention will preferably be taken from a P. pastoris protein-coding gene, which may be the same or different from the P. pastoris gene from which the promoter of the DNA according to the invention are taken, and which drive the transcription of the animal pre-lysozyme coding segment and polyadenylation signal and site coding segments. In the preferred case, both the terminator segment and the promoter will control

transcription of the DNA segment encoding the animal pre-lysozyme c be from the P. pastoris AOX1 gene.

In a DNA according to the present invention, the DNA segment encoding an animal pre-lysozyme c can be any DNA segment having a sequence free of introns, comprising a translation start site coding triplet ( hereafter referred to as "translation start triplet") and a translation stop signal-encoding triplet (hereafter referred to as "translation stop triplet") and which codes for, starting with the translation start triplet and ending with the triplet adjacent to the translation stop triplet (in the 5' direction from the stop triplet), a complete animal pre-lysozyme c. An example of such a DNA segment, with the sequence of a bovine pre-lysozyme c2, is approximately 4 60 bp EcoRI site-terminated segments constructed as described in Example 2 below. Of course, another segment, which differs from this segment constructed as described in example 2 by one or two nucleotide changes, which do not change the length or the amino acid sequence of the encoded polypeptide, is also a bovine pre-lysozyme c2 coding segment which can be used in a DNA according to the invention.

In accordance with procedures well known in the art of molecular biology, DNA segments with sequences encoding pre-lysozyme c other than bovine lysozyme c2 can be isolated. E.g. such DNA segments can be isolated by using probes, based on the amino acid sequence of the mature lysozyme c of interest, to screen a cDNA library of the species involved to isolate a suitable cDNA, comprising the DNA segment of interest. Reference is also made to the following examples. Besides the bovine lysozymes c, the human lysozymes are preferred.

A DNA according to the invention which comprises a segment which codes for bovine pre-lysozyme c2 or the signal segment of bovine pre-lysozyme c2 can be any DNA which (A) has a segment which has a sequence of 441 base pairs which codes for bovine pre-lysozyme c2 (with either histidine or lysine, but preferably histidine, at position 9 8 of the mature protein part) or a sequence of 54 base pairs encoding the signal segment of bovine pre-lysozyme c2 (see the sequence in the last full the paragraph in example 1 below); and (B) is (i) capable, upon transformation into a host, of being expressed to form pre-lysozyme c2 or the signal segment thereof (fused to a mature animal lysozyme c2, or any other desired protein); or (ii) is capable of being ligated into another DNA capable of causing expression of a protein as described in step (B)(i) above. Thus, e.g. a DNA according to the invention which comprises a segment which codes for the signal segment of bovine pre-lysozyme c2, be a DNA which comprises a segment which codes for the fusion polypeptide in which the signal segment of the bovine pre-lysozyme c2 is fused to the amino terminus of mature human milk lysozyme.

In the DNA according to the present invention with promoter and terminator segments delimiting a pre-lysozyme c-coding segment, the animal pre-lysozyme c-coding segment is located and oriented, with respect to the P. pastoris promoter and terminator -segments, operative for transcription of the animal pre-lysozyme c coding segment under the control of the promoter of said promoter segment into a transcript capable of providing expression in P. pastoris of the animal pre-lysozyme c. Those skilled in the art those skilled in the art will understand how to perform such positioning and orientation, operative to provide expression in P. pastoris of the animal pre-lysozyme c from the DNA of the invention, provided that the animal pre-lysozyme c coding segment is transcribed from said promoter. As understood in the art, the animal pre-lysozyme c coding segment, comprising its translation start and translation stop triplets, must be downstream from the transcription initiation site in terms of the direction of transcription from said promoter, and upstream from the polyadenylation signal and polyadenylation site-encoding subsegment of the terminator segment, which, in turn, must be upstream of the transcription termination site of the terminator segment. The segment coding for the animal pre-lysozyme c must be oriented with the translation start triplet upstream of the translation stop triplet. Preferably, no transcription terminator site will be located between the promoter and the polyadenylation site upstream from the transcription terminator of the terminator segment on the downstream end of the DNA according to the invention. Preferably, in a DNA in accordance with the invention, in the direction of transcription from the promoter which drives the transcription of the animal lysozyme c-encoding segment, between said promoter and the transcription terminator which terminates said transcription from said promoter, there will only be a single, long open reading frame having the sequence that codes for an animal pre-lysozyme c. Likewise, this transcript will preferably have a single polyadenylation signal and site.

The terms "downstream" and "upstream" in a DNA according to the invention mean respectively "downstream" and "upstream" with respect to the direction of transcription from the promoter driving transcription of the animal pre-lysozyme c coding segment. Construction of a variety of transformation DNAs in accordance with the invention, including pSL12A, pBL11 and PHLZ103, is described in the following examples.

Of these three plasmid DNAs, Clal-(BamHI/Bglll) is the fragment of pSL12A, which includes the Clal-site terminated bovine pre-lysozyme c2 expression cassette and the P. pastoris HIS4 gene, and Clal-(BamHI/ HpaI) fragment of pBLll, comprising the ClaI site-terminated bovine pre-lysozyme c2 expression cassette and the S. cerevisiae ARG4 gene, and the ClaI-(BamHI/Bglll) fragment of PHLZ103, comprising the ClaI site-terminated human the pre-lysozyme c expression cassette and the P. pastoris HIS4 gene, also DNAs in accordance with the invention.

A transforming DNA according to the invention can include elements that are necessary for its selection and replication in bacteria, especially E. coli, whereby the production of large amounts of the DNA during replication in bacteria will be simplified. In this respect, a preferred DNA according to the invention is a plasmid which includes a segment comprising the origin of replication and ampicillin resistance or tetracycline resistance genes of plasmid pBR322.

A DNA according to the invention may, after transformation into P. pastoris, be maintained as an episomal DNA (eg closed circular plasmid), provided it includes an origin of replication or autonomous replication sequence (ARS) functional for episomal maintenance in P. pastoris. A number of DNA segments comprising replication origins and ARSs functional in Pichia pastoris are known in the art.

A DNA according to the invention can integrate via homologous recombination into the P. pastoris genome in a certain part of the cells. Such integration can be accomplished by transforming P. pastoris cells with linearized or circularized plasmids, or linearized fragments of both, consisting of homologous DNA fragments. Thus, in the preferred P. pastoris cultures according to the invention, DNA according to the invention will be retained in the cells as part of the cells' genomes. Methods for providing integration of heterologous DNA into yeast genomes, including those of P. pastoris, are well known in the art and can be used for DNAs according to the invention. See e.g. European patent application with publication number 0 226 7 52. In particular, as illustrated in the examples that follow, the possibility of integration of a DNA into the P. pastoris genome is increased by the absence, from the DNA, of any origin of replication or ARS functional in P. pastoris to maintain the DNA in episomal form. Furthermore, targeting of the integration site to preferred sites in the P. pastoris genome is carried out by incorporation into the transforming DNA "targeting segments", which are segments, usually at the two ends of a linearized DNA according to the invention, whose segments have sequences which are homologous to the desired sites of integration into the genome. If the transforming DNA is a plasmid, it can be linearized, or cut into linearized fragments, usually by cutting with one or more restriction enzymes that cut at one or more appropriate sites, to give a linear transforming DNA according to the invention with targeting segments on the ends. Appropriate targeting segments will have a length of at least about 200 bp. Examples of the treatment of transforming plasmid DNA<1>s according to the invention in this way are given in the examples. For example, a linearized transforming plasmid DNA is obtained by cutting, with SacI, (an isoschizomer of Sstl), a derivative of pAO804, which has an animal pre-lysozyme c coding segment (lacking a SacI site) inserted into the EcoRI site, and a linearized transforming fragment of a transforming DNA is obtained by cutting, with Bglll, a derivative of pAO804, which has an animal pre-lysozyme c coding segment (lacking a Bglll site) inserted into EcoRI - the seat.

By using the native animal lysozyme c signal sequences (or the human lysozyme c or bovine lysozyme c2 signal sequence), the present invention allows an animal lysozyme c to be secreted with unexpected efficiency from P. pastoris cells into the culture media. Surprisingly, the animal lysozyme c secreted into the media has the same biological activity as the naturally occurring enzyme and the pre-enzyme is correctly processed at the junction of the signal sequence with the mature protein. Thus and unexpectedly, the animal pre-lysozyme c signal sequences are correctly recognized and processed in the P. pastoris secretion track.

An animal lysozyme c isolated from culture media of cultures in accordance with the invention is judged to be the same as the authentic lysozyme c by means of a number of criteria. First, the N-terminal sequence of the mature secreted protein is identical to that of the naturally occurring enzyme. Subsequently, Western blot analyzes of the secreted lysozyme c show a single immunoreactive species with the same molecular weight as the naturally occurring mature enzyme. Finally, in bioassays based on the ability of the secreted lysozyme c to lyse Micrococcus luteus (formerly Micrococcus lysodeikticus) cells, the protein secreted into the media from P. pastoris cells has a specific activity that is essentially the same as that for lysozyme c isolated from the sources in which it occurs naturally.

The surprising and advantageous result that an animal lysozyme c produced in accordance with the present invention is not contaminated with significant amounts of fragments of the secreted enzyme (or incorrectly processed protein with a molecular weight greater than that of the mature enzyme), has been established by electrophoresis and amino-terminal sequence analyses, which indicate the absence of such contaminating fragments or proteins larger than the mature enzyme.

The animal lysozyme c present in the media of a P. pastoris culture according to the invention can be easily purified by techniques well known in the art of protein purification, due to the high enzyme concentration and the low concentration of contaminating proteins and protein fragments in the media. A simple two-step cleaning procedure is described in the following examples.

The animal lysozymes c provided by means of the present invention can be used as is known within the state of the art for such lysozymes in general. E.g. can the lysozymes c be used for degradation of E. coli to isolate cloned, genetically engineered plasmids. See e.g. Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, US (1982). Like other lysozymes, the lysozymes c provided by the present invention can be used, either alone or in combination with other components, as ripening agents for cheese, as antimicrobial agents for cosmetics, detergents and various food products, as preservatives for certain food products, such as cheese, sausage and marine products, and as a means of treating microbial infections in animals, including humans. However, when lysozyme c is used in conjunction with other antibiotics, it is important to avoid aminoglycosidic antibiotics, such as neomycin B, gentamicin c^a, kanamycin A or dihydrostreptomycin, whose structures are related to the saccharide substrate for the enzyme. Other reported uses for the lysozymes c provided by the method according to the invention include the treatment of various pain conditions, allergies and inflammations, colitis, herpes zoster, rheumatic fever, rheumatoid arthritis, as well as in pediatrics (maternalization of bovine milk by adding lysozyme) . See e.g. Jolles et al., Mol. Cell. Biochem. 63, 165 (1984).

Another possible use for the lysozymes c provided by the present invention, particularly the bovine lysozymes c, is as an additive to feed for ruminants, to increase the efficiency with which such animals use the feed for growth. Rumen bacteria are an important source of nutrition for ruminants. An important factor in their applicability, however, is the animal's ability to break down the bacteria. As mentioned earlier, the latter are surrounded by peptidoglycans containing glycosidic bonds that can be cleaved by lysozyme. Consumption of a lysozyme c, as an additive to f6r, may result in an increased level of the lysozyme in the fundus region of the ruminant abomasum, which, in turn, may increase the amount of digested microbial mass entering such region of the stomach from the rumen . Increased digestion of this microbial mass can result in the ruminant having available an increased amount of bacterial content for energy production and growth.

The following examples describe and illustrate the present invention in more detail.

All patents and publications to which reference is made in the present application are incorporated by reference in the application.

EXAMPLE 1

ISOLATION AND CHARACTERIZATION OF A FULL-LENGTH cDNA CLONE ENCODING BOVINE LYSOZYME c

A partial bovine lysozyme c genomic clone, designated pLl, and its DNA sequence was obtained from Dr. Gino Cortopassi.

Fresh bovine abomasum tissue was obtained from Talone Meat Packing Co., Escondido, California, USA. XgtlO and E. coli strain C600HF1 were obtained from Clontech Labs, Inc. 4055 Fabian Way, Palo Alto, California 94303. The kit used for XgtlO was prepared according to Maniatis et al., supra. Restriction and DNA modification enzymes were obtained from Boehringer Mannheim Biochemicals, Inc.

(Indianapolis, Indiana), and New England Biolabs, Inc.

(Beverly, Massachusetts), and was used as recommended by the manufacturers.

After total intact RNA was isolated from approximately 20 g of bovine abomasum tissue, by modification of the method of Shields and Blobel, Proe. Nati. Acad. Pollock. USA 74:2059 (1977), polyadenylated RNA species were separated from non-polyadenylated species by a standard technique using an oligo-dT column.

As the fundus region of the ruminant abomasum contains a high concentration of pancreatic ribonuclease, speed and correct processing of the tissue, including maintaining a low temperature, is essential to preserve the RNA.

Light frozen bovine abomasum tissue was pulverized in a manual stainless steel pulverizer in the presence of liquid nitrogen. The powdered tissue was added to 100 ml proteinase K buffer (.14 M NaCl, .05 M Tris/7.4, .01 M EDTA/8.0, 1% sodium dodecyl sulfate (SDS)) containing 400 µg/ml proteinase K (Boehringer Mannheim Biochemicals, Inc.). The mixture was immediately shaken well and then incubated at room temperature for 15 minutes.

After incubation, the preparation was extracted with an equal volume of PCIA (phenol:chloroform:isoamyl alcohol, 25:24:1) followed by extraction with an equal volume of CIA (chloroform:isoamyl alcohol, 24:1). The obtained DNA/RNA mixture was precipitated by setting the NaCl concentration to 0.25 M, adding two volumes of ethanol, and the solution was placed at -20°C overnight. After centrifugation at 5000 x g for 60 min, the DNA/RNA was resuspended in 20 ml ETS buffer (0.1 M Tris, pH 7.6, 0.01 M EDTA, 0.2% SDS). PCIA and CIA extractions, and DNA/RNA precipitations were carried out as described above. The DNA/RNA precipitate was collected by centrifugation and resuspended in 32 ml of 10 mM Tris, 10 mM EDTA, pH 7.4.

After resuspension, the RNA present in the solution was enriched by dividing the preparation into four tubes, with a 4 ml CsCl (1 g CsCl/ml) substrate placed over each tube. The preparation was then centrifuged (Beckman SW41 rotor) at 37,000 x g for 20 hours. After centrifugation, the supernatant was carefully removed and the RNA pellets were resuspended in 8 ml of ETS buffer, ethanol-precipitated twice (by addition of NaCl to a final concentration of 0.25 NaCl followed by the addition of two volumes of 95% ethanol), and resuspended in 5 ml ETS buffer. Polyadenylated (poly(A)+) RNA was selected from the solution by affinity chromatography on oligodeoxythymidylated cellulose columns, as described by Aviv et al., Proe. Nati. Acad. Pollock. USA 69:1408 (1972). After the RNA was bound to the oligo-dT column in 0.5 M NETS buffer (0.5 M NaCl, 0.01 M Tris, 0.01 M EDTA and 0.2% SDS, pH 7.6), the column washed with 30 ml of 0.5 M NETS. This was followed by elution of polyadenylated RNA with 5 ml ETS buffer and ethanol precipitation.

Ten µg of the polyadenylated RNA was denatured in 2 mM Cl₂HgOH (Alpha Products, Danver, Mass.) for 5 min at room temperature. Following the procedure of Huynh et al., DNA Cloning: A Practical Approach (D. Glover, ed.), IRL Press, Oxford (1984), this RNA was subjected to a cDNA synthesis reaction and a cDNA library was developed from total polyA -mRNA and engineered into XgtlO (Gubler et al., Gene 25:263 (1983); Lapeyer et al., Gene 37:215 (1985)). MMLV reverse transcriptase was carefully used to generate a cDNA from the mRNA. This was followed by RNase H treatment and DNA polymerase I-mediated synthesis of the second cDNA strand to form a double-stranded cDNA. After synthesis of the second strand, the cDNA was blunt-ended with S]_ nuclease and the ends were further treated with E. coli DNA polymerase I Klenow fragment (Telford et al., Proe. Nati. Acad. Sei. 76:2590

(1979)). EcoRI adapters (Wood et al., Nature 312:330 (1984)) were ligated to the double-stranded cDNA allowing

the need for methylation and subsequent EcoRI cutting of cDNA is avoided. Excess adapters were removed by chromatography on a Sepharose CL-4B column equilibrated with 10 mM Tris, pH 7.4, 1 mM EDTA. The column fractions containing cDNA<1>s with more than 400 bp were collected and ligated into the cDNA bacteriophage cloning vector Xgt10, which had been EcoRI-cut and phosphatase-treated. Ligations were carried out in a volume of 5 µl and incubated at 16°C for 18 hours. The reaction mixture contained lyg XgtlO vector (final concentration = 200 µg/ml) and 50-100 ng of cDNA (2- to 4-fold molar excess of insert relative to vector).

The obtained cDNA was packaged, using a lambda packaging kit, and the obtained vectors were plated on E. coli strain C600HF1, and plaques were screened with radioactively labeled pL1. The plaques identified as containing cDNA encoding at least a portion of bovine lysozyme c were plaque-purified (Maniatis et al., supra) and screened with both pL1 and an oligonucleotide probe synthesized from the DNA sequence information provided by Dr. Cortopassi. The sequence of such a probe is the sequence of a single strand of a DNA segment at the 5' end of exon 2 of the bovine lysozyme c gene, which comprises sequences coding for amino acids 29-38 of mature bovine lysozyme C2> and is as following:

Plaque transfers were performed essentially as described by Benton et al., Science 196:180 (1977). Nitrocellulose filters were prehybridized for 4 h at 42°C in 5 x SSPE (0.9 M NaCl, 0.04 M NaoH, 0.05 M NaH 2 PO 4 -H 2 O, 0.00 5 M EDTA, pH 7.0), 5 x Denhardt's [50 x Denhardt's: 5 g Ficoll 400 (Pharmacia, Inc., Piscataway, New Jersey, catalog no. 17-0400-01, average molecular weight approximately 400,000 daltons), 5 g polyvinylpyrrolidone PVP-360 (Sigma Chemical Co. , St. Louis, Missouri, average molecular weight approximately 360,000 daltons), 5 g bovine serum albumin in H2O to bring the volume to 500 ml], 50%, (v/v) formamide, 0.2% (w/v) SDS and 200 µg/ml sheared herring sperm DNA (Boehringer Mannheim Biochemicals, Inc.). After the nick-translated probe pL1 was added directly to the prehybridization solution at 1x10<6>cpm/ml, the filters were hybridized for 16 hours at 42°C and then washed several times, for 15 minutes each. wash, in 0.1 x SSPE, 0.1% (w/v) SDS at 65°C, and autoradiographed. For oligonucleotide screening, the filters were prehybridized in 6 x SSPE, 5 x Denhardt's, 25% formamide, 0.2% (w/v) SDS and 200 µg/ml herring sperm DNA for 2 h at 42°C. They were then hybridized for 3 hours in the same buffer at 42°C and washed several times in 1x SSPE at 45°C.

One of the plaque-purified clones that hybridized to the oligonucleotide was designated XBL3, and the size of its inserted cDNA was determined, after EcoRI restriction enzyme cutting of phage "mini-preps" (Benson et al., Biotechniques, 23, 126 (1984 )), to be 950 bp.

The nucleotide sequence of the cDNA insert of clone XBL3 was determined using the subclone M13 template and the dideoxynucleotide protocol described by Sanger et al., Proe. Nati. Acad. Sei., USA 74:5463 (1977) and the M13 Cloning/Dideoxy Sequencing Manual, Bethesda Research Laboratories, Inc., Gaithersburg, Maryland (1980) to be:

The results indicated that the XBL3 cDNA insert consists of a coding region of 436 bp of bovine lysozyme c2 and 482 bp of 3'-untranslated non-coding sequence. The 3<1> non-coding sequence in XBL3 does not contain a polyadenylation signal or a poly (A)<+> tail. DNA sequencing of the 5' terminus of the cDNA insert in XBL3 indicated that the insert contains 450 bp encoding the C-terminal part of the protein signal sequence, but does not contain the ATG triplet corresponding to the translation initiation codon of pre-lysozyme c2 mRNA. Thus, the cDNA insert codes for sixteen amino acids amino-terminal to the amino-terminal end of the mature protein.

The amino acid sequence of the protein corresponding to the cDNA insert of X-BL3, derived from the nucleotide sequence, and indicated in the preceding sequence below the nucleotide sequence, has a histidine at amino acid position 98 in the mature protein, while the published protein sequence (Jolles et al., J. Biol. Chem. 259, 11617 (1984)) indicates a lysine at this position. Presence of the histidine residue at this position was confirmed by N-terminal analysis of the C-terminal CNBr fragment of the protein expressed from the insert. Furthermore, sequencing of the lysozyme c2 from bovine abomasum tissue showed that the amino acid in position 98 is actually histidine, at least in the animal used as RNA source in this work.

Subsequent sequencing of bovine lysozyme mRNA was performed using the variations of the dideoxy sequencing protocol recommended in Karanthanasis, Bethesda Res. Lab. Focus 4:3, 6 (1982)

(Bethesda Research Laboratories, Inc.). Four µg of abomasum poly (A)<+>RNA was used as a template and a synthetic 27-residue oligonucleotide with sequence

which was copied from the DNA sequence at the 5' end of the bovine lysozyme gene and which was synthesized using standard phosphoramidite chemistry on an Applied Biosystems synthesizer (Model 380A), was used to initiate the extension by reverse transcriptase. The sequencing of the mRNA showed that the XBL3 insert was missing 5 bp of the coding sequence at its 5' end, including an initiation ATG codon. In addition, the RNA sequence indicated that the base at position 2 of the XBL3 insert should be a G rather than an A. Such sequencing further revealed the amino acid sequence of the amino-terminal end of the bovine lysozyme c signal sequence, including the position of the initiating methionine. One such signal sequence was found to be the following: MKALVILGFLFLSVAVQG. By comparison with known sequences (Jolles et al., supra), the bovine lysozyme c encoded by the cDNA insert of clone XBL3 is very similar to bovine lysozyme c2, the most abundant of the three bovine lysozyme c. The only difference between this and the sequence for bovine lysozyme c2 reported by Jolles et al. is a lysine to histidine change at position 98 of the mature protein, as discussed above. A greater number of amino acid changes and/or a difference in the total number of amino acids excludes the possibility that the protein coded for by the cDNA insert in XBL3 is lysozyme cl or c3. The simple, conservative amino acid change between the lysozyme encoded by XBL3 and bovine lysozyme c2, as reported by Jolles et al., which results from two nucleotide changes, can most likely be explained by allelic differences. In any case, as described in the following examples, lysozyme c2 with histidine in position 9 8 has essentially the same properties as the lysozyme c2 isolated from bovine abomasum tissue.

EXAMPLE 2

CONSTRUCTION OF EXPRESSION VECTORS

Two in vitro M13 mutagenesis procedures were performed, one at each of the 5' and 3' ends of the bovine pre-lysozyme c2 coding region of XBL3, prior to insertion of the coding region into the Pichia pastoris expression vector pAO804 (described below), to modify the coding region to make the region compatible with the expression vector and to make certain other desired changes. The 3' end of the coding region was mutagenized to remove the 482 bp of non-coding sequence and to introduce an AsuII restriction site (5'-TTCGAA-3'), and an EcoRI restriction site (5'-GAATTC -3'), immediately 3' to the TAA triplet encoding the translational stop codon. The 5' end of the insert, in a clone (designated pBL4C) identified as having these changes at the 3' end, was mutagenized to introduce a seven base pair sequence (5'-ATGAAGG-3') which is the correct sequence as indicated by RNA sequencing. This modification at the 5' end complemented the coding sequence for the N-terminus of the signal sequence (Met-Lys-Ala...) and added another EcoRI restriction site directly before the Met codon encoding ATG. Clone pBLI6C was identified as having both of the desired mutagenized ends.

In the first in vitro mutagenesis procedure, a mutagenic oligomer of sequence 5'-dGAGCTGAAGAATGATATTACAGGGTGCAACCC-3' was synthesized and used as a primer for mutagenesis of the 3' end of the bovine lysozyme gene insert in XBL3 and for screening. Four of the positive plaques from another hybridization screen were used to prepare template DNA for sequencing. One of the template DNAs, pBL4C, was found to contain the correct sequence, with the EcoRI site 3' of the TAA encoding the translation termination codon.

Another mutagenic oligomer, with sequence

was used to initiate from pBL4c template DNA for the second mutagenesis. For this modification, a synthetic oligonucleotide with sequence 5'-dTAACGAGAGCCTTCATGAATTC-3' was used for screening. A clone pBLI6C, which was identified as having the desired sequence, comprising the EcoRI site, the ATG triplet encoding the translation initiation codon and

the sequence specifying the next two amino acids, lys and ala, at the 5' end was used to form the template DNA.

Base pair matching of mutagenic oligonucleotides, initiator extensions and alkaline sucrose gradient separations were carried out using the procedures described in Zoller et al., Methods in Enzymology, Academic Press, New York (1983). Screening for positives was performed using solution hybridization and electrophoresis through agarose gels, as described by Hobden et al., Anal. Biochem. 144:75 (1985).

The generalized Pichia pastoris expression cassette vector pA0804, illustrated in FIG. 1, has two Bglll seats which delimit a fragment which has, moving clockwise in fig. 1 from the Bglll site approximately 100 bp from a ClaI site, (1) a fragment of approximately 900 base pairs (bp) (designated "5'-AOXl" in the figures) which is a "promoter segment" in accordance with the invention and is from the P. pastoris major alcohol oxidase (AOX1) gene site, including the promoter and transcription initiation site, and ending in an EcoRI linker, which was added immediately upstream of the translation initiation codon of the AOX1 gene product (Ellis et al., Mol. Cell. Biol. 5:1111 (1985)) and which provides the only EcoRI site in pAO804; (2) a fragment of approximately 300 bp (designated "3'-AOX1" in the figures), which is a "terminator segment" in accordance with the invention and is from the P. pastoris AOX1 gene, said fragment having the EcoRI linker on the 5' end, and the ClaI site at the 3' end, and including the polyadenylation signal and site coding segments and the transcription terminator of the AOX1 gene; (3) a Bglll fragment of approximately 2700 bp (in the orientation as indicated in Fig. 1) encompassing the P. pastoris histidinol dehydrogenase (HIS4) gene, to provide a selectable marker for His4 strains of P. pastoris (Cregg et al., supra), said fragment being inserted into the BamHI site of pBR322 used to generate pAO804; and (4) an approximately 800 bp fragment (designated "3'-from-AOX1" in the figures) of 3' sequence taken downstream from the transcriptional terminator of the P. pastoris AOX1 gene site, said fragment terminating at one end with a Bglll site (formed by modification of pBR322 at its PvuII site) and at the other end with the residue from combining an XhoI site with the pBR322 SalI site. pAO804 also includes a number of fragments from pBR322: (1) the approximately 350 bp segment from the ClaI site at the end of the fragment labeled "3-AOX1" to the remainder of the BamHI site at one end of the segment with the P. pastoris HIS4 gene; (2) the approximately 280 base pairs from the remainder of the BamHI segment at the other end of the segment with the P. pastoris HIS4 gene to the remainder of the Sali site at one end of the "3'-from-AOX1" segment; and (3) the approximately 2320 base pair fragment from the remainder of the pBR322 PvuII site (not shown in the figures) within a few bases of the Bglll site at one end of the "3<1->from-AOXl" segment (and outside this segment) to The Clal site near the Bglll site on one end of the "5'-AOXl" segment. The 2320 bp pBR322 segment has been modified to eliminate the pBR322 EcoRI site and includes the pBR322 origin of replication and the beta-lactamase gene (conferring ampicillin resistance in bacteria transformed with the plasmid).

Construction of pAO804 is described in Example 15.

■ An "expression unit" bound at the Bglll sites (or, e.g., the ClaI-Bglll fragment that includes the fragment bound at the Bglll sites) shown for pAO804 in Figure 1 is formed by inserting, at the EcoRI site, an intron-free DNA segment whose transcript, together with the polyadenylation signal and the site provided by the 3'-AOX1 segment from the AOX1 promoter, will be translated into the protein of interest.

The Bglii-EcoRI segment of approximately 900 bp, comprising the A0X1 promoter and the transcription start site, and the genome segment of approximately 800 bp from 3' of the AOX gene ("3'-A0Xl" in the figures) are used for site-directed integration of the Bglll -site-terminated expression unit into the AOX1 site in P. pastoris cells transformed with the plasmid.

With the bovine pre-lysozyme c2 coding fragment bound at EcoRI sites from pBLI6C inserted into the unique EcoRI site of pAO804 in such a way that the lysozyme sequence is oriented operatively for transcription from the A0X1 promoter, pre-lysozyme c2 will be expressed, under the transcriptional control of the A0X1 promoter, when P. pastoris cells transformed with the plasmid

(or the Bglll site-terminated expression unit thereof)

grown with methanol as a carbon source so that the promoter is active in transcription.

Studies of P. pastoris have shown that certain methanol-regulated promoters from P. pastoris, such as that of the major alcohol oxidase gene (A0X1) or the p76 gene (dihydroxyacetone synthase (DAS), European patent application publication number 0 183 071) , are particularly favorable for heterologous gene expression in industrial-scale processes. Such promoters provide transcription at a high level, even in single copy, and are tightly regulated, thus allowing the period of expression to be limited. In ethanol-, glycerol-, or glucose-grown wild-type cells, alcohol oxidase is absent. However, it constitutes as much as 30% of total cell protein in cells grown on methanol.

A0X1 "deletion" mutants produce approximately 15% wild-type alcohol oxidase enzyme activity (from the minor alcohol oxidase (AOX2) gene), while AOX2 "deletion" strains produce wild-type levels of alcohol oxidase when cells are grown on methanol.

The P. pastoris AOX1 promoter is among the strongest and most tightly regulated known promoters. When P. pastoris cells are grown on glucose or glycerol, the AOX1 promoter is repressed and alcohol oxidase mRNA is not formed and alcohol oxidase is not expressed. In contrast, when such cells are grown on methanol, the AOX1 promoter is induced and the alcohol oxidase expressed from the AOX1 gene can account for as much as 30% of total cell protein under certain conditions. The AOX1 gene, including its promoters, has been isolated and extensively characterized. Studies indicate that regulation of A0X1 occurs at the transcriptional level and that, when the gene is transcribed, A0X1 mRNA is the predominant species in the "steady state" mRNA.

Although the P. pastoris A0X2 gene is also methanol-regulated, it is not as strongly transcribed as A0X1. Accordingly, although able to grow on methanol, A0X1-deficient mutants, which are A0X2 normal, have a significantly longer growth time on methanol than wild-type strains.

Double-stranded DNA from the replicative form of pBLI6C was cut with EcoRI, and the resulting, approximately 460 bp, EcoRI fragment was ligated into EcoRI-cut and alkaline phosphatase (Boehringer Mannheim Biochemicals, Inc.)-treated pAO804 DNA. One of the two resulting expression plasmids, designated pSL12A and shown in FIG. 2, has the correct 5' to 3' orientation of the bovine lysozyme coding sequence regarding the position and orientation of the AOX1 promoter and the transcription start site in the "5'AOX1" promoter segment and the "3'-AOX1" terminator segment .

EXAMPLE 3

TRANSFORMATION OF GS115 CELLS WITH pSL12A; STRAIN A37

Pichia Pastoris strain GS115 ((His4~); Cregg et al., supra), a histidine-requiring auxotrope of P. pastoris, previously determined to be deficient in histidinol dehydrogenase (His4) and to give a reversion frequency to histidine prototropy of less than 10<->^, was used as the gene expression host for transformation with pSL12A. P. pastoris GS115 is grown efficiently on methanol in a defined minimal medium supplemented with histidine, to a high cell density and is desirable as a host system for single cell protein production. The strain contains no bacterial replicons, antibiotic resistant genes or other heterogeneous sequences that could be considered a potential biological hazard. Plasmid pSL12A was cut with restriction endonuclease BglII and the resulting mixture of DNA fragments was used to transform P. pastoris strain GS115 using the whole-cell LiCl yeast transformation system. Details of the transformation procedure are given below.

Cutting pSL12A with BglII releases a DNA fragment with ends homologous to regions 5' and 3' of the P. pastoris AOX1 gene, as described above. When this fragment is transformed into a His4" P. pastoris strain and maintained there under selective conditions (weighted in the absence of histidine), a replacement-type integration is performed at the AOX1 site in the Bglll site-terminated, expression cassette-containing fragment (including the His4 gene) in some cells.

This integration results in cells with a phenotype designated Mut<+>/~ (for "methanol utilization +/-"), which is due to loss of the A0X1 gene product but retention of the minor alcohol oxidase gene product (A0X2), and allows slow growth on methanol. This one-step gene replacement technique, which results in chromosomal integration of the heterologous gene, avoids difficulties associated with plasmid instability, distribution and copy number and provides a highly accurate expression system. The technique also results in the incorporation of a minimal amount of heterologous DNA into the P. pastoris genome. Cells carrying the correct integration will be His<+> and can be distinguished, by means of a reduced growth rate on methanol, from cells in which the integration has occurred at sites other than the A0X1 site. In cells in which integration has not occurred, or in which integration has not occurred at the A0X1 site, the A0X1 gene remains functional and the ability of such cells to grow on methanol is not impaired.

A whole-cell lithium chloride yeast transformation system, modified from that described for Saccharomyces cerevisiae (Ito et al., Agric. Biol. Chem. 48:341 (1984)), was used to introduce the linear DNA fragments into in GS115. As such a method does not require the formation and maintenance of speroplasts, it is more suitable and less time-consuming than the speroplast method. The Speroplast technique which is exactly as described in Cregg et al., Mol. Cell. Biol. 5.3376 (1985) with the one exception that the regeneration agar does not contain sorbital, but contains 0.6 M KCl instead, and which can also be used to transform the P. pastoris cells, is however preferred in that such a method gives a larger number of transformants.

A 50 ml shake bottle culture of P. pastoris strain GS115 was grown in YPD (10 g Bacto yeast extract, 20 g Bacto peptone, 20 g dextrose, 20 g Bacto agar, 1000 ml distilled water) at 30°C with shaking to an OD of approximately 1.0 (5 x 10<7>

b U U

cells/ml). The density at harvest can be from 0.1 to 2.0 OD600 units- After the cells were washed once in 10 ml of sterile H2O, and after pelleting by centrifugation at approximately 1600 x g for 3-5 min, the cells were pelleted again by the same procedure and then washed once in 10 ml of sterile TE buffer (10 mM Tris-HCl, pH 7.4, 1 mM EDTA). The cells were resuspended in 20 ml of sterile lithium chloride + TE buffer (0.1 M LiCl, 10 mM Tris-HCl, pH 7.4, 1 mM EDTA) and incubated at 30°C, with random shaking, for one hour.

A sterile polypropylene tube of 12 x 75 mm, containing 10 µg of Haelll-cut E. coli carrier DNA, 2 µg of Bglll-cut DNA from plasmid pSL12A and 0.1 ml of competent P. pastoris cells in TE buffer with LiCl was incubated in a water bath at 30°C for 30 min. About 0.1-20 µg of vector DNA can be used to transform the cells. 0.7 ml of 40% polyethylene glycol (PEG-^-^q, Fischer Scientific, Fair Lawn, New Jersey) in lithium chloride + TE buffer was added, and the tube was gently vortexed and incubated again in a water bath for 30 min. at 30°C. After the cells had received a heat shock for 5 min. at 37°C, the mixture was centrifuged and then resuspended in 0.1 ml of sterile H 2 O. The cells were then spread on selective plates (0.67% yeast-nitrogen base, without amino acids, 2% glucose, 2% agar) and then incubated for 3 days at 30°C. Transformants with the Mut+^ phenotype were identified as follows: His<+> transformants were plated to obtain colonies derived from single cells. Individual colonies were transferred to master plates with minimal glucose (2%). After overnight incubation at 30°C, the master plates were replica-transferred to minimal low glucose (0.2%) plates and again incubated overnight at 30°C. The following day, the low glucose plates were replica transferred to minimal methanol plates (0.5%) and then incubated at room temperature for 2 to 5 days. Colonies showing visible growth were labeled as Mut<+> and those without visible growth were labeled as Mut+// .

Transformant colonies were recovered from the surface of the agar plates by adding 5 ml of sterile H2O to the plate and suspending the cells with a spreader, after which, after dilution, the cells were sonicated and plated to obtain colonies of single cells. The sonication step was necessary to separate P. pastoris cells, as such cells tend to grow in clumps.

About 20% of the obtained 276 His<+>transformants grew slowly on methanol and consequently revealed the above Mut"1"^ phenotype. Southern blot analyzes of 9 Mut<+/>transformants showed identical hybridization traces and confirmed that the predicted integration into the AOX1 site had occurred in the cells. One of these AOX1<_> transformants, designated A37, was selected for further analysis.

Seven of the Mut+^~ strains were grown in 100 ml shake flask cultures. Six of the strains contained the lysozyme gene in the correct orientation and one strain had the gene in the opposite orientation. Shake flask cultures were first grown in 0.67% yeast-nitrogen base (Difco Labs, Detroit, Michigan) and 2% glycerol as the sole carbon and energy source at 30°C and with shaking for approximately 1 day. After centrifugation and washing of the cell pellet in sterile water, the cells were transformed into media containing 0.5% methanol, instead of glycerol, as the sole carbon source. Incubation at 30°C with shaking was then continued and the samples were harvested after 1 and 4 days of growth in methanol.

After the cells from the seven Mut+/^ strains were removed from the samples by sterile filtration, 200 µg of each medium sample was applied to a nitrocellulose filter using a slit-printer. The nitrocellulose filter was processed by a standard Western blotting procedure, using a 1:2500 dilution of antiserum from rabbit #156 (described in Example 6).

The results of the Western blotting experiment, after application of 200 µl medium samples from strains after growth in glycerol and from strains after 1 and 4 days in methanol, as well as dilutions of a purified bovine lysozyme c2 standard, were as follows : (1) cells with the bovine pre-lysozyme c2 insert in the incorrect orientation and cells grown in glycerol secreted no detectable bovine lysozyme; (2) significant amounts of bovine lysozyme c2 were secreted into the media by the cells that had the bovine lysozyme insert in the correct orientation and that were grown in methanol; (3) with respect to the cells described in (2), the lysozyme concentration in the media increased tenfold between 1 day of cell growth in methanol and 4 days of growth in methanol (lysozyme levels of approximately 125 µg secreted/ml were measured on day 1 while lysozyme levels of approximately 1.25 mg secreted/ml were measured on day 4); (4) there was very little interspecific variation in the secretion levels among the strains with the lysozyme coding segment in the correct orientation.

EXAMPLE 4

SCALE-UP OF THE PRODUCTION OF PICHIA PASTORIS STRAIN A37 FROM SHAKE-BOTTLE CULTURES TO LARGE FERMENTATION CULTURES AND CHARACTERIZATION OF THE FERMENTATION PRODUCT

As mentioned earlier, the transcription from the alcohol oxidase promoter is strongly repressed by growth in glycerol and induced by growth in methanol. In fermentation cultures, transformants carrying expression cassettes in AOX1 are instead grown in a simple 2-stage production system. In the first step, cell mass is developed in the absence of expression of the heterologous gene using glycerol as a carbon source. When glycerol is used up, a methanol supply is initiated. As strains lacking A0X1 grow slowly on methanol, and the doubling time is about ten times longer on methanol compared to wild-type strains, there is a longer production phase of 30 to 200 hours with a minimum of cell division. By growing an expression host initially on repressive carbon sources, such as glucose or glycerol, a large cell mass can be formed without significant selection for mutants lacking heterologous gene expression. Then, by allowing the cells to use the repressive carbon source and adding methanol, a high level of heterologous gene expression can be initiated.

For large-scale production of bovine lysozyme c2 from GS115 strain A37, a two-stage host fermentation system with high cell density was used. In the first step or growth step, A37 was grown in a defined minimal medium with glycerol as a carbon source. The desired cell density to be achieved was established by adjusting the initial glycerol concentration in the medium. When glycerol was used up, the culture, then at the desired density, was transferred to the second stage or production stage by adding methanol.

Fermentation cultures were grown in an inorganic salt-based medium prepared from commercially supplied analytical reagents consisting of the following: inorganic salts in final concentrations of 0.3 M H 3 PO 4 , 4.4 mM CaSO 4 -2H 2 O, 65 mM K 2 SO 4 and 38 mM MgSO 4 ; trace salts at final concentrations of 0.4 uM CuS04-5H2, 2.5 uM Kl, 9.0 uM MnS04-H20, 4.0 p Na2Mo04-2H20, 1.5 uM H3BO3, 35 uM ZnS04~7H20 and 89 uM FeCl3 <->6H2O, prepared as a 200x stock solution and filter sterilized; 2 mg/ml biotin; and either 2% glycerol for the 2 liter New Brunswick Bioflo fermenter or 6% glycerol for the 15 liter Biolafitte fermenter. NH 3 was added to adjust the pH of the medium to 5.5 before inoculation and to maintain this pH during the fermentation. When glycerol was used up, a methanol feed was initiated to maintain the methanol concentration between 0.2 and 1.0%. To prevent a lack of trace salts during the fermentations, a new solution of trace salts was added to the cultures at regular intervals.

Results from a series of 2 liter and 15 liter (1 liter and 10 liter working volume) fermentation experiments with strain A37 indicated that strain A37 is clearly capable of maintaining a high production and secretion level of bovine lysozyme c2 when cultured for 5 to 7 days on methanol in 2 liter and 15 liter cultures. In fermentation run #182, bovine lysozyme c2 concentrations of approximately 200 mg/l were measured in the cell-free fermentation medium, at an OD60Q_<0>76.

In another case, a cell-free fermentation medium from fermentation run No. 207 was cultured for 7 days on methanol to a cell concentration of OD,- =195, measured by

0 u u

using radioimmunoassay (RIA), as described in Example 6, to have 200 mg/l bovine lysozyme c2. The specific production capacity measured for fermentation-grown cultures was within the range observed for A37 cells grown in 100 ml shake-flask cultures. Thus, there was no loss of cell production capacity in upscaling to 21 and 151 fermenters.

A medium sample of 124 ml from fermentation run No. 182 was adjusted with concentrated acetic acid to pH=4 and placed in a water bath at 100°C for 3 to 5 min. After a second adjustment to pH=5 with NH 4 OH, the bovine lysozyme c2 was purified on a cation exchange resin (Whatman CM-52), as described by Dobson et al., J. Biol. Chem. 259:11607 (1984); 12.5% denaturing polyacrylamide gels indicated a purity of at least 95%. The stained gel indicated that bovine lysozyme c2 comprised approximately 50% of the protein present in the fermentation medium and that all other proteins were present in very small amounts relative to bovine lysozyme c2. Moreover, the recombinant bovine lysozyme c2 and the native bovine lysozyme c2 appear to comigrate precisely. From the stained gel of the cell extract, no band corresponding to bovine lysozyme c2 was evident.

A purified sample of recombinant bovine lysozyme c2 (250 pmol) secreted from P. pastoris A37 grown in fermentation run #182 was analyzed by automated, N-terminal sequence analysis (Applied Biosystems 470A, Foster City, California). The sequence, which was verified through fourteen cycles, with very little accompanying background, showed that more than 88% of the partially purified bovine lysozyme c2 was correctly processed at the junction between the signal sequence and the mature protein. The first fourteen amino acids of the recombinant material were identical to the first fourteen amino acids of the published protein sequence of the mature enzyme.

Brief exposure of Western blots of Laemmli gels of a sample of secreted bovine lysozyme c2 taken from fermentation run No. 250, a 10 liter fermentation of strain A37, showed a single immunoreactive species with the correct molecular weight. In the soluble and insoluble cell fractions, an immunoreactive band migrating to the same position as the standard was seen, although in a much smaller amount than in the medium. Material that migrated to the same extent was not seen from control Aoxl<+/>^ cells that carried no expression cassette.

A number of other runs tested the effect of restricted cell growth on secretion of bovine lysozum c2. Limiting amounts (2.5 ml/l, 5 ml/l and 10 ml/l) of trace salts were used respectively. passages No. 239, No. 240 and No. 241, to limit cell growth. The curve for lysozyme secreted in the media was parallel to the cell growth curve. Based on these data, it appears that accumulation of bovine lysozyme c2 i

the medium is associated with vigorous cell growth.

EXAMPLE 5

CHARACTERIZATION AND QUANTIFICATION OF RECOMBINANT BOVINE LYSOZYME c2 PRODUCTION BY POLYACRYLAMIDE GEL ELECTROPHORESIS OF A FERMENTATION SAMPLE

Parallel runs were performed on 15% polyacrylamide-Laemmli gels (Laemmli, Nature 227:680 (1970)). One gel was stained with silver stain and the other was treated as an immunoprint. Runs were performed on both gels with (1) bovine lysozyme c2 purified from bovine gastric mucosa; (2) bovine lysozyme c2 purified, as described in Example 9, from the media of an A0X1<+>lysozyme-secreting strain; and (3) cell extract and media from a 10 liter mid-pass fermenter of P. pastoris strain A37 (passes. 250). For media samples, cells with a density of 308 g/cells/l and which had been on methanol for 111.5 hours were purified by low speed centrifugation. Cell extracts were prepared by lysing the cells using glass beads in a buffer consisting of 10 mM sodium phosphate, pH 7.5, 500 mM sodium chloride, 0.1% Triton X-100, and 2 mM phenylmethylsulfonyl fluoride (PMSF). With respect to the media samples and cell extract samples, the amount of cell extract applied to the stained gel was volumetrically equivalent to 0.1 times the amount of medium added. For the immunoprint, cell extract in equal amounts and 0.1 times the amount of medium was applied. Antiserum for the immunoprint was prepared, as described in Example 6, in rabbits against the bovine lysozyme purified from the abomasum and used in a dilution of 1:2500.

From the gels, it was evident that native and recombinant bovine lysozyme c2 comigrated, that bovine lysozyme c2 comprises at least 50% of the protein in crude fermentation medium, and that no significant amounts of lysozyme degradation products were

present.

Relative amounts of bovine lysozyme c2 in the cell extract and in the culture medium can be estimated from the immunoprint on which volumetrically equivalent samples based on cell density were applied. Although it was impossible to calculate how efficient the secretion was from these data, which is due to the constant accumulation of bovine lysozyme c2 in the fermenter, it appeared, from these data, and from concentration data generated by means of RIA, as described in example 6, that less than 5% of the bovine lysozyme c2 formed by the cell remained in the cell.

EXAMPLE 6

IMMUNOASSAYS FOR BOVINE LYSOZYME C2

Bovine lysozyme c2 was purified according to the procedure described by Dobson et al. supra, from 25 g of frozen bovine abomasum tissue. As the starting material was 15 times smaller than that used by Dobson et al., the column size and flow rate were resp. discounted. As reported by Dobson et al., three distinct peaks corresponding to the bovine gastric lysozymes c1, c2 and c3 were evident by absorbance at 280 nm after column chromatography on Whatman CM5 2.

Each of the three bovine gastric lysozyme peaks contained lysozyme activity, as measured in a spectrophotometric assay, described in Example 7, and approximately 2 mg of protein. The purity of the bovine lysozyme c2 protein was estimated to be greater than 95% by polyacrylamide gel electrophoresis (Laemmli, supra) on a 15% polyacrylamide gel.

All experimental results rely on quantification with a sample of the purified bovine lysozyme dialyzed against 1 x PBS and stored at -20°C. For the results described below, the standard is quantified using the published extinction coefficient for a 1% solution measured at 280 E=28.2 (Jolles et al., Mol. and Cell.Biochem, 63 165

(1984)).

Antisera raised in rabbits against bovine lysozyme c2 were prepared using standard procedures. Briefly, two young male New Zealand white rabbits were each immunized with 250 µg bovine lysozyme c2, purified as described above and dialyzed against PBS prior to the injections. Each of the rabbits was again given an injection with 20 µg of lysozyme c2 30 days later and given a second injection 10 days later with a further 100 µg of protein. While the initial immunizations were performed with lysozyme c2 emulsified with Freund's complete adjuvant (Difco Labs, Detroit, Michigan), Freund's incomplete adjuvant (Difco Labs, Detroit, Michigan) was used for the new injections. Blood samples were taken from the rabbits to obtain antisera one week after the last additional injection.

Antisera from both rabbits were tested by placing different amounts of native and denatured purified bovine lysozyme c2 and bovine serum albumin (Sigma Chemical Co., St. Louis, Missouri) in phosphate-buffered saline, and binding to nitrocellulose filters that had been incubated at various dilutions of the antisera, using a "slit-print" apparatus (Schleicher and Schuell, Inc., Keene, New Hampshire), and using a standard immunoprinting procedure (Towbin et al., J. Proe. Nat. Acad. Sci USA 76:4350 (1979)). The buffer used for filter-

125

blocking and washing, and the antibody and I-protein A dilution, was composed of 1 x PBS (140 mM sodium chloride, 3 mM potassium chloride, 10 mM disodium phosphate, 2 mM monopotassium phosphate, pH 7.2), 0, 25% gelatin (Bio-Rad, Inc. Richmond, Calif.), 0.05% Tween-20 (Sigma Chemical Co., St. Louis, Missouri), and 0.02% sodium azide (Sigma Chemical Co., St. Louis, Missouri). Serum from a rabbit, designated No. 156, was chosen because of the substantially lower cross-

reactivity of this sera with bovine serum albumin. Using a 1:2500 dilution of the serum from rabbit #156, 2.5 ng of the lysozyme standard was readily detectable on a slit-print, and 25 ng gave a very strong signal.

Radio-iodination of lysozyme, for use as a marker in RIA,

TM

was carried out using the IODOBEAD method. Briefly, it became a lyser (Pierce Chemical Co., Rockville, Illinois)

125 added to a reaction mixture containing 1 mCi Na I (New England Nuclear, Boston, Massachucetts), 50 µg of purified lysozyme c2 in 50 µl, and 10 µl of 0.5 M sodium phosphate buffer, pH 7.3, and incubated in 30 min. on ice. Then, the contents of the reaction tube were transferred to a prepacked G-25 desalting column (PD-10 from Pharmacia, Piscataway, New Jersey) and eluted with 50 ml of 0.05 M sodium phosphate buffer, pH 7.3, containing 0, 05% crystalline bovine serum albumin. The fractions were counted in a Micromedic Gamma Counter, and

125

the position of I-lysozyme was determined. The iodinated preparation was tested by precipitation in 10% trichloroacetic acid (TCA)

125

to estimate the proportion of intact peptide. Typically, the I-lysozyme preparations used in RIA were more than 98% TCA precipitate-bar.

The RIAs by which the concentration of bovine lysozyme c2 in P. pastoris culture samples was determined were carried out by a standard procedure involving the incubation of varying amounts of standard or unknown samples with a final dilution of 1:25,000 of rabbit anti-lysozyme antibody,

125

10,000-20,000 pulses of I-lysozyme in a final volume of 500 µl test buffer (50 ml sodium phosphate, 0.1 M NaCl, 25 mM EDTA, 0.1% sodium azide, 0.1% bovine serum albumin (Fraction V), 0 .1% Triton X-100, pH 7.4). After overnight incubation at 4°C, 100 µl of a 1:40 dilution of Pansorbinv (R)'

(Calbiochem, San Diego, California) suspension of S. aureus cells coated with protein A added and incubated for 15 min. at room temperature. The tubes were centrifuged for 60 min. at 23 60 x g after addition of 2 ml of an ice-cold wash buffer (0.9% NaCl, 5 mM EDTA and 0.1% Triton X-100). The supernatant was decanted

125

and the pellets were counted for I-lysozyme. Below these

125

conditions, approximately 50% of total I-lysozyme was recovered in the pellet in the reference tube and 10% in the non-specific binding tube, i.e. in the presence of excess unlabelled lysozyme. The sensitivity range for the experiment was 0.2-20 ng with an EDcb n U of approximately 2 ng. The unknown samples were measured by three dilutions of each sample, in duplicate.

EXAMPLE 7

BIOASSAYS FOR SECRETED BOVINE LYSOZYME c2

Microcuccus luteus cells were obtained from Sigma Chemical Company, St. Louis, Missouri.

Two types of bioassays (spectrophotometric assay and halo assay) were used to analyze the bioactivity of the secreted bovine lysozyme c2 (Grosswics et al., Meth. Biochem. Analys. 29:435 (1983)). Each experiment measured the ability of the lysozyme to lyse Microcuccus luteus cells. Activity tests were carried out at pH 5.0, rather than at pH 7.0, which is standard for egg white lysozyme, due to the varying pH optimum for the enzymes.

Halo assay is an in vitro assay that results in a halo of lysis of Micrococcus luteus cells on agar plates. Using the halo assay, 10 µl samples were added to 2 mm wells punched in an agarose plate consisting of 1% agarose and 1.2 mg/ml dried M. luteus cells in 0.1 M phosphate-buffered saline. is, pH 5.0. The samples can be impure medium, lysozyme purified from medium or lysozyme standard. The diameters of the resulting halos were measured after a 16 hour incubation at room temperature and quantified by comparison with a semilog plot of halo diameter versus amount, derived using the lysozyme standard (bovine lysozyme c2 purified from bovine abomasum tissue). The plot was linear from 100 ng to 10 ug. The lower sensitivity limit of the assay is approximately 50 ng and, using this assay, the minimum detectable concentration was 5 mg/l purified bovine lysozyme c2.

In the second experiment, a spectrophotometric assay measuring in vitro lysis of dried M. luteus cells, different amounts of purified lysozyme were added to a buffered suspension of M. luteus cells. Samples were added to a 0.3 mg/ml suspension of dried Micrococcus luteus cells in 0.1 M phosphate buffer, pH 5.0. Cell lysis was measured by measuring the reduction in absorbance at 450 nm every 15 sec. for 2 min. The slope of the curve was calculated using linear regression and quantified by comparing this slope with a curve where slope versus concentration of the purified standard was plotted. The smallest detectable concentration in the spectrophotometric test was 5 mg/l.

EXAMPLE 8

TRANSFORMATION OF GS115 CELLS WITH pSL12A; TRIBE LI

An A0X1<+>, His<+>strain, designated LI, which is a single-copy integrant of bovine lysozyme c2 expressed from the AOX1 promoter in A0X1<+> cells, resulted after GS115 cells were transformed, as described in Example 3, with pSL12A, cut with Sali (which cuts in the P. pastoris HIS4 gene region) to integrate at the HIS4 site.

Cells were grown in shake flasks for 4-5 days in 100 mM sodium-phosphate buffered minimal medium containing 0.2% glycerol and 1.0% methanol, to a density between 4 and 5 OD,nA

b U U units/ml. The final concentration of bovine lysozyme c2 accumulated from the cell was between 0.24 and 0.56 mg/l (defined by RIA).

From single-cell protein production experiments, it is known that the expression of alcohol oxidase is highly dependent on the dilution rate. In a steady state condition, the dilution rate (D) is directly related to the generation time (td) by the equation D=0.69/td.

Dilution rate experiments carried out with LI in continuous fermentations, where the growth rate could be controlled, showed that the expression levels were dependent on the growth rate.

Data from run no. 255, a methanol-limited continuous fermentation carried out in a 1 liter fermenter with a 33% MeOH feed, in which the dilution rate varied between 0.lt<_1> and 0.04t_1 (td = 6.9 and 17 .25 hours per generation) showed that the dilution rate had a marked effect on the specific productivity (amount of product/cells/hour) of bovine lysozyme c2 from strain LI. Three samples taken after three fermenter volumes at each dilution rate were measured to determine steady state values. The maximum specific productivity of 20 ug/g wet cells/h seen at a dilution rate of 0.065 h, or a generation time of 10.6 h, is significantly greater than the specific productivity of 7 ug/g wet cells/h for AOX1 <->stem, A37.

During the dilution rate experiment, a marked decrease in the concentration of bovine lysozyme c2 in the fermenter was observed after 600 h of continuous cultivation. During the first 550 h, the production of bovine lysozyme c2 varies as a function of the dilution rate. Southern blot analyzes of DNA extracted from the cell samples taken from the fermenter at 550, 643 and 717 h showed, at the last two time points, that a significant proportion of the cells in the fermenter showed an altered restriction map. The change was an approximately 300 bp deletion in the DNA fragment that includes the bovine lysozyme c2 coding sequence and the alcohol oxidase promoter. The new fragment containing the deletion was present in about 50% of the cells in the fermenter after 643 h and in about 90% of such cells after 717 h.

EXAMPLE 9

TRANSFORMATION OF GS115 WITH pSL12A; STRAIN C6

An AOX1<+>, His4<+> strain, designated C6, which is a single-copy integrant of a heterologous gene expressing bovine pre-lysozyme c2 from the AOX1 promoter, resulted after the GS115 cells were transformed, as described in example 3, with pSL12A cut with Sstl (or SacI at the same site), in the 5'A0X1 region, indicated in the figures, upstream of the promoter (with respect to the direction of transcription from the promoter). Some of the transformants had pSL12A integrated at the AOX1 site, 5" of the AOX1 promoter.

Cells were grown in shake flasks for 4-5 days in 100 mM sodium phosphate-buffered minimal medium containing 0.2% glycerol and 1% methanol to a density between 4 and 5 OD,An

bUU units/ml. The final concentration of bovine lysozyme c2 accumulated from the cells in the medium was 0.24 and 0.42 mg/l.

EXAMPLE 10

CLEANING PROCEDURE ON A LARGE SCALE; TRIBE LI

Purification procedures applicable to large-scale production were used to purify recombinant bovine lysozyme c2 isolated from strain LI in run #253.

Cells from 80 L fermentation culture in passage #253 were removed using a Ceraflow cartridge (1 micron, Amicon Corp., Danvers, Mass.), and the resulting filtrate (80 liters) was concentrated with a 3,000 molecular weight fuse-coil cartridge (Amicon). The concentrate was diafiltered with distilled water to reduce the conductivity to below 4 ms. Two and a half liters of the diafiltrate was pumped at a flow rate of 15 mg/min. on a ZetaChrom SP-100 capsule (Western Analytical, Temecula, Calif.) pre-equilibrated with 50 mM sodium acetate, pH 5.8. After application was complete, the capsule was washed with equilibrated buffer and the bovine lysozyme c2 was eluted with a linear gradient consisting of 1 1 50 mM sodium acetate, pH 5.8, and 1 1 300 mM sodium acetate, pH 8, 0. The bovine lysozyme c2 that eluted in the first third of the peak contained small amounts (no more than 15%) of contaminating proteins as eluted by SDS-gel electrophoresis and high-pressure liquid chromatography (HPLC). These contaminants were removed by repeated binding, washing and elution from the SP-100 capsule. HPLC analyzes were performed using an ubondapak C-18 column (Waters Associates, Milford, Massachusetts) and an acetonitrile gradient in aqueous trifluoroacetic acid. This HPLC method was also used to quantify lysozyme during the fermentation and purification procedure. The lysozyme activity was determined by the spectrophotometric method as described in example 7.

A unit is defined as a 1% change in OD^g per my. at 25°C. Protein concentrations were determined either by the method of Lowry et al., J. Biol. Chem. 193:265 (1951) after precipitation with trichloroacetic acid (TCA) or by quantitative amino acid analyzes using norleucine as an internal standard.

Under this three-step procedure, the bovine lysozyme c2 secreted by Pichia pastoris can be purified to near homogeneity. Total enzyme yield from the cell-free culture is approximately 60% of the initial amount of lysozyme measured in impure medium and is increased to 75% when impure fractions are re-chromatographed on the sulfopropyl plate. The purity is greater than 90%, as judged by SDS electrophoresis and HPLC.

EXAMPLE 11

TRANSFORMATION OF PPF1 CELLS WITH PLASMIDS pBLll and pSL12A;

STRAINS CSBL11 AND CSBL3

P. pastoris cells, of strain PPF1 (His4~Arg4~), were transformed with an uncut plasmid, designated pBLll, in the manner described in Example 3. An A0X1<+>, Arg<+>transformant, designated CSBL11, was selected for further processing.

Plasmid pBL11, which is illustrated in FIG. 3, is similar to plasmid pSL12A, except that it carries an HpaI site-terminated segment with the S. cerevisiae ARG4 gene cloned into the pBR322 BamHI site of pSL12A for selection instead of the Bglll site-terminated fragment of P. pastoris The HIS4 gene. The S. cerevisiae ARG4 gene is functional in P. pastoris.

The 5'-AOX1 sequence on pBL11 directed circular integration at the 5' end of the AOX1 site in some of the transformants. Transformants were first screened for Arg<+>phenotype. Arg<+>, AOX1<+>transformants were shown by Southern hybridization to have integrated the plasmid at the 5' end of the AOX1 site.

CSBL11 cells were retransformed with uncut pSL12A DNA. His<+> transformants were selected and screened by Southern hybridization. The transformants were double-copy integrants of the bovine pre-lysozyme c2, AOX1 promoter-driven expression cassette. The localization of pSL12A DNA in the genome of the double-copy integrants remains unknown, although it is not at the HIS4 or A0X1 site. An A0X1<+>, His<+>, Arg<+>transformant, designated CSBL3, was selected for further processing.

A fermenter run (run #274) with strain CSBL3 resulted in a specific productivity of 33ug/g wet cells/hr at a dilution rate of

0 081~~

and a specific productivity of 28 ug/g wet cells/hour at a dilution rate of 0.06t<_1>.

Authentic and recombinant lysozymes c were found to have comparable biological activity when tested in parallel using the same assay.

EXAMPLE 12

CONSTRUCTION OF HUMAN LYSOZYME EXPRESSION VECTOR pHLZ10 3

A plasmid, designated by applicants as pHLZ100, was prepared using standard techniques. See European patent application, publication no. 0,222,366 and Castanon et al., Gene 66, 223-234 (1988). Plasmid pHLZ100 consists of pUC9 (Vieira and Messing, Gene 19, 259 (1982)) with an insert, between the SalI and HindIII sites, comprising a segment of 435 base pairs which encodes, in addition to a translation stop signal, the entire sequence, except for the four N-terminal amino acids of the signal peptide, of the human pre-lysozyme c of placental origin. The mature lysozyme c corresponding to this pre-lysozyme c has the same amino acid sequence as human milk lysozyme. See Jolles and Jolles (1984), supra.

Using the Sanger dioxy method, the sequence of this 4 35-bp segment was found to be as indicated in the following Figure XII:

By simply using the genetic code for the 3' 393 base pairs indicated in fig. XII, the amino acid sequence can be deduced for the 130-amino acid, mature lysozyme c, where all but the 4 N-terminal amino acids of the corresponding pre-lysozyme c are coded for by the DNA with the sequence in fig. XII. As indicated in the description of the site-directed mutagenesis of pHLZ101 to produce pHLZ102 below, the sequence of the four amino acids of the N-terminus of pre-lysozyme c, for which the nucleotide sequence coding for the 114 carboxyl-terminal amino acids and the translational stop codon given in fig. XII, MKAL.

The 435-bp segment, for which the sequence is given in FIG. XII, except for differences in two base pairs, has the same sequence as the 4 35-bp cDNA encoding all but the 4 N-terminal amino acids of the signal peptide of the human pre-lysozyme c that was prepared from mRNA isolated from a human histiocytic lymphoma cell line U-937 (Sundstrøm and Nilsson, Intl. J. Cancer 117, 565-577 (1976)).

The amino acid sequences in pre-lysozyme c of placental origin and of cell line U-9 37 origin are the same, as the two differences in nucleotide sequence in the cDNA ends that code for pre-lysozyme c do not change the amino acid sequence. Castanon et al., Gene 66, 223-234 (1988). The positions of the two differences in nucleotide sequence are indicated by underlining in the sequence in fig. XII. In the U-937 cell line-derived cDNA, there is a T instead of C in GTC in FIG. XII which codes for Val in position -3 in the signal peptide and there is an A instead of G in CAG in fig. XII which codes for Gin in position -2 of the signal peptide.

In pHLZlOO, between the 5' end of the 435-bp segment indicated in

fig. XII and 5'-GTCGAS-3<*>in the SalI site, there is a segment with a sequence 5'-CTGCA{C}-3', in which 5'-CTGCA-3' is the residue in the Pstl site of pUC9 and {C} denotes a polydC block (of not precisely known length) resulting from polydC extensions of

polydC-extended, Pstl-cut pUC9 where a polydG-extended

cDNA comprising the 4 3 5-bp segment was ligated into to form pHLZ100. Furthermore, in pHLZ100, between the 3' end of the 435-bp segment indicated in FIG. XII and 5'-AAGCTT-3' in the HindIII site, there is a segment with the sequence 5'-CTCCAGAATTTT{G}TGCAGCC-3<1>, where {G} denotes a polydG block (of not exactly known length) which is due, at least in part, to polydG extension of the cDNA comprising the 435-bp segment prior to ligation of the cDNA into polydC-extended pUC9 to form pHLZlOO and optionally in part from a block of one or more dGs on the 3'- the end of this cDNA before it is polydG-extended; 5'-CTCCAGAATTTT-3' corresponds to the first 12 untranslated ribonucleotides after the translational stop codon in the lysozyme-encoding mRNA from which the cDNA used to generate pHLZlOO was prepared; and 5'-TGCAGCC-3' corresponds to (i) 5'-GCC-3' immediately preceding the HindIII site of pUC9 and (ii) 5'-TGCA-3' which is due to the filling of the gap, between the 3'-end of polydG -extended cDNA base pairs extended to the 31 end of polydC-extended, Pstl-cut pUC9, in the preparation of pHLZ100.

Approximately 1 ng of HLZ-100 was transformed into E. coli strain MC1061. Transformants were identified by resistance to ampicillin. Plasmid was prepared from transformants and cut with BamHI and HindIII according to the manufacturer's instructions. The correct plasmid was indicated by a pattern of approximately 2750 bp (HindIII-BamHI segment of pUC9) and approximately 540 bp (BamHI-HindIII segment comprising the 435-bp segment indicated in Fig. XII).

The SalI-HindIII site-linked human pre-lysozyme c insert in pHLZ100 was inserted into M13mpl8 for site-directed mutagenesis as follows, to add the 12 nucleotides required at the 5' end of the 435-bp segment in Fig. XII to code for the first four amino acids of pre-lysozyme c and to add EcoRI ends just before ATG which codes for the translation start and the first amino acid of the signal peptide and just after TAA which codes for the translation stop codon. Thus, M13mpl8 was cut with HindIII and SalI and was phosphatase treated. Likewise, a HindIII/SalI cut was performed on pHLZ100 and the approximately 530-bp fragment containing the 435-bp, partial pre-lysozyme c-encoding segment was isolated on a 1.0% agarose gel. 750 ng of vector and 250 ng of fragment were ligated together in a standard ligation reaction and the ligation mixture was used to transform E. coli JM103 cells. White plaques were selected and plasmid was isolated from there. A HindIII/SalI cut of the plasmid yielded fragments of approximately 7240 bp and 530 bp, indicating the correct plasmid.

The 3<1-> end of the insert in M13mpl8 was then modified to add an EcoRI site immediately after the translation-stop-coding TAA triplet. To accomplish this, site-directed mutagenesis was carried out in accordance with the procedure of Zoller and Smith, Methods in Enzymology 100, 468 (1983) and using the oligonucleotide with the sequence:

Plaques were screened with the screening oligonucleotide with the sequence:

and a mini-template preparation was carried out on the positives.

E. coli JM103 cells were transformed with the 3<1>-end-modified template from the mini-preparation and plaques were again screened with the same screening oligonucleotide. Positives in this second screening round were used to prepare the template for sequencing. Sequencing was carried out by the Sanger dideoxy method using the universal initiator with sequence:

A plasmid (RF form of M13mpl8) in which the 3' end of the pre-lysozyme coding segment had the desired modification was identified and designated pHLZ101.

The 5'-end of the 435-bp, pre-lysozyme-encoding segment in pHLZlOl was then modified, following the same site-directed mutagenesis procedure as for the 3'-end, except that the mutagenic oligonucleotide had the sequence:

and the screening oligonucleotide had the sequence: A plasmid was identified, by Sanger dideoxy sequencing using the universal primer and two primers with sequences

to have both ends of the pre-lysozyme c coding segment modified as desired. The plasmid was designated pHLZ102.

Plasmid (RF form M13mpl8) pHLZ102 was isolated using a CsCl gradient.

To construct the P. pastoris expression vector, the approximately 450-bp, pre-lysozyme-c-encoding, EcoRI site-terminated fragment was removed from pHLZ102 by EcoRI digestion and isolated on a 1.2% agarose gel . Plasmid pA0804 was then cut with EcoRI and the ends were phosphatase treated. 25 ng of cut, phosphatase-treated pAO804 and 250 ng of the pre-losozyme c coding fragment were ligated together in a standard ligation reaction and the ligation mixture was used to transform E. coli MC1061. Transformants were identified by means of ampicillin resistance and a transformant with a plasmid of the correct structure was identified by means of a mini-prepared plasmid DNA from the transformant, the mini-prepared DNA was cut with Pstl and the fragment sizes of the cut plasmid DNA were analyzed. A plasmid with the pre-lysozyme coding segment inserted in the correct orientation, relative to the direction of transcription from the AOX1 promoter, will have a Pstl fragment approximately 2100 bp in length. A plasmid with the pre-lysozyme coding segment inserted in the incorrect orientation, relative to the direction of transcription from the AOX1 promoter, will have a Pstl fragment approximately 1960 bp in length. A plasmid with the correct structure was found and designated pHLZ103.

EXAMPLE 13

HUMAN P. PASTORIS STRAINS FOR EXPRESSION OF LYSOZYME

Plasmid pHLZ103 was purified on a CsCl gradient.

5 µg of Sac1-cut pHLZ103 was transformed into P. pastoris strain GS115 (ATCC No. 20864) using the spheroplast transformation method (Cregg et al., Mol. Cell. Biol. 5, 3376-3385 1985). (SacI is an isoschizomer of Sstl). His<+>Mut<+> cells were identified and DNA from several transformants was characterized by Southern blot hybridization analyses. Thus, the DNAs were cut with EcoRI and probed with plasmid pAO803 DNA. Fragments of 2000 and 11200 bp, with loss of a wild-type 5700 bp fragment, indicated that in one transformant the linearized pHLZ10 3 plasmid had integrated by insertion into the AOX1 site 5' of the AOX1 coding sequence, leaving the transformant Mut<+>. Such a transformant was selected for further refinement and designated G+HLZ103S.

To develop Mut<+/>human lysozyme expression strains, plasmid pHLZ103 was cut with BglII and 5 µg fragment was transformed into GS115 cells by the spheroplast method. His<+> cells were identified and screened for their Mut phenotype.

Screening for the Mut phenotype was performed by plating His<+> transformants onto minimal-glycerol (2%) master plates to obtain colonies derived from single cells. After 2 days' incubation at 30°C, the master plate replica was transferred to minimal glycerol plates and plates that did not contain any carbon source to which methanol had been added in the vapor phase. This was accomplished by adding a drop, approximately 200 µl, of methanol to the underside of the lid of the Petri dish containing the plate gel. The plates were then incubated at 30°C for 2 days with further addition of methanol in vapor phase every day. Colonies showing visible growth were labeled as Mut<+> and those with no visible growth were labeled as Mut<+/>~.

Mut+^ transformants were characterized by Southern blot hybridization assays. DNA in the transformants was cut with EcoRI and probed with plasmid pA0803 DNA. Fragments of 2000 bp and 5100 bp (with loss of the vi11type 5700 bp fragment) indicated integration of the expression cassette by breaking the AOX1 site. One such Mut<+//>transformant was selected for further processing and designated G-HLZ103S.

EXAMPLE 14

ADDITIONAL PROCEDURES FOR THE CULTIVATION OF LYSOZYME-SECRETING P.

PASTORIS TRIBES

The procedure described in this example is advantageously used with bovine lysozyme c2 secreting P. pastoris strains A37 (Mut+// ) and LI (Mut+). The procedures can be used to form human lysozyme with P. pastoris strains G-HLZ103S and

G+HLZ103S.

The different strains have been maintained on yeast nitrogen base (YNB) without amino acids (Difco Laboratories, Inc., Detroit, Michigan, USA) + 2% glucose agar plates with transfers every month. Inocula for procedures described in this example were grown overnight at 30°C with shaking at 200 rpm in YNB without amino acids + 2% glucose + phosphate buffer (PH 6.0). 1. Start of fermentation and general operation.

2-liter fermenters (L.H. Fermentation, Hayward, California, USA; or Biolafitte, LSL Biolafitte, Princeton, New Jersey, USA) were autoclaved at a volume of 700 ml containing 225 ml of 10X basal salts (42 ml/l 85% phosphoric acid, 1.8 g/l calcium sulfate-2H20, 28.6 g/l potassium sulfate, 23.4 g/l magnesium sulfate-7H20, 6.5 g/l potassium hydroxide) and 30 g glycerol. After sterilization, 3 ml of a YTM4 trace salt solution (5.0 ml/l sulfuric acid, 65.0 g/l ferrous sulfate-7H20, 6.0 g/l copper sulfate-5H20, 20.0 g/l zinc sulfate-7H20, 3.0 g/l manganese sulfate-H 2 O, 0.1 g/l biotin) added and the pH was adjusted to 5.0 with the addition of concentrated ammonium hydroxide. The fermenters were then inoculated with a 10-50 ml volume of inoculum. When the initial amount of glucose was used up, a glycerol supply was started as described below in sections 2, 3 or 4 of this example. During a fermentation, the pH was controlled to 5.0 with the addition of 20% ammonium hydroxide solution containing 0.1% Struktol J673 antifoam (Struktol Co., Stow, Ohio, USA) and vigorous

foam was observed using a foam probe and controlled by the addition of 2% Structol J67 3 antifoam. Dissolved oxygen during the fermentation was maintained above 20% air saturation by increasing the air flow rate up to 3 1/min. and stirring speed up to 1500 rpm during the fermentation.

10-liter fermentations (in a 14-liter Biolafitte fermenter) were started with a 7.0-liter volume containing

1.9 1 10X basal salts and 300 g glycerol for the Mut<->mixed-supply, methanol-non-limiting, batchwise supply, or a 5.0 liter volume containing 2.4 liters 10X basal salts and 360 g glycerol for the Mut <+>methanol aliquot, or an 8-liter volume containing 3.2 1 10X basal salts and 480 g glycerol for the Mut<->methanol aliquot. After the sterilization, 29 ml of a YTM4 trace salt solution was added and the pH was adjusted and then controlled to 5.0 by adding ammonium gas during the fermentation. Strong foaming was controlled by adding 10% Struktol J67 3 antifoam. The fermenter was inoculated with an inoculum with a volume of 200-500 ml. When the initial amount of glycerol was used up, a feed was started as indicated in paragraphs 2, 3 or 4 of this example. The dissolved oxygen was maintained above 20% by increasing the air flow rate up to 40 l/min., stirring up to 1000 rpm and/or the pressure in the fermenter up to 1.5 bar during the fermentation.

2. Mut+^ cells: Mixed-feed, methanol non-limiting, portion-fed-fermentation

After the glycerol aliquot phase was complete (i.e. when the initial glycerol amount was used up), a 50 wt% glycerol feed containing 12 ml/l YTM4 trace salts was started at 5.4 ml/h for the 2-liter fermenter or 4 ml/h for the 10-litre fermenter. After feeding glycerol for 6 h, the glycerol feed was decreased to 3.6 ml/h (36 ml/h to the 10-liter fermenter) and a methanol feed containing 12 ml/l YTM4 trace salts was initiated at 1.1 ml/h for 2- liter fermenter or 11 ml/h for the 10-litre fermenter. After 5 hours, the methanol feed was adjusted to give a residual methanol concentration up to about 1%, preferably between 0.2 and 0.8%. The fermentation is carried out for 40-50 hours on the methanol and glycerol feeds.

3. Mut cells: Fermentation with batchwise addition of methanol

After the initial amount of glycerol was used up, a 50% glycerol feed containing 12 ml/l YTM4 trace salts was started at 12 ml/h for the 2-liter fermenter or 200 ml/h for the 10-liter fermenter and run for a total of 7 hours. After 6 hours of the glycerol feed, the methanol feed, containing 12 ml/l YTM4 trace salts, was started at 1.1 ml/h for the 2-liter fermenter and 11 ml/h for the 10-liter fermenter for 5 min. When an increase in dissolved oxygen was recorded after the methanol supply was shut off, the methanol supply was turned on again for a further 5 min. The latter process was repeated several times until an immediate response with respect to dissolved oxygen was observed when the methanol supply was stopped. When this happened, the methanol supply was increased by 20% per hour in 30 minute intervals. The methanol feed was increased until a feed rate of 7.6 mL/h was achieved for the 2-liter fermenter or 126 mL/h for the 10-liter fermenter. The fermentation was then carried out for 40-60 hours for the 2-liter fermenter or 25-35 hours for the 10-liter fermenter.

4. Mut<+//> cells: Fermentation with batchwise additions of methanol

After the initial amount of glycerol was used up, methanol was added to the fermenter to maintain a residual methanol concentration between 0.2% and 0.8%. For run-throughs in the 10-liter fermenter, YTM4 trace salts were not used, but instead 40 ml of IM^_ trace salts (5 ml/l sulfuric acid, 4.8 g/l ferric chloride-2H 2 O, 2.0 g/l zinc sulfate- H20, 0.02 g/l boric acid, 0.2 g/l sodium molybdate, 0.3 g/l manganese sulfate-H20, 0.08 g/l potassium iodide, 0.06 g/l copper sulfate-5H20) and 2 mg/l biotin injected into the fermenter every other day. The fermentation was carried out up to 192 hours for the 2-liter fermenter or up to approximately 144 hours for the 10-liter fermenter.

Additional information regarding procedures for growing lysozyme c-secreting P. pastoris strains is provided in US Patent Application Serial No. 249,446, filed Sep. 26, 1988, and which is incorporated by reference herein.

EXAMPLE 15

PREPARATION OF PLASMIDS pAO803, PAO804, AND pA0811

The procedure in this example was carried out using standard techniques, such as by Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, New York (1986) and Maniatis et al., supra.

Plasmid pBR322 was modified as follows to eliminate the EcoRI site and insert a BglII site into the PvuII site: pBR322 was cut with EcoRI, the overhanging ends being filled in with the Klenow fragment of E. coli DNA polymerase I, and the resulting DNA was re-ringed using T4 ligase. Recirculated DNA was used to transform E. coli MC1061 to ampicillin resistance and transformants were screened to have a plasmid of approximately 4.37 kbp in size without an EcoRI site. One such transformant was selected and grown to yield a plasmid, designated pBR322 RI, which is pBR322 with the EcoRI site replaced by the sequence:

pBr322 RI was cut with PvuII and the linker, with sequence

were ligated to the resulting blunt ends using T4 ligase. The DNAs obtained were annealed again, also with T4 ligase, and then cut with BglII and again annealed using T4 ligase to eliminate multiple BglII sites due to ligation of more than one linker to the PvuII-cut pBR322 RI. The DNAs, processed to eliminate multiple BglII sites, were used to transform E. coli MC1061 to ampicillin resistance. Transformants were screened for an approximately 4.38 kbp plasmid with a BglII site. One such transformant was selected and grown to yield a plasmid, designated pBR322 RIBG, for further processing. Plasmid pBR322 RIBG is the same as pBR322 RI except that pBR322 RIBG has the sequence

instead of the PvuII site in pBR322 RI.

pBR322 RIBG was cut with sal and BglII and the large fragment (approximately 2.97 kbp) was isolated. Plasmid pBSAGI5I, which is described in European patent application publication number 0 226 752, was cut completely with BglII and XhoI and a fragment of approximately 850 bp from a region of P pastoris A0X1 site downstream of the A0X1 gene transcription terminator (relative to the direction of transcription from AOX1 the promoter) was isolated. This BglII-XhoI fragment from PBSAGI5I and the approximately 2.97 kbp SalI-BglII fragment from pBR322 RIBg were combined and subjected to ligation with T4 ligase. The ligation mixture was used to transform E. coli MC1061 to ampicillin resistance and transformants were screened for a plasmid of the expected size (approximately 3.8 kbp) with a BglII site. This plasmid was designated

PAO801. The overhanging end of the SalI site from the pBR322 RIBG fragment was ligated to the overhanging end of the XhoI site of the 850 bp pBSAGI5I fragment and, in the process, both the SalI site and the XhoI site of pAO801 were eliminated.

pBSAGI5I was then cut with ClaI and the approximately 2.0 kbp fragment was isolated. The 2.0 kbp fragment has an approximately 1.0 kbp segment encompassing the P. pastoris AOX1 promoter and transcription initiation site, an approximately 700 bp segment encoding the heptatitis B virus surface antigen ("HBsAG") and a segment of approximately 300 bp encompassing the P. pastoris AOX1 gene polyadenylation signal and site coding segments and transcription terminator. The HBsAg-encoding segment of the 2.0 kbp fragment is terminated, at the end adjacent to the 1.0 kbp segment with the AOX1 promoter with the EcoRI site and, at the end adjacent to the 300 bp segment with the AOX1 transcription terminator, with a Stul site, and has its subsegment encoding HBsAG oriented and positioned, with respect to the 1.0 kbp promoter-containing and 300 bp transcription terminator-containing segments, operative for expression of HBsAG by transcription from the AOX1 promoter. The EcoRI site connecting the promoter segment to the HBsAG coding segment occurs immediately upstream (with respect to the direction of transcription from the AOX1 promoter) from the translation initiation signal coding triplet of the AOX1 promoter.

For further details regarding the promoter and terminator segments of the 2.0 kbp, ClaI site-terminated fragment of pBSAGI5I, see European Patent Application Publication No. 0 226 846 and Ellis et al., Mol. Cell. Biol. 5, 1111 (1985).

Plasmid pAO801 was cut with the approximately 2.0 kbp ClaI site-terminated fragment from pBSAGI5I. The ligation mixture was used to transform E. coli MCC1061 to ampicillin resistance, and transformants were screened for a plasmid of the expected size (approximately 5.8 kbp) which, when cut with ClaI and BglII, yielded fragments of approximately 2.32 kbp ( with the origin of replication and ampicillin resistance gene from pBR322) and approximately 1.9 kbp, 1.48 kbp and 100 bp. Upon cutting with Bglll and EcoRI, the plasmid yielded a fragment of approximately 2.48 kbp and with the 300 bp terminator segment from the AOX1 gene and the HBsAG-coding segment, a fragment of approximately 900 bp containing the segment upstream of the AOX1 protein-coding segment of the AOX1 gene in the AOX1 site, and a fragment of approximately 2.42 kbp containing the origin of replication and the ampicillin resistance gene from pBR322 and a ClaI-Bglll segment of approximately 100 bp of the AOX1 site (further upstream from the AOX1 coding segment than the first mentioned 900 bp EcoRI-Bglll segment). Such a plasmid had the ClaI fragment from pBSAGI5I in the desired orientation; and in the opposite undesired orientation there will be EcoRI-Bglll fragments of approximately 3.3 kbp, 2.38 kbp and 900 bp.

One of the transformants containing the desired plasmid, designated pAO802, was selected for further processing and was grown to yield this plasmid. The desired orientation of the ClaI fragment from pBSAGI5I in pAO802 had the AOX1 site fragments delimiting the HBsAG coding segment and the approximately 800 bp Bglll site-terminated fragment from downstream of the AOX1 gene in the AOX1 site oriented correctly to lead to the correct integration into the P. pastoris genome at the AOX1 locus of the linearized plasmid prepared by cutting at the Bglll site at the terminal end of the 800 bp fragment from downstream of the AOX1 gene at the AOX1 locus.

pAO802 was then processed to remove the HBsAG coding segment terminated by an EcoRI site and a Stul site. The plasmid was then cut with Stul and a linker with sequence:

were ligated to the blunt ends using T4 ligase. The mixture was then treated with EcoRI and again subjected to ligation using T4 ligase. The ligation mixture was then used to transform E. coli MC1061 to ampicillin resistance and the transformants were screened for a plasmid of the expected size (5.1 kbp) with EcoRI-Bglll fragments of approximately 1.78 kbp, 900 bp and 2.42 kbp and Bglll-Clal fragments of approximately 100 bp, 2.32 kbp, 1.48 kbp and 1.2 kbp. This plasmid was designated pAO803. A transformant with the desired plasmid was selected for further processing and was grown to yield pAO803.

Plasmid pAO804 was then generated from pAO803 by inserting, into the BamHI site from pBR322 in pAO803, an approximately 2.75 kbp BglII fragment from the P. pastoris genome containing the P. pastoris HIS4 gene. See e.g. Cregg et al., Mol. Cell. Biol. 5, 3376 (1985) and European Patent Application Publication Nos. 0 180 899 and 0 188 677. pAO803 was cut with BamHI and combined with the HIS4 gene-containing BglII site-terminated fragment and the mixture subjected to ligation using T4 ligase. The ligation mixture was used to transform E. coli MC1061 to ampicillin resistance and transformants were screened for a plasmid of the expected size (7.85 kbp), cut with SalI. One such transformant was selected for further processing, and the plasmid containing it was designated pAO804.

PAO804 has a SalI-Clal fragment of about 1.5 kbp and another of about 5.0 kbp and a Clal-Clal fragment of 1.3 kbp; this indicates that the direction of transcription of the HIS4 gene in the plasmid is the same as the direction of transcription of the ampicillin-resistance gene and opposite to the direction of transcription from the A0X1 promoter.

The orientation of the HIS4 gene in pAO804 is not critical for the effect of the plasmid or its derivatives with cDNA-encoding segments inserted at the EcoRI site between the AOX1 promoter and terminator segments. Thus, a plasmid with the HIS4 gene in an orientation opposite to that of the HIS4 gene in pAO804 will also be effective for use in accordance with the present invention.

It is worth noting that other plasmids, similar to pAO804 but with selectable marker genes different from the P. pastoris HIS4 gene and capable of providing selection to P. pastoris, can be constructed by inserting a fragment into the BamHI site of pAO80 3 comprising a selectable marker gene that is different from the P. pastoris HIS4 gene. Among such fragments known in the prior art are fragments with the S. cerevisiae ARG4 gene, the P. pastoris ARG4 gene and the S. cerevisiae HIS4 gene.

To form a plasmid, designated pA0811, which is the analogue of pAO804 with a S. cerevisiae ARG4 gene instead of the P. pastoris HIS4 gene, and which was used to form plasmid pBLll according to the invention, e.g. the following procedure was followed: pAO80 3 was cut with BamHI and the overhanging ends were filled in using the Klenow fragment of E. coli DNA polymerase I. Then the linearized, filled-in plasmid was combined with an HpaI fragment of approximately 2.1 kbp from S. cerevisiae genome which contains the S. cerevisiae ARG4 gene. See Tschumper and Carbon, J. Mol. Biol. 156, 293 (1982); European patent application with publication number 0 211 455. The HpaI fragment is also available from a plasmid, designated pYM25, which is available from the Northern Regional Research Laboratory depository of the US Department of Agriculture in Peoria, Illinois, USA, where the plasmid is deposited in E. coli MC1061 under NRRL accession number B-18015. The fragments were butt-ligated using T4 ligase and the ligation mixture was used to transform E. coli MC1061 to ampicillin resistance. Transformants were then screened for plasmids of the expected size (approximately 7.2 kbp). One such transformant was selected for further processing. The plasmid it contains was designated pA0811. Although the orientation of the ARG4 gene in pA0811 has not been determined, it should be noted that there is a Bglll site in the HpaI site-terminated fragment that is approximately 400-500 bases from an HpaI site on a fragment terminus and that is near the 3' end of the ARG4 gene.

The P. pastoris ARG4 gene is provided in European patent application with publication number 0 211 455. A plasmid, which includes a fragment with the P. pastoris ARG4 gene, is available from the Northern Regional Research Laboratory depository in Peoria, Illinois, where it is deposited in E .coli MC1061 under NRRL accession number B-18016.

The S. cerevisiae HIS4 gene is given in Donahue et al., Gene 18:47

(1982). A plasmid, designated pBPGl-1, comprising a fragment of the S. cerevisiae HIS4 gene, is described in European patent application publication number 0 226 7 52 and is available from the Northern Regional Research Laboratory depository in Peoria, Illinois, where it is deposited in E. coli MC1061 under NRRL accession number B-18020.

DEPOSITS

Cultures of P. pastoris strains GS115 and PPF1 and E. coli K-12 strain MC1061 containing plasmid pBSAGI5I have been deposited with the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Maryland 20852 USA, on the following dates and with the corresponding ATCC accession number:

Said deposits were carried out and accepted by ATCC in accordance with the conditions of "the Budapest Treaty on the International Recognition of the Deposits of Microorganisms for Purposes of Patent Procedures and the Regulations promulgated under said Treaty". Samples of said depositions will be available, in accordance with said convention and provisions, to patent offices and other persons and institutions legally entitled to receive them under the patent laws and regulations of the United States and, if applicable, other national or international organizations in which the present application, or an application with the same priority as the present application is submitted. All restrictions regarding the general availability of samples of said deposits will be irrevocably removed, at the latest upon the issuance of a US patent for this application.

While the various aspects of the present invention are described in detail herein, the person skilled in the art will perceive the modifications and variations that are within the inventive concept. These modifications and variations are within the scope of the present invention which is described and protection sought for herein.

Claims (30)

1. A DNA characterized in that it comprises (1) a promoter segment of a first P. pastoris gene and a terminator segment of a second P. pastoris gene, said first and second genes being the same or different; and (2) a DNA segment encoding an animal pre-lysozyme c, said segment being oriented and positioned, with respect to said promoter and terminator segments, operative for transcription in P. pastoris, from the promoter of said promoter segment, of said pre-lysozyme c coding segment and the polyadenylation signal coding and polyadenylation site coding segments of said terminator segment. 2. A DNA as stated in claim 1, characterized in that said first P. pastoris gene and said second P. pastoris gene are the same and are the P. pastoris A0X1 gene. 3. A DNA as specified in claim 2, characterized in that the pre-lysozyme c coding segment codes for a pre-lysozyme c from a mammalian species. 4. A DNA as specified in claim 3, characterized by-that the species is human. 5. A DNA as stated in claim 4, characterized in that the human pre-lysozyme c-encoding segment has the sequence of the pre-lysozyme c-encoding, EcoRI-site-terminated segment of plasmid pHLZ102 or the sequence which is that of said pre-lysozyme c-encoding, EcoRI-site-terminated segment of plasmid pHLZ102 altered to have the triplet 5'-GTT-3' which codes for Val in position -3 of the signal peptide of pre-lysozyme c and the triplet 5'-CAA-3' which codes for Gin in position -2 of the signal peptide of pre -the lysozyme c. 6. A DNA as specified in claim 3, characterized in that the species is bovine. 7. A DNA as specified in claim 6, characterized in that the bovine pre-lysozyme c-encoding segment has the sequence of the pre-lysozyme c2-encoding, EcoRI site-terminated segment of plasmid pBLI6C. 8. A DNA as stated in claim 1, characterized in that it is capable of transforming P. pastoris cells to express said pre-lysozyme c and comprising a gene to provide a selectable marker to P. pastoris cells containing said DNA. 9. A DNA as stated in claim 7, characterized in that the gene which provides the selectable marker is selected from the group consisting of the P. pastoris HIS4 gene, the P. pastoris ARG4 gene, the S. cerevisiae ARG4 gene and the S. cerevisiae HIS4 gene. 10. A DNA as stated in claim 9, characterized in that said first P. pastoris gene and said second P. pastoris gene are the same and are the P. pastoris AOX1 gene. 11. A DNA as stated in claim 10, characterized in that the pre-lysozyme c-encoding segment codes for a pre-lysozyme c from a mammalian species. 12. A DNA as stated in claim 11, characterized in that the species is human. 13. A DNA as stated in claim 12, characterized in that the human pre-lysozyme c-encoding segment has the sequence of the pre-lysozyme c-encoding, EcoRI site-terminated segment of plasmid pHLZ102 or the sequence of said pre-lysozyme c- coding EcoRI site-terminated segment of plasmid pHLZ102 altered to have the triplet 5'-GTT-3' coding for Val in position -3 of the signal peptide of pre-lysozyme c and the triplet 5'-CAA-3' coding for Gin in position -2 of the signal peptide of the pre-lysozyme c. 14. A DNA as stated in claim 1, characterized in that it is plasmid pHLZ103. 15. A DNA as stated in claim 11, characterized in that the species is bovine. 16. A DNA as stated in claim 15, characterized in that the bovine pre-lysozyme c-encoding segment has the sequence of the pre-lysozyme c2-encoding, EcoRI site-terminated segment of plasmid pBL16C. 17. A DNA as stated in claim 16, characterized in that it is selected from the group consisting of pSL12A and pBL11. 18. A culture of P. pastoris characterized in that it is transformed with a DNA as stated in claims 8-17. 19. A culture as stated in claim 18, characterized in that said culture is of a P. pastoris strain selected from the group consisting of G-HLZ103S and G+HLZ103S. 20. A culture as stated in claim 18, characterized in that said culture is of a P. pastoris strain selected from the group consisting of A37, LI, C6, CSBL11 and CSBL3. 21. Method for the production of an animal lysozyme c, characterized by cultivation of P. pastoris cells which have a gene capable of expression of the corresponding pre-animal lysozyme c in P. pastoris, said cultivation being carried out under conditions such that said gene is transcribed in said cells. 22. Method as stated in claim 21, characterized in that the cells are from a culture, or a subculture of a culture, which has been transformed with a DNA as stated in claims 8-17 which is capable of expression of the corresponding pre-lysozyme c i P. pastoris. 23. Method as stated in claim 22, characterized in that the cells are of a strain selected from the group consisting of G-HLZ103S and G+HLZ103S. 24. Method as stated in claim 22, characterized in that the cells are of a strain selected from the group consisting of A37, LI, C6, CSBL11 and CSBL3. 25. Method as stated in claims 21 - 24, characterized in that said cultivation is carried out in a medium containing methanol which is a carbon source. 26. A polypeptide with 18 amino acids, characterized in that it comprises the sequence MXALVILGFLPL5VAVQG. 27. A DNA, characterized in that it comprises a segment selected from the group consisting of a 54-bp segment in a sequence that codes for the polypeptide with the sequence MKALVILGFLPLSVAVQG, and the 441-bp segments in sequences encoding a polypeptide of sequence wherein Xgg is K or H, and a 444-bp segment of a sequence encoding the fusion polypeptide wherein the polypeptide of sequence MKALVILGFLFLSVAVQG is fused to the amino terminus of mature human milk lysozyme. 28. A DNA as stated in claim 27, characterized in that it comprises a 54-bp segment in a sequence that codes for the polypeptide with the sequence 29. A DNA as stated in claim 28, characterized in that it comprises a segment of 441 bp in a sequence that codes for the polypeptide with the sequence: 30. A DNA as stated in claim 28, characterized in that it comprises a segment of 444 bp in a sequence that codes for the fusion polypeptide in which the polypeptide with the sequence MKALVILGFLFLSVAVQG is fused to the amino terminus of the mature human milk lysozyme.