AU2825489A - Production of animal lysozyme c via secretion from pichia pastoris and composition therefor - Google Patents

Description

PRODUCTION OF ANIMAL LYSOZYME C VIA SECRETION FROM PICHIA PASTORIS AND COMPOSITION THEREFOR

BACKGROUND OF THE INVENTION

The present invention generally relates to a microbiological process for producing an animal lysozyme c utilizing recombinant DNA technology and, more particularly, is concerned with the production of an animal lysozyme c by culturing Pichia pastoris yeast cells which contain a gene which is capable in such cells of expressing the pre-form of the lysozyme in such cells, the DNAs employed to transform such cells to express the pre-form of the lysozyme, and cultures and subcultures of the transformed cells. Lysozymes are basic enzymes which exhibit anti-bacterial action directly, as a result of their ability to lyse bacterial cells, and indirectly, as a result of their ability to produce a stimulatory effect upon the phagocytic activity of polymorphonuclear leukocytes and macrophages (Jolles et al., Mol. and Cell. Biochem. 63, 165 (1984)). Lysozymes exist in many tissues and secretions of humans, other vertebrates and invertebrates, as well as in plants, bacteria and phage. In performing their primary function of protecting organisms against bacterial infection, lysozymes, which are also known as 1,4-β-N-acetylmuramidases, cleave the glycosidic bond between the C-l of N-acetylmuramic acid and the C-4 of N-acetylglucosamine in the bacterial peptidoglycan, a polysaccharide of amino sugars attached to short cross-linked peptides which is a component of bacterial cell walls. Gram-negative bacteria have cell walls which contain mono- or bi-layered peptidoglycan whereas gram-positive bacteria possess cell walls which contain highly complex multi-layered peptidoglycan. As a result of the above-described cleavage, all such bacteria lyse and, consequently, die. Some lysozymes also display a more or less pronounced chitinase activity. corresponding to a random hydrolysis of 1,4-β-N-acetyl- glucosamine linkages in chitin, and consequently have the additional capacity of protecting organisms against a large number of chitin-covered pathogens. A slight esterase activity of lysozymes has also been reported.

Due to their bacteriolytic activity, lysozymes are employed, by themselves and in combination with other components, such as lactotransferrin, which inhibits the _Q growth of certain microorganisms by chelating iron, complement, antibodies, vitamins, other enzymes and various antibiotics, such as tetracyclin and bacitracin, as antimicrobial agents, as preservatives for foods, such as cheese, sausage and marine products, as ripening ₅ agents for cheese, and in various other applications. As lysozymes also possess the ability to indirectly stimulate the production of antibodies against a variety of antigens, such enzymes may also be employed to enhance resistance against infection. o Lysozymes of the c, or "chicken", type contain

129-130 amino acids in their mature, secreted forms. Forty of these 129-130 have been found to be invariant among different species. Two of the several carboxyl groups of lysozyme c's (corresponding to the Glu-35 and ₅ Asp-52 of the chicken egg white lysozyme amino acid sequence) which participate in the enzyme's catalytic activity, and which are essential for lysozyme activity, occur in similar positions in all c-type lysozymes. A third carboxyl group (corresponding to Asp-101 in chicken ₀ egg white lysozyme) , which is involved in a substrate binding interaction, occurs in most c-type lysozymes. The eight half-cysteine residues of all of the c-type lysozymes are invariant. The disulfide bonds formed by the cysteines play an important role in the formation and 5 maintenance of the enzymes' secondary and tertiary structures. The three-dimensional structures, and processes of folding to form these structures upon translation of mRNAs, are thought to be closely similar for all lysozyme c's. The complete primary structures are known for the mature lysozyme c's obtained from the following sources: (1) hen, quail, turkey, guinea fowl, duck, pheasant, chachalaca and chicken egg whites; (2) human milk and urine; (3) moth; (4) baboon, rat, and bovine stomach; and (5) T2 and T4 phage. DNA sequences encoding mature human milk lysozyme c are known. See European Patent Application Publication Nos. 0181 634, 0 208 472, and 0 222 366.

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

Although lysozymes of both the c and g types have acidic amino acid residues as part of their active sites, several differences exist between the two types. The g-type lysozymes contain about 185 amino acids in their mature forms, exhibit low activity on N-acetylglucosamine polymers, and do not cross-react immunologically with lysozymes of the c type. Lysozymes of the g type have an unusually high occurrence of paired amino acids (i.e., the same amino acid occurring at neighboring positions in the sequence) in the molecules, and all of the four half-cysteine residues in the g type molecules are situated in the N-terminal half of the chain, c-type lysozymes are equally active on peptide-substituted or unsubstituted peptidoglycan, and are active as well on chitin oligosaccharides. g-type lysozymes, which have activity against the linear peptidoglycan similar to that of the c-type enzymes, do not act on chitin oligosaccharides. Furthermore, in contrast to the c-type lysozymes, which are capable of both hydrolysis and transglycosylation, g-type enzymes act oniy as hydrolases. The existence of other distinct types of lysozymes, which differ from the c and g types on the basis of structural, catalytic and immunological criteria, have also been reported.

A number of mammalian species are known which have a foregut fermentation and which utilize lysozyme in their digestive systems. Domestic cattle of the species Bos taurus and other cud-chewing mammals in the order Artiodactyla (i.e., ruminants) have developed a symbiotic relationship with microbes that live in the rumen (foregut) and digest cellulose and other dietary components. Such mammals have an unusually high level of lysozyme in the stomach ucosa. As a result of this digestive mode, large numbers of microbes enter the fundic region (anterior part) of the abomasum (stomach) , where lysozymes are secreted by the mucosal lining, and become digested by the lysozymes, thus allowing the ruminant to utilize the nutritional content of the microbes. Metabolic balance studies which have been performed suggest that the ruminant animal utilizes the lysed bacteria as a source of carbon, nitrogen, and phosphorus for energy and growth.

The three forms of lysozyme which are present in the abomasum of domestic cattle constitute approximately 10% of the total protein that can be extracted -from the abomasum mucosa. These three, nonallelic lysozymes, which are of the c type (and designated cl, c2 and c3) , are closely related to one another antigenically and in amino acid composition.

Jolles et al., J. Biol. Chem. 259, 11617 (1984), determined the complete 129 amino acid sequence of a mature bovine lysozyme c2, the most abundant of the three lysozymes found in the bovine stomach, to be as follows: 1 10 20

K-V-F-E-R-C-E-L-A-R-T-L-K-K-L-G-L-D-G-Y-K-G-V-S-L-A-N- -

30 40 50

L-C-L-T-K- -E-S-S-Y-N-T-K-A-T-N-Y-N-P-S-S-E-S-T-D-Y-G-I-

60 70 80

F-Q-I-N-S-K- -W-C-N-D-G-K-T-P-N-A-V-D-G-C-H-V-S-C-S-E-L-

90 100 110

M-E-N-D-I-A-K-A-V-A-C-A-K-K-I-V-S-E-Q-G-I-T-A-W-V-A- -K-

120

S-H-C-R-D-H-D-V-S-S-Y-V-E-G-C-T-L.

The three lysozyme c's present in the abomasum of domestic cattle differ in certain respects from other lysozyme c's in that (1) the pH optimum for enzymatic activity of lysozyme c's present in bovine abomasum is approximately 5, instead of 7 for other lysozyme c's; and (2) the lysozyme d s present in bovine abomasum are more stable in acidic environments, such as that of the abomasum, and are more resistant to proteolytic enzymes, such as pepsin, which occur in the abomasum, than other lysozyme c's.

The production of heterologous proteins by secretion to the culture medium mediated by signal sequences has been described for many organisms, including various Aspergillus species, Saccharomyces cerevisiae, and various types of mammalian cells. In these species, both native (i.e., intra-generic) and mammalian signal sequences have been demonstrated to be capable of directing secretion of certain heterologous proteins into the growth media. However, each of these host systems has various disadvantages.

For example, Aspergillus strains secrete large quantities of endogenous proteins into the growth media, thus significantly increasing the complexity and the expense of purifying a desired heterologous protein product. The productivity of heterologous protein production by secretion from S. cerevisiae appears to be severely limited, for those proteins which can be secreted at all. A major disadvantage associated with mammalian cell hosts is the difficulty of, and large expense associated with, maintaining such host systems and culturing such hosts on a large scale.

Some polypeptides which are expressed in, and secreted from, heterologous hosts exhibit no, or reduced, levels of biological activity in comparison 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 system's secretory apparatus without activity-reducing degradation or cleavage and to properly fold and form disulfide bonds during the secretion process.

Yeasts offer certain advantages over other host systems with respect to the large-scale production of heterologous proteins in biologically active form.

Yeasts can generally be grown to higher cell densities 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 favorable for use as a host system for the large-scale production of those heterologous proteins which it is capable of secreting into its culture media at significant levels in biologically active form. P. pastoris is readily adaptable to continuous industrial-scale fermentation processing, whereby the yeasts grow to high cell densities in a defined and inexpensive fermentation medium. With P. pastoris, production levels usually scale up from shake-flask cultures to large fermenter cultures. Simple culture media which are inexpensive and free of undefined ingredients, which can be potential sources of pyrogens and toxins, can be used for this yeast. Moreover, as many critical functions of yeasts, such as oxidative phosphorylation, are performed within organelles, such functions are not, as they are in prokaryotic hosts, directly exposed to the possible deleterious effects of production of polypeptides foreign to the host cells. Also, since yeasts are eukaryotes, their intracellular environment tends to be more suitable than that of prokaryotes for the correct folding of eukaryotic proteins. In addition, the cultivation of yeasts, particularly P. pastoris, is easier than that of most other host systems. Contamination of P. pastoris cultures growing on methanol can more easily be prevented than that of cultures of other types of hosts, thereby increasing the reliability and safety of the heterologous polypeptide products.

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

Although P. pastoris is a particularly favorable host system for the production of heterologous proteins which it does happen to secrete to the medium, it cannot be predicted that a particular type of protein, e.g., lysozyme c's, will be secreted by the yeast in a biologically active form and, if secreted at all, what the efficiency of secretion will be. However, once a particular protein is found to be secreted efficiently from the yeast, it is likely that other proteins that are similar in sequence and three dimensional structure will be secreted also.

Even for the yeast S. cerevisiae, which has been considerably more extensively studied than P. pastoris, secretory systems for proteins remain ill-understood. Secretion to the medium of a mature protein in biologically active form requires that, upon expression, the mature protein have a "signal sequence" (also referred to as "signal-peptide") appended to its amino-terminus. This signal sequence has an ill-understood role in leading a protein into the cell's secretory apparatus and is cleaved in the course of translation to yield the mature protein.

SUMMARY OF THE INVENTION

0 The present invention provides a novel, surprisingly and unexpectedly efficient method of producing large quantities of a biologically active and easily-purified animal lysozyme c by culturing P. pastoris cells, which contain a gene which is capable of expressing the pre-form of the animal lysozyme c

(i.e., the animal lysozyme c with its naturally occurring signal sequence appended to the N-terminus of the mature protein) in such cells, under conditions such that the gene is transcribed in the cells. o In accordance with the present invention, high levels 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 fermenter cultures. Virtually all of the animal lysozyme c which is secreted into and recovered from the media is correctly processed at the junction of the signal sequence with the mature protein, has disulfide bonds formed correctly to yield the correctly folded _Q protein, and is biologically active. Further, the secreted mature protein has essentially the same immunologic properties and specific activity as the naturally-occurring animal lysozyme c. In addition, the secreted animal lysozyme c comprises at least 50% of the 5 protein present in the media and proteins other than animal lysozyme c are present in small quantities relative to the animal lysozyme c. The present invention also encompasses DNAs, 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.

The P. pastoris cells which have been transformed with the DNAs of the invention are cultured to carry out the method of the invention for making an animal lysozyme c. Finally, the present invention entails the discovery of the signal peptide, of bovine lysozyme c2 and of bovine pre-lysozyme c2; and the invention encompasses DNAs with sequences encoding these polypeptides.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGURE 1 is a restriction map of the generalized Pichia pastoris expression vector pA0804. Major functional features and restriction sites of the plasmid are indicated and are described in the Examples.

FIGURE 2 provides a restriction map of bovine pre-lysozyme c2 expression plasmid pSL12A and illustrates construction of the plasmid from plasmid pAO804 and the EcoRI site-terminated, bovine pre-lysozyme c2-encoding segment of plasmid pBLI6C (derived from clone ΛBL3) . Major functional features and restriction sites of pSL12A are indicated in the Figure and are described in the Examples that follow. FIGURE 3 is a restriction map of the bovine pre-lysozyme c2 expression plasmid pBLll. Major functional features and restriction sites of the plasmid are indicated and are described in the Examples that follow. In the Figures, sites with restriction enzymes indicated in parentheses are sites at which one fragment, at an end thereof produced with one of the indicated enzymes, was joined with another fragment, at an end thereof produced with the other of the indicated enzymes, and, as a result of the joining of the fragments at the site, sites for both of the indicated enzymes were eliminated.

DETAILED DESCRIPTION OF THE INVENTION

In one aspect, the present invention involves a method of producing an animal lysozyme c comprising culturing P. pastoris cells, which have a gene, which is capable of expressing the corresponding pre-lysozyme c in P. pastoris, said culturing being under conditions such that the gene is transcribed in the cells.

The invention further entails a DNA which comprises (1) a promoter segment of a first P. pastoris gene, said segment comprising the promoter and transcription initiation site of said first gene, and a terminator segment of a second P. pastoris gene, said terminator segment comprising the polyadenylation signal-encoding and polyadenylation site-encoding segments and the transcription termination signal of said second gene, said first and second genes being the same or different and said terminator segment oriented, with respect to the direction of transcription from the promoter of said first gene in said promoter segment, operatively for termination of transcription from said promoter at said transcription terminator of said second gene in said terminator segment; and (2) a DNA segment encoding an animal pre-lysozyme c oriented and positioned, between said promoter and terminator segments, operatively for transcription, from said promoter of said first gene of the animal pre-lysozyme c encoding DNA segment and the polyadenylation signal-encoding and polyadenylation site-encoding segments of said second gene in said terminator segment. In yet another aspect, the invention entails a DNA, which is capable of transforming P.pastoris cells to express an animal pre-lysozyme c and which has the attributes of a DNA of the invention described in the preceding paragraph, and which comprises, in addition, a gene to provide a selectable marker to cells which harbor the DNA.

In still another aspect, the invention encompasses a culture of P. pastoris cells transformed with a DNA according to the invention.

In a further aspect, the invention entails a polypeptide with the sequence of bovine pre-lysozyme c2, a polypeptide with the sequence of the signal peptide of bovine pre-lysozyme c2, and a DNA segment which comprises a segment which encodes bovine pre-lysozyme c2 or the signal peptide thereof.

The present invention provides a novel, surprisingly and unexpectedly efficient method of producing large quantities of a biologically active and easily-purified animal lysozyme c.

Various terms used in this application are defined generally as follows:

(1) The term "culture" means a propagation of cells in a medium conducive to their growth, and all subcultures thereof.

(2) The term "subculture" means a culture of cells grown from cells of another culture (source culture) , or any subculture of the source culture, regardless of the number of subculturings which have been performed between the subculture of interest and the source culture.

(3) The phrase "animal pre-lysozyme c" means the pre-form of the animal lysozyme c protein, which consists of the naturally occurring signal peptide of the lysozyme c fused to the amino-terminus of the mature lysozyme c. Pre-bovine lysozyme c2 made in accordance with the invention has the 147 amino acid sequence indicated in Example 1, including the 18 amino acid signal peptide and the 129 amino acid mature protein. Pre-human lysozyme c made in accordance with the invention has the 148 amino acid sequence described in Example 12, including the 18 amino acid signal peptide and the 130 amino acid mature protein. (4) The amino acids, which occur in the various amino acid sequences appearing herein, may be identified according to the following three-letter or one-letter abbreviations:

(5) The nucleotides, which occur in the various nucleotide sequences appearing herein, have their usual single-letter designations used routinely in the art; (6) The phrase "animal lysozyme c" means a lysozyme c from an organism of the kingdom Animalia and of either the class Aves or the class Mammalia (birds and mammals) .

Methods of transforming Pichia pastoris with DNAs, including vectors comprising genes for expression of heterologous proteins, are known in the art. Similarly, methods are known for culturing P. pastoris cells, which have a gene for an heterologous protein, in order to express the heterologous protein from such a gene. Further, methods are known for isolating from the medium of such cultures of P. pastoris heterologous protein that is secreted from the cells into the medium. These known methods can be employed to make cultures of P. pastoris according to the invention and to carry out the method of the invention with such cultures to make an animal lysozyme c. Certain of these methods are described in some detail in the examples which follow. In a DNA according to the invention which comprises a selectable marker gene, any selectable marker gene may be employed which is functional in P. pastoris cells to allow cells transformed with the DNA of the invention to be distinguished from cells not so transformed. 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 which complements an auxotrophic 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 providing complementation for auxotrophic mutations are the P. pastoris HIS4 gene (for transformation of His4~ strains of P. pastoris) , the S. cerevisiae HIS4 gene (for transformation of His4~ strain of P. pastoris) , the P. pastoris ARG4 (arginosuσcinate lyase) gene (for transformation of 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 of the invention, including the DNAs for transforming P. pastoris to make an animal lysozyme c (referred to herein as "transforming DNAs of the invention") , the promoter of any P. pastoris gene can be employed to drive transcription of the animal pre-lysozyme c-encoding DNA segment of the DNA of the invention. Preferably, the promoter driving transcription of the animal pre-lysozyme c-encoding segment will be the promoter of a P. pastoris gene whose transcription is tightly regulated by factors easily varied in P. pastoris cultures, e.g., the carbon source for culture growth. Among such P. pastoris genes, a preferred one is the major alcohol oxidase gene (AOX1 gene) , the promoter of which is essentially completely inactive unless methanol is present in the culture medium but which is highly active, yielding high levels of the gene product, when methanol is present. In the promoter segment of a DNA of 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 of the invention (including the transforming DNAs) has a subsegment which encodes a polyadenylation signal and polyadenylation site in the transcript, from the promoter of the DNA of the invention, which transcript includes the transcript of the animal pre-lysozyme c-encoding DNA segment of the DNA of the invention, and a subsegment which provides a transcription termination signal for transcription from said promoter. The entire "terminator segment" of a transforming DNA of the invention will be preferably taken from one P. pastoris protein-encoding gene, which may be the same as, or different from, the P. pastoris gene from which the promoter of the DNA of the invention is taken, which drives transcription of the animal pre-lysozyme-encoding segment and polyadenylation signal- and site-encoding segments. In the preferred case, both the terminator segment and the promoter controlling transcription of the DNA segment encoding the animal pre-lysozyme c will be from the P. pastoris AOX1 gene.

In a DNA according to the invention, the DNA segment encoding an animal pre-lysozyme c can be any DNA segment which has a sequence that is free of introns, that includes a translation-start-site-encoding triplet (referred to herein as "translation-start triplet") and translation-stop-signal-encoding triplet (referred to herein as "translation stop triplet") and that encodes, 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 a sequence for a bovine pre-lysozyme c2, is the approximately 460 bp EcoRI site-terminated segment 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 more nucleotide changes, which do not alter the length or the amino acid sequence of the encoded polypeptide, is also a bovine pre-lysozyme c2-encoding segment that could be employed in a DNA according to the invention.

Following procedures well-known in the molecular biological arts, DNA segments with sequences encoding pre-lysozyme c's other than bovine lysozyme c2 can be isolated. For example, such DNA segments can be isolated using probes, based on the amino acid sequence of the mature lysozyme c of interest, to screen a cDNA library of the involved species to isolate a suitable cDNA, which includes the DNA segment of interest. See, also, the examples that follow. Preferred among the lysozyme c's, other than the bovine, are the human.

5 A DNA of the invention which comprises a segment which encodes 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 encodes bovine pre-lysozyme c2 (with either __Q histidine or lysine, but preferably histidine, at position 98 of the mature protein portion) or a sequence of 54 base pairs which encodes the signal segment of bovine pre-lysozyme c2 (see sequence- in last full paragraph of Example 1 below) ; and (B) is (i) capable,

■_jc upon transformation into a host, of being expressed to make the pre-lysozyme c2 or the signal segment thereof (fused to a mature animal lysozyme c2, or any other desired protein) , or (ii) capable of being ligated into another DNA which has the capability to effect expression

2o of a protein as described in the preceding clause

(B) (i) . Thus, for example, a DNA of the invention which comprises a segment which encodes the signal segment of bovine pre-lysozyme c2 can be a DNA which comprises a segment which encodes the fusion polypeptide wherein the

₂_ signal segment of bovine pre-lysozyme c2 is fused to the amino-terminus of mature human milk lysozyme.

In the DNA according to the invention with promoter and terminator segments bracketing a pre-lysozyme c-encoding segment, the animal pre-lysozyme

₃₀ c-encoding segment is positioned and oriented, with respect to the P. pastoris promoter and the terminator segments, operatively for transcription of the animal pre-lysozyme c-encoding segment under control of the promoter of said promoter segment into a transcript which is capable of providing expression in P. pastoris of the animal pre-lysozyme c. Persons of skill understand how to effect such positioning and orientation, operative for providing expression in P. pastoris of the animal pre-lysozyme c from the DNA of the invention, provided that the animal pre-lysozyme c-encoding segment is transcribed from said promoter. As understood in the art, the animal pre-lysozyme c-encoding segment, including its translation start and translation stop triplets, must be downstream of the transcription-initiation site with respect to the 0 direction of transcription from said promoter, and upstream from the polyadenylation signal- and polyadenylation site-encoding subsegment of the terminator segment, wiύch, in turn, must be upstream of the transcription termination site of the terminator 5 segment. The segment encoding the animal pre-lysozyme c must be oriented with the translation start triplet upstream from the translation stop triplet. Preferably, no transcription terminator site will be located between the promoter and the polyadenylation site upstream from o the transcription terminator of the terminator segment at the downstream end of the DNA of the invention. Preferably, in a DNA according to the invention, in the direction of transcription from the promoter which drives transcription of the animal lysozyme c-encoding segment, 5 between said promoter and the transcription terminator which terminates said transcription from said promoter, there will be only a single, long open reading frame which has the sequence encoding an animal pre-lysozyme c. Preferably, as well, this transcript will have a _Q single polyadenylation signal and site.

Reference to "downstream" and "upstream" in a DNA of the invention means "downstream" and "upstream," respectively, with respect to the direction of transcription from the promoter which drives 5 transcription of the animal pre-lysozyme c encoding segment. Construction of several transforming DNAs according to the invention, including pSL12A, pBLll, and pHLZ103, is described in the examples that follow. Of these three plasmid DNA's, the

ClaI-(BamHI/BglII) fragment of pSL12A, which includes the Clal-site terminated bovine pre-lysozyme c2 expression cassette and the P. pastoris HIS4 gene, and the ClaI-(BamHI/HpaI) fragment of pBLll, which includes the Clal-site-terminated bovine pre-lysozyme c2 expression cassette and the S. cerevisiae ARG4 gene, and the ClaI-(BamHI/BglII) fragment of pHLZ103, which includes the Clal-site terminated human pre-lysozyme c expression cassette and the P. pastoris HIS4 gene, are also DNAs according to the invention.

A transforming DNA according to the invention may include elements necessary for its selection and replication in bacteria, especially E. coli, whereby the production of large quantities of the DNA by replication in bacteria will be facilitated. In this regard, a preferred DNA of 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 of the invention can, after transformation into P. pastoris, be maintained as an episomal DNA (e.g., 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 origins of replication and ARS's functional in Pichia pastoris are known in the art.

A DNA of the invention may integrate via homologous recombination into the P. pastoris genome in a certain proportion of cells. Such integration can be accomplished by transformation of P. pastoris cells with linearized or circularized plasmids, or linearized fragments of either, comprised of homologous DNA sequences. Thus, in the preferred P. pastoris cultures of the invention, the DNA of the invention will be maintained in the cells as part of the cells' genomes. Methods for causing integration of heterologous DNA into yeast genomes, including those of P. pastoris, are well known in the art and may be applied with the DNAs of the instant invention. See, e.g., European Patent Application Publication Number 0 226 752. In particular, as illustrated in the examples that follow, the probability 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. Further, targeting of the site of integration to preferred sites in the P. pastoris genome is accomplished by incorporating in the transforming DNA "targeting segments," which are segments, usually at the two ends of a linearized DNA according to the invention, which segments have sequences 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, conveniently by cutting with restriction enzyme(s) that cut(s) at a suitable site or sites, to yield a linear transforming DNA of the invention with targeting segments at its ends. Suitable targeting segments will be at least about 200 bp in length. Examples of treating transforming plasmid DNAs of the invention in this way are provided in the Examples. For example, a linearized transforming plasmid DNA is obtained by cutting with Sad (an isoschizomer of SstI) a derivative of pAO804, which has an animal pre-lysozyme-c-encoding segment (which lacks a Sad site) inserted at 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-encoding segment (which lacks a Bglll site) inserted at the EcoRI site.

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 to 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, unexpectedly, the animal pre-lysozyme c signal sequences are correctly recognized and processed in the P. pastoris secretory pathway. An animal lysozyme c isolated from culture media of cultures of the present invention is judged to be the same as the authentic lysozyme c by several criteria. First, the N-terminal sequence of the mature, secreted protein is identical to that of the naturally-occurring enzyme. Second, Western blot analysis of the secreted lysozyme c reveals a single immunoreactive species of the same molecular weight as the naturally-occurring, mature enzyme. Finally, in bioassays based upon the ability of the secreted lysozyme c to lyse Micrococcus luteus (formerly named Micrococcus lysodeikticus) cells, the protein secreted to the media from P. pastoris cells has a specific activity essentially the same as that of the lysozyme c isolated from the sources in which it occurs naturally. The surprising and advantageous result that an animal lysozyme c produced according to the invention is not contaminated with significant quantities of fragments of the secreted enzyme (or incorrectly processed protein of molecular weight greater than that of the mature enzyme) is established by electrophoretic 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 protein purification art, because of the high concentration of the enzyme and low concentration of contaminating proteins and protein fragments in the media. A simple two-step procedure for purification is described in the Examples below. The animal lysozyme c's provided by the present invention can be used as known in the art for such lysozymes generally. For example, the lysozyme c's can be used for digestion of E. coli to isolate therefrom 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, USA (1982) . Like other lysozymes, the lysozyme c's provided by the 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 an agent for treatment of microbial infections of animals, including humans. When the lysozyme c is used in association with other antibiotics, however, it is important to avoid aminoglycosidic antibiotics, such as neomycin B, gentamicin c_la, kanamycin A or dihydrostreptomycin, the structures of which are related to the saccharidic substrate of the enzyme. Other reported uses for the lysozyme c's provided by the methods of the present invention include the treatment of various pains, allergies, and inflammations, colitis, herpes zoster, rheumatic fever, rheumatoid arthritis, as well as in pediatrics (maternalization of bovine milk by the addition of lysozyme). See, e.g., Jolles et al., Mol. Cell. Biochem. 63, 165 (1984). Another possible use for the lysozyme c's provided by the present invention, especially the bovine lysozyme c's, is as an additive to feed for ruminants, to increase the efficiency with which such animals utilize feed for growth. Rumen bacteria are an important nutrient source for ruminants. However, an important factor in their utilization is the ability of the animal to break open the bacteria. As previously discussed, the latter are surrounded by peptidoglycans which contain glycosidic bonds which can be split by lysozyme. Consumption of a lysozyme c, as a food additive, may result in an increased level of the lysozyme in the fundic region of the ruminant abomasum, which, in turn, may increase the level of digestion of the microbial mass which enters such region of the stomach from the rumen. Increased digestion of this microbial mass may result in availability to the ruminant of an increased quantity of bacterial contents for energy production and growth. The following examples describe and illustrate the present invention in greater detail.

All of the patents and publications referred to in this application are hereby incorporated by reference into the application.

EXAMPLE 1

ISOLATION AND CHARACTERIZATION OF FULL-LENGTH cDNA

CLONE ENCODING BOVINE LYSOZYME C

A partial bovine lysozyme c genomic clone, designated pLl, and its DNA sequence were obtained from Dr. Gino Cortopassi. Fresh bovine abomasum tissue was obtained from the Talone Meat Packing Co. , Escondido, California, U.S.A. ΛgtlO and E. coli strain C600HF1 were obtained from Clonetech Labs, Inc., 4055 Fabian Way, Palo Alto, California 94303. The packaging-kit used to package λgtlO 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 were used as recommended by the suppliers.

After total intact RNA was isolated from approximately 20g of bovine abomasum tissue, by a modification of the method of Shields and Blobel, Proc. Natl. Acad. Sci. U.S.A. 74:2059 (1977), polyadenylated RNA species were separated from the nonpolyadenylated species by a standard technique employing an oligo-dT column.

Since the fundic region of ruminant abomasum contains a high concentration of pancreatic ribonuclease,speed and proper handling of the tissue, including maintenance of a low temperature, is essential in order to preserve the RNA.

Briefly, frozen bovine abomasum tissue was powdered in a stainless steel manual pulverizer in the presence of liquid nitrogen. The powdered tissue was added to 100 ml of 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 allowed to incubate at room temperature for 15 minutes.

After incubation, the preparation was extracted with an equal volume of PCIA (phenol:chloroform: isoamylalcohol, 25:24:1) followed by extraction with an equal volume of CIA (chloroform:isoamylalcohol, 24:1). The resulting DNA/RNA mixture was precipitated by adjusting the NaCl concentration to .25 M, adding 2 volumes of ethanol, and placing the solution at -20°C overnight. After centrifugation at 5000 x g for 60 minutes, the DNA/RNA was resuspended in 20 ml of ETS buffer (0.1 M Tris, pH 7.6, .01 M EDTA, 0.2% SDS). PCIA and CIA extractions, and DNA/RNA precipitations were performed as described above. The DNA/RNA precipitate was collected by centrifugation and was resuspended in 32 ml of 10 mM Tris, 10 mM EDTA, pH 7.4.

Following resuspension, the RNA present in this solution was enriched by aliquoting the preparation into 4 tubes, layering it over a 4 ml CsCl (lg CsCl/ml) cushion in 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 0.25M NaCl and then adding two volumes of 95% ethanol) , and resuspended in 5 ml of ETS buffer. The polyadenylated (poly (A)+) RNA was selected from the solution by affinity chromatography on oligodeoxythymidylate cellulose columns, as described by Aviv et al., Proc. Natl. Acad. Sci. U.S.A. 69:1408 (1972). After the RNA was bound to the oligo-dT column in 0.5 M NETS buffer (.5 M NaCl, .01 M Tris, .01 M EDTA and 0.2% SDS, pH 7.6), the column was washed with 30 ml of 0.5 M NETS. This was followed by elution of polyadenylated RNA with 5 ml of ETS buffer and ethanol precipitation.

Ten μg of the polyadenylated RNA was denatured in 2mM CH₃HgOH (Alpha Products, Danver, Massachusetts) for 5 minutes at room temperature. Following the protocol 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 generated from total polyA-mRNA and constructed into ΛgtlO (Gubler et al., Gene 25:263 (1983); Lapeyer et al., Gene 37:215 (1985)). Briefly, MMLV reverse transcriptase was used to make a cDNA from the mRNA. This was followed by RNase H treatment and DNA polymerase I-mediated second cDNA strand synthesis to generate double-stranded cDNA. Following second-strand synthesis, the cDNA was blunt-ended with S.^ nuclease and the ends further polished with E. coli DNA polymerase I Klenow fragment (Telford et al., Proc. Natl. Acad. Sci. 76:2590 (1979)). EcoRI adaptors (Wood et al., Nature 312:330 (1984)) were ligated to the double-stranded cDNA eliminating the need for methylation and subsequent EcoRI digestion of the cDNA. Excess adaptors were removed by chromatography over a Sepharose CL-4B column equilibrated with lOmM Tris, pH 7.4, ImM EDTA. Those column fractions containing cDNAs in excess of 400 bp were pooled and ligated into cDNA bacteriophage cloning vector ,λgtl0, which had been EcoRI-digested and phosphatase-treated. Ligations were performed in a 5 μl volume and incubated at 16"C for 18 hours. The reaction contained 1 μg of gtlO vector (final concentration = 200 μg/ml) and 50-100 ng of cDNA (2- to 4-fold molar excess of insert over vector) . The resulting cDNA was packaged, using a lambda packaging kit, and the resulting vectors were plated on E. coli strain C600HF1, and plaques were screened with radiolabelled pLl. Those plaques which were identified as containing cDNA coding at least part of bovine lysozyme c were plaque-purified (Maniatis et al., supra) and screened with both pLl and an oligonucleotide probe synthesized from the DNA sequence information provided by Dr. Cortopassi. The sequence of such probe is that of one strand of a DNA segment at the 5'-end of exon 2 of the bovine lysozyme c gene, which encompasses sequences coding for amino acids 29-38 of mature bovine lysozyme c₂, and is as follows: 3'-AAC ACA AAC TGG TTT ACC CTT TCG TCA ATA-5'.

Plaque lifts were performed essentially as described by Benton et al.. Science 196:180 (1977).

Nitrocellulose filters were prehybridized for 4 hours at 42°C in 5 x SSPE (.9 M NaCl, .04 M NaOH, .05 M NaH₂P0₄-H₂0, .005 M EDTA, pH 7.0), 5 X Denhardt's [50 x Denhardt's: 5g Ficoll 400 (Pharmacia, Inc., Piscataway, New Jersey, catalog No. 17-0400-01, average molecular weight approximately 400,000 daltons), 5g polyvinylpyrrolidone PVP-360 (Sigma Chemical Co. , St. Louis, Missouri, average molecular weight approximately 360,000 daltons), 5g bovine serum albumin in H₂0 to bring 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 pLl was added directly to the prehybridization solution at 1 x 10⁶ cpm/ml, the filters were hybridized for 16 hours at 42^βC and then washed several times, for 15 minutes per wash, in .1 x SSPE, .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 hours at 42^βC. They were then hybridized for 3 hours in the same buffer at 42^βC and washed several times in 1 x SSPE at 45^βC.

One of the plaque-purified clones which hybridized to the oligonucleotide was designated ΛBL3, and its cDNA insert size was determined, after EcoRI restriction enzyme digestion of phage "mini-preps"

(Benson et al., Biotechniques, 2.3, 126 1984)), to be 950 bp.

The nucleotide sequence of the cDNA insert of clone /\BL3 was determined using subcloned M13 templates and the dideoxynucleotide protocol described by Sanger et al., Proc. Natl. Acad. Sci., U.S.A. 74:5463 (1977) and the M13 Cloning/Dideoxy Sequencing Manual, Bethesda Research Laboratories, Inc., Gaithersburg, Maryland (1980) to be:

10 20

G A A T T C A T G T C T T A C G G T C A A G G G A C T EcoRI Adaptor T 30 40 50 C T C G T T A T T C T G G G G T T T C T C T T C C T L V I L G F L F L

60 70 80

T C T G T C G C T G T C C A A G G C.A A G G T C T T T S V A V Q G K V F

Mature

Protein

Starts >

90 100

G A G A G A T G T G A G C T T G C C A G A A C T C T G E R C E L A R T L

no 120 130

A A G A A A C T T G G A C T G G A C G G C T A T A A G K K L G L D G Y K

140 150 160 G G A G T C A G C C T G G C A A A C T G G T T G T G T

V N W

170 180

T T G A C C A A A T G G G A A A G C A G T T A T A A C L T K W E S S Y N

190 200 210

A C A A A A G C T A C A A A C T A C A A T C C T A G C T K A T N Y N P S

220 230 240

A G T G A A A G C A C T G A T T A T G G G A T A T T.T

250 260 270

C A G A T C A A C A G C A A A T G G T G G T G T A A T Q I N S K W W C N

280 290

G A T G G C A A A A C C C C T A A T G C A G T T G A C D G K T P N A V D 300 310 320 G G C T G T C A T G T T C C T G C A G C G A A T T A G C H V S C S E L

330 340 350

A T G G A A A A T G A C A T C G C T A A A G C T G T A M E N D I A K A V

360 370

G C G T G T G C A A A G C A T A T T G T C A G T G A G C A K H I V S E

*

380 390 400

C A A G G C A T T A C A G C C T G G G T G G C A T G G Q G I T A W V A W

410 420 430

A A A A G T C A T T G T C G A G A C C A T G A C G T K S H C R D H D V

440 450 A G C A G T T A C G T T G A G G G T T G C A C C C T G S S Y V E G C T L

460 470 480

T A A C T G T G G A G T T A T C A T T C T T C A G C T ***

490 500 510

C A T T T T A T C T C T T T T T C A T A T T A A G G A

520 530 540

A G T G A T A G T T G A A T G A A A G T T T A T A C C

550 560

A C C A T T G T T T C A A A C A A A T A A C A T T T T

570 580 590

T A C A G A A G C A G G A G C A T G T G G T C T T T C

600 610 620

T T C T A A G A A G C C T A A T G T T T A T C T A A T 630 640

G T G T T A A T T G T T T G A T A T T A G G C C T A C

650 660 670

A A T A T T T T T C A G T T T G C T A A T G A A A C T

680 690 700

A A T C C T G G T G A A T A T T T G T C T A A A C T C

710 720

T T A A T T A T C A A A T A T G T C T C C A G T A C A

730 740 750

T T C A G T T C T T A A T T A A A G C A A G A T C A T

760 770 780

T T A T G T G C C T T G C T G A T C A T G A A G G A A

790 800 810

T A T A A A G A G G G A T T A G A T G A G C T G T T T

820 830

C T T T T C C T T A A T T T T A T T A G C A T A G A T

840 850 860

C A T G C A T T A T G A C C A A A T T T A G A G G C

870 880 890

A G A T A A G T A T T G A A A T A A C T A A C C A C A

900 910

G A T A T G A A A T T A T G C A T G C T G T A A A A A

920 930 940

A T A C A A A C A T T T T C A T T A A A G G C

950 960

C C T T G A C C G T A A G A C A T G A A T T C EcoRI Adaptor The results indicated that the vBL3 cDNA insert consists of a 436 bp coding region of bovine lysozyme c2 and 482 bp of 3'-untranslated noncoding sequence. The 3'-noncoding sequence in ^BLS does not contain a polyadenylation signal or a poly (A)⁺ tail. DNA sequencing at the 5'-terminus of the cDNA insert in Λ^βk3 indicated that the insert contains 49 bp encoding the C-terminal portion of the protein signal sequence, but 0 does not contain "the ATG -triplet corresponding to the translation initiation codon of the pre-lysozyme c2 mRNA. Thus, the cDNA insert encodes sixteen amino acids amino-terminal to the a ino-ter inus of the mature protein. The amino acid sequence of the protein corresponding to the cDNA insert of _/ΛBL3, derived from the nucleotide sequence, and indicated in the foregoing sequence below the nucleotide sequence, has a histidine at amino acid position 98 of the mature protein, whereas o the published protein sequence (Jolles et al., J. Biol. Chem. 259, 11617 (1984)) indicates a lysine at this position. The presence of the histidine residue at this position was con irmed by N-terminal analysis of the C-terminal CNBr fragment of the protein expressed from 5 the insert. Furthermore, sequencing of lysozyme c2 from bovine abomasum tissue showed that the amino acid at position 98 is in fact histidine, at least in the animal used as the source of RNA for this work.

Subsequent sequencing of "the bovine lysozyme _Q mRNA was conducted using the variations of the dideoxy sequencing protocol recommended in Karanthanasis, Bethesda Res. Lab- Focus 4s3, 6 (1982) (Bethesda Research Laboratories, Inc.) _. Four μg of abomasum poly (A)⁺ RNA was used as a template and a synthetic 27-residue ₅ oligonucleotide of sequence

5'-dGCAAGCTCACATCTCTCAAAGACCTTG-3', which was copied from the DNA sequence of the 5'-end of the bovine lysozyme gene and which was synthesized by standard phosphoramidite chemistry on an Applied Biosystems synthesizer (Model 380A) , was used for priming the extension by reverse transcriptase. The sequencing of the mRNA revealed that the A^BL3 insert lacks 5 bp of coding sequence at its 5'-end, including an initiating ATG codon. In addition, the RNA sequence indicated that the base at position 2 of the >BL3 insert should be a G rather than an A. Such sequencing further revealed the amino acid sequence of the amino-terminus of the bovine lysozyme c signal sequence, including the position of the initiating methionine. Such signal sequence was found to be as follows: M K A L V I L G F L F L S V A V Q G. By comparison to known sequences (Jolles et al., supra.), the bovine lysozyme c encoded by the cDNA insert of clone \BL3 closely resembles bovine lysozyme c2, the most abundant of the three bovine lysozyme c's. The only difference between it 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 encoded by the cDNA insert in ΛBL3 is lysozyme cl or c3. The single, conservative amino acid change between the lysozyme encoded by A^{BL3 and} bovine lysozyme c2, as reported by Jolles et al., which results from two nucleotide changes, can be explained most probably by allelic differences. Regardless, as described in the subsequent examples, the lysozyme c2 with histidine at position 98 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, on one each of the 5'- and 3'-ends of the bovine pre-lysozyme c2 coding region of ABL3, prior to insertion of the coding region into Pichia pastoris 0 expression vector pA0804 (described below) , in order to modify the coding region to make the region compatible with the expression vector and to make some other desired changes. The 3'-end of the coding region was mutagenized to remove the 482 bp of noncoding sequence and to 5 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, o was mutagenized to introduce a seven-base-pair^"sequence (5'-ATGAAGG-3') that is the correct sequence as indicated by RNA sequencing. This modification at the 5'-end completed the coding sequence for the N-terminus of the signal sequence (Met-Lys-Ala...) and added a second EcoRI 5 restriction site directly before the Met codon-encoding ATG. Clone pBLI6C was identified as possessing both of the desired mutagenized ends.

In the first in vitro mutagenesis procedure, a mutagenic oligomer, of sequence 5'-dGAGCTGAAGAATGATATTA- _Q CAGGGTGCAACCC-3', was synthesized and used as a primer for mutagenesis of the 3'-end of the bovine lysozyme gene insert in ΛBL3 and for screening. Four of the positive plaques from a second hybridization screen were used to prepare template DNA for sequencing. One of the template 5 DNAs, pBL4C, was found to contain the correct sequence, with the EcoRI site 3' of the TAA encoding the translation termination codon. A second mutagenic oligomer, of sequence 5'-dCCAGAATAACGAGAGCCTTCATGAATTCGAGCTCGGTACCCGGGG-3', was used to prime from pBL4C template DNA for the second mutagenesis. For this modification, a synthetic oligonucleotide of sequence 5'-dTAACGAGAGCCTTCATGAATTC-3' was used for screening. A clone, pBLI6C, which was identified as having the desired sequence, including 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 make template DNA.

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

The generalized Pichia pastoris expression cassette vector pAO804, illustrated in Figure 1, has two Bglll sites which bracket a fragment which has, moving clockwise in Figure 1 from the Bglll site about 100 bp from a Clal site, (1) an approximately 900 base pair (bp) fragment (designated "5'-AOXl" in the Figures) , which is a "promoter segment" according to the invention and is from the P. pastoris major alcohol oxidase (AOX1) gene locus, including the promoter and the 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) an approximately 300 bp fragment (designated "3'-AOXl" in the Figures), which is a

"terminator segment" according to the invention and is from the P. pastoris AOX1 gene, said fragment having at its 5'-end the EcoRI linker, having at the 3'-end a Clal site, and including the polyadenylation signal- and site-encoding segments and the transcriptional terminator of the AOX1 gene; (3) an approximately 2700 bp Bglll fragment (in the orientation indicated in Figure 1) comprising the P. pastoris histidinol dehydrogenase (HIS4) gene, to provide a selectable marker to His4~ strains of P. pastoris (Cregg et al., supra), said 0 fragment inserted at the BamHI site of pBR322 used to make pAO804; and (4) an approximately 800 bp fragment (designated "3'-from-A0Xl" in the Figures) of 3'-sequence taken from downstream from the transcriptional terminator of the P. pastoris A0X1 gene locus, said fragment 5 terminated at one end with a Bglll site (made by modifying pBR322 at its PvuII site) and at the other end with the remnant of combining an Xhol site with the pBR322 Sail site. pAO804 also includes several fragments from pBR322: (1) the approximately 350 bp segment from o the Clal site at the end of the fragment labeled "3-AOX1" to the remnant of the BamHI site at one end of the segment with the P. pastoris HIS4 gene; (2) the approximately 280 bp from the remnant of the BamHI segment at the other end of the segment with the 5 P. pastoris HIS4 gene to the remnant of the Sail site at one end of the "3'-from-AOXl" segment; and (3) the approximately 2320 bp fragment from the remnant of the pBR322 PvuII site (not shown in the Figures) within a few bases of the Bglll site at one end of the "3'-from-AOXl" _Q segment (and outside that segment) to the Clal site near the Bglll site at 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 beta-lactamase gene (providing ampicillin _ resistance to bacteria transformed with the plasmid) .

Construction of pAO804 is described in Example 15. An "expression unit" bounded by the Bglll sites (or, e.g., the Clal-Bglll fragment which includes the fragment bounded by the Bglll sites) shown for pAO804 in Figure 1 is made by inserting at the EcoRI site an intron-free DNA segment the transcript of which, together with the polyadenylation signal and site provided by the 3'-AOXl segment, from the AOX1 promoter will be translated into the protein of interest. The approximately 900 bp Bglll-EcoRI segment, comprising the AOX1 promoter and transcription start site, and the approximately 800 bp genomic segment from 3' of the AOX1 gene ("3'-from-AOXl" in the Figures) are used for site-directing integration of the Bglll-site- terminated expression unit into the AOX1 locus of P. pastoris cells transformed with the plasmid.

With the bovine pre-lysozyme c2 coding fragment bounded by EcoRI sites from pBLI6C inserted into the unique EcoRI site of pAO804 in such a manner that the lysozyme sequence is oriented operatively for transcription from the A0X1 promoter, pre-lysozyme c2 will be expressed, under transcriptional control of the AOX1 promoter, when P. pastoris cells transformed with the plasmid (or the Bglll-site-terminated expression unit thereof) are grown with methanol as a carbon source, so that the promoter is active in transcription.

Studies of P. pastoris have revealed that certain methanol-regulated promoters from P. pastoris, such as that of the major alcohol oxidase gene (AOX1) 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 high level transcription, even in single copy, and are tightly regulated, thus permitting the expression period to be limited. In ethanol-, glycerol-, or glucose-grown wild-type cells, alcohol oxidase is absent. However, it constitutes as much as 30% of the total cellular protein in cells grown on methanol.

AOXl deletion mutants produce approximately 15% of wild- ype alcohol oxidase enzyme activity (from the minor alcohol oxidase (A0X2) gene), whereas A0X2 deletion strains produce wild-type levels of alcohol oxidase when cells are grown on methanol.

The P. pastoris AOXl promoter is among the 0 strongest and most tightly regulated promoters known. When P. pastoris cells are grown on glucose or glycerol, the AOXl promoter is repressed and alcohol oxidase mRNA is not made and alcohol oxidase is not expressed. In contrast, when such cells are grown on methanol, the AOXl 5 promoter is induced and the alcohol oxidase expressed from the AOXl gene can constitute as much as 30% of the total cellular protein under certain conditions. The AOXl gene, including its promoter, has been isolated and extensively characterized. Studies indicate that o regulation of AOXl occurs at the transcriptional level and that, when the gene is transcribed, the AOXl mRNA is an abundant species in steady state mRNA.

Although the P. pastoris AOX2 gene is also methanol-regulated, it is not as highly transcribed as 5 AOXl. Consequently, although they are able to grow on methanol, AOXl-defective mutants, which are AOX2 normal, have a significantly longer generation time on methanol than wild-type strains.

. Double-stranded DNA from the replicative form of 0 pBLI6C was cut with EcoRI, and the resulting, approximately 460 bp, EcoRI fragment was ligated into EcoRI-cut and alkaline phosphatase (Boehringer Mannheim Biochemicals, Ine.)-treated pAO804 DNA. One of the two resulting expression plasmids, which is designated pSL12A 5 and which is illustrated in Figure 2, has the correct 5' to 3' orientation of the bovine lysozyme coding sequence relative to the position and orientation of the AOXl promoter and transcription start site in the "5'-AOXl" promoter segment and the "3'-AOXl" terminator segment.

₅ EXAMPLE 3

TRANSFORMATION OF GS115 CELLS WITH pSL12A; STRAIN A37

Pichia pastoris strain GS115 ((His4~); Cregg

20 et al., supra), a histidine-requiring auxotroph of P. pastoris, which was previously determined to be defective in histidinol-dehydrogenase (HIS4) and to possess a reversion frequency to histidine prototrophy of less than 10~⁸, was used as the gene expression host for

-c transformation with pSL12A. P. pastoris GS115 grows . efficiently on methanol in a defined minimal medium supplemented with histidine at a high cell density and is desirable as a host system for purposes of single-cell protein production. The strain contains no bacterial

20 replicons, antibiotic resistance genes, or other heterogenous sequences that might be considered a potential biological hazard.

Plasmid pSL12A was digested with restriction endonuclease Bglll and the resulting mixture of DNA

25 fragments was used to transform P. pastoris strain GS115 employing the whole-cell LiCl yeast transformation system. Details of the transformation procedure are provided below.

Digestion of pSL12A with Bglll releases a DNA

_3n fragment with ends homologous to regions 5' and 3' of the P. pastoris AOXl gene, as described above. When this fragment is transformed into a Hisjfc^" P. pastoris strain and maintained there under selective conditions (growth in the absence of histidine) , a replacement-type

__[. integration at the AOXl locus of the Bglll-site- terminated, expression-cassette-containing fragment (including the HIS4 gene) is effected in some cells. This integration results in cells having a phenotype designated Mut⁺/~ (for "methanol utilization +/-"), .due to loss of the AOXl gene product, but retention of the minor alcohol oxidase gene product (AOX2) , allowing slow growth on methanol. This one-step gene replacement technique, resulting in chromosomal integration of the heterologous gene, avoids difficulties associated with plasmid instability, distribution, and copy number and 0 provides a high fidelity expression system. The technique also results in the incorporation of a minimum amount of heterologous DNA into the P. pastoris genome. Cells bearing the proper integration will be His⁺ and can be distinguished, by a lessened growth rate on 5 methanol, from cells in which the integration has occurred at sites other than the AOXl locus. In cells in which integration has not occurred, or in which integration has not occurred at the AOXl locus, the AOXl gene remains functional and the ability of such cells to o 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 5 fragments into GS115. Since such method does not require the generation and maintenance of spheroplasts, it is more convenient and less time-consuming than the spheroplast method. However, the spheroplast technique, which is exactly as described in Cregg et al., Mol. Cell. 0 Biol. 5,3376 (1985) with the one exception that the regeneration agar does not contain sorbital, but contains 0.6 M KC1 in its place, and which may also be employed to transform the P. pastoris cells, is preferred in that such method yields a greater number of transformants. 5 A 50 ml shake-flask culture of P. pastoris strain GS115 was grown in YPD (lOg Bacto-yeast extract, 20g Bacto-peptone, 20g dextrose, 20g Bacto-agar, 1000 ml distilled water) at 30"C with shaking to an ODg_0Q of approximately 1.0 (5 x 10⁷ cells/ml). The density at harvest can be from 0.1 to 2.0 ODg₀₀ Units. After the cells were washed once in 10 ml of sterile H₂0, and after pelleting by centrifugation at approximately 1500 x g for 3-5 minutes, they 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 occasional shaking, for one hour.

A sterile 12 x 75 mm polypropylene tube, containing 10 μg of Haelll-digested E. coli carrier DNA, 2 μg of the 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 minutes. Approximately 0.1-20 μg of vector DNA can be used to transform the cells. 0.7 ml of 40% polyethylene glycol (PEG₃₃₅₀, Fisher Scientific, Fair Lawn, New Jersey) in lithium chloride + TE buffer was added, and the tube was vortexed briefly and again incubated in a water bath for 30 minutes at 30°C. After the cells were heat shocked for 5 minutes at 37°C, the mixture was centrifuged and then resuspended in 0.1 ml sterile H₂0. The cells were then spread on selective plates (0.67% Yeast Nitrogen Base, without amino acids, 2% glucose, 2% agar) and then incubated for three days at 30°C. Transformants with the Mut⁺' phenotype were identified as follows: His⁺ transformants were plated to obtain colonies originating from single cells. Individual colonies were transferred to minimal glucose (2%) master plates. After overnight incubation at 30^βC, the masters were replica-plated to minimal, low-glucose (0.2%) plates and again incubated overnight at 30°C. The following day, the low-glucose plates were replica-plated to minimal methanol (0.5%) plates and then incubated at room temperature for 2-5 days. Colonies showing visible growth were scored at Mut⁺ and those with no visible growth were scored as Mut⁺' .

Transformant colonies were recovered from the surface of the agar plates by adding 5 ml of sterile H₂0 to the plate and suspending the cells with a spreader, and then sonicating and plating the cells, after dilution, to obtain single-cell colonies. The sonication step was necessary to separate P. pastoris cells, since such cells tend to grow in clumps.

Approximately 20% of the 276 His⁺ transformants recovered grew slowly on methanol and, consequently, revealed the above-described Mut⁺'~ phenotype. Southern blot analysis of 9 Mut⁺/~ transformants showed identical hybridization patterns and confirmed that the predicted integration in the AOXl locus had occurred in the cells. One of these Aoxl^" 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 grown first in 0.67% Yeast Nitrogen Base (Difco Labs, Detroit, Michigan) and 2% glycerol as the sole carbon and energy source at 30^βC with shaking for approximately one day. After centrifugation and washing of the cell pellet in sterile water, the cells were transformed to media containing 0.5% methanol, in place of glycerol, as the sole carbon source. Incubation at 30^βC with shaking was then continued and samples were harvested after 1 and 4 days of growth in methanol.

After 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 slot blot apparatus. The nitrocellulose filter was processed through a standard Western blot protocol, using a 1:2500 dilution of antisera from rabbit #156 (described in Example 6) .

The results of the Western slot blot assay, after application of 200 μl of media samples from strains after growth in glycerol and from strains after 1 and 4 days in methanol, as well as dilutions of a bovine lysozyme c2 purified standard, were as follows: (1) Cells with the bovine pre-lysozyme c2 insert in the incorrect orientation and cells grown in glycerol did not secrete any detectable bovine lysozyme; (2) Significant amounts of bovine lysozyme c2 were secreted into the media by cells which had the bovine lysozyme insert in the correct orientation and were grown in methanol; (3) With respect to the cells described in (2) , lysozyme concentration in the media increased ten-fold 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 whereas lysozyme levels of approximately 1.25 mg secreted/ml were measured on day 4) ; (4) There was very little interstrain variability in the secretion levels among the strains with the lysozyme-encoding segment in the correct orientation.

EXAMPLE 4

SCALE-UP OF PICHIA PASTORIS PRODUCTION STRAIN A37

FROM SHAKE-FLASK CULTURES TO LARGE FERMENTER CULTURES AND CHARACTERIZATION OF

FERMENTER PRODUCT

As previously discussed, transcription from the alcohol oxidase promoter is strongly repressed during growth in glycerol and induced during growth in methanol. In fermenter cultures, transformants bearing expression cassettes at the AOXl locus are grown in a simple two-stage production scheme. In the first stage cell mass is developed in the absence of expression of the heterologous gene by using glycerol as a carbon source. Upon glycerol exhaustion, a methanol feed is initiated. Since AOXl deficient strains grow slowly on methanol, and the doubling times are about ten-fold greater on methanol compared to wild-type strains, there is a lengthy production phase of 30-200 hours with a minimum of cell division. By growing an expression host initially on repressing carbon sources, such as glucose or glycerol, a large mass of cells can be generated without significant selection for mutants defective in heterologous gene expression. Then, by allowing the cells to deplete the repressing carbon source and adding methanol, high level heterologous gene expression can be initiated.

For large-scale production of bovine lysozyme c2 from GS115 strain A37, a two-stage high cell-density batch fermentation scheme was employed. In the first, or growth, stage, A37 was cultured in defined minimal medium with glycerol as carbon source. The desired cell density to be achieved was established by adjusting the initial concentration of glycerol in the medium. Upon depletion of glycerol, the culture, then at the desired density, was shifted to the second, or production stage, by addition of methanol.

Fermenter cultures were grown in an inorganic salt-based medium prepared from commercially supplied analytical reagents consisting of the following: inorganic salts at final concentrations of 0.30 M H₃P0₄, 4.2 mM CaS0₄-2H₂0, 65 mM K₂S0₄, and 38 mM MgS0₄; trace salts at final concentrations of 0.4 μM CuS0₄-5H₂0, 2.5 μM KI, 9.0 μM MnS0₄-H₂0, 4.0 μM Na₂Mo0₄-2H₂0, 1.5 μM H3BO3, 35 μM ZnS0₄-7H₂0, and 89 μM FeCl₃-6H₂0, prepared as a 200x stock and filter sterilized; 2 mg/ml of biotin; and either 2% glycerol for the 2 liter New Brunswick Bioflo fermenter or 6% glycerol for the 15 liter Biolafitte fermenter. NH₃ was added to adjust the pH of the medium to pH 5.5 prior to inoculation and to maintain that pH during the fermentation. Upon glycerol exhaustion, a methanol feed was initiated to maintain the methanol concentration between 0.2% and 1.0%. To prevent nutritional limitation for trace salts during the fermentations, cultures were periodically supplemented with additional trace salt solution. Results of several 2 liter and 15 liter (1 liter and 10 liter working volume) fermentation runs of strain A37 indicated that strain A37 is clearly capable of maintaining a high level of production and secretion of bovine lysozyme c2 when grown for 5-7 days on methanol in 2L and 15L cultures. In fermenter run #182, bovine lysozyme c2 levels of approximately 200 mg/L were measured in the cell-free fermentation medium, at an OD₆₀₀=76. In another instance, cell-free fermentation broth from fermenter run #207, grown for seven days on methanol to a cell concentration of ODg₀₀=195, ^was assayed by radioimmunoassay (RIA) , as described in Example 6, to have 200 mg/L of bovine lysozyme c2. The specific productivities measured for the fermenter-grown cultures were within the range observed for A37 cells grown in 100 ml shake-flask cultures. Thus, there was no loss of per cell productivity in scaling up to the 2L and 15L fermenters.

A 124 ml aliquot of broth from fermenter run #182 was adjusted with concentrated acetic acid to pH=4 and placed in a 100*C water bath for 3-5 minutes.

Following a second adjustment to pH=5 with NH₄0H, 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 about 50% of the protein present in the fermenter medium and that any other proteins were present in minute amounts relative to bovine lysozyme c2. Moreover, the recombinant bovine lysozyme c2 and native bovine lysozyme c2 appeared to comigrate exactly. From the stained gel of the cell extract, no band corresponding to bovine lysozyme c2 was obvious.

A purified sample of recombinant bovine lysozyme c2 (250 pmoles) secreted from P. pastoris A37 grown in fermenter 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, revealed 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 14 amino acids of the recombinant material were identical to the first 14 amino acids of the published protein sequence of the mature enzyme. Short exposure of Western blots of Laemmli gels of a sample of secreted bovine lysozyme c2 taken from fermenter run #250, a 10L fermentation of strain A37, revealed a single immune-reactive species of the correct molecular weight. In the soluble and insoluble cell fractions, an immune-reactive band migrating at the same position as the standard was seen, although at a much lower amount than in the media. Material migrating at the same size was not seen from control Aoxl ' cells carrying no expression cassette. Several other runs tested the effect of restricted cell growth on bovine lysozyme σ2 secretion. Limiting amounts (2.5 ml/1, 5 ml/1, and 10 ml/1) of trace salts were used in runs #239, #240, and #241, respectively, to restrict cell growth. The curve of lysozyme secreted into the media paralleled that of the cell growth curve. From this data, it appears that accumulation of bovine lysozyme c2 in the medium is tied to healthy cell growth.

EXAMPLE 5

CHARACTERIZATION AND QUANTITATION OF RECOMBINANT

BOVINE LYSOZYME c2 PRODUCTION AFTER POLYACRYLAMIDE

GEL ELECTROPHORESIS OF FERMENTER SAMPLE

Parallel 15% polyacrylamide Laemmli gels were run (Laemmli, Nature 227:680 (1970)). One gel was stained with silver stain and the other was treated as an im unoblot. Both gels were run with (1) bovine lysozyme c2 purified from bovine stomach mucosa; (2) bovine lysozyme c2 purified, as described in Example 9, from the media of an Aoxl⁺ lysozyme secreting strain; and (3) cell extract and media from the midpoint of a 10 liter fermenter run of P. pastoris strain A37 (run #250) . For media samples, cells which were at a density of 308 g/cells/L and had been on methanol for 111.5 hours, were cleared by a low-speed centrifugation step. Cell extracts were made by breaking cells using glass beads in a buffer composed of 10 mM sodium phosphate, pH 7.5, 500 mM sodium chloride, 0.1% Triton X-100 and 2 mM phenylmethyl sulfonyl fluoride (PMSF) . Regarding the media and cell extract samples, the amount of cell extract loaded on the stained gel was equivalent volumetrically to 0.1 times the amount of media added. For the immunoblot, cell extract in equal amounts and 0.1 times the amount of media was loaded. Antisera for the immunoblot was raised, as described in Example 6, in rabbits against the bovine lysozyme purified from abomasum and used at a 1:2500 dilution. From the gels, it was apparent that native and recombinant bovine lysozyme c2 comigrate, that bovine lysozyme c2 comprises at least 50% of the protein in crude fermenter media, and that no significant amounts of lysozyme degradation products were present.

Relative proportions of bovine lysozyme c2 in the cell extract and in the growth medium can be estimated from the immunoblot on which volumetrically equivalent samples based on cell density were loaded. Although it was impossible to calculate efficiencies of secretion from these data, due to the constant accumulation of bovine lysozyme c2 in the fermenter, it appeared from these data, and from concentration data generated by RIA, as described in Example 6, that less than 5% of the bovine lysozyme c2 produced 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 25g of frozen bovine abomasum tissue. Since the starting material was fifteen times less than that used by Dobson et al., the column size and flow rate were scaled down accordingly. As reported by Dobson et al., three distinct peaks corresponding to bovine stomach lysozymes, cl, c2 and c3, were evident by absorbance at 280 nm after column chromatography on Whatman CM52.

Each of the three bovine stomach lysozyme peaks contained lysozyme activity, as measured in a spectrophotometric assay, described in Example 7, and approximately 2 mg of protein. Purity of the bovine lysozyme c2 protein was estimated at greater than 95% after polyacrylamide gel electrophoresis (Laemmli, supra) on a 15% polyacrylamide gel. All of the assay results are dependent upon quantitation with an aliquot of the purified bovine lysozyme dialyzed against 1 x PBS and stored at -20^βC. For the results described below, the standard has been quantified using the published extinction coefficient for a 1% solution measured at 280_nm, E=28.2 (Jolles et al., Mol. and Cell. Biochem, 63, 165 (1984)).

Antisera raised in rabbits against bovine lysozyme c2 were prepared by standard protocols.

Briefly, two young, male, white. New Zealand rabbits were each immunized with 250 μg of bovine lysozyme c2, purified as described above and dialyzed against PBS before the injections. The rabbits were each boosted with 100 μg of lysozyme c2 30 days later and boosted again ten days later with another 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 boosts. The rabbits were bled for antisera one week after the last boost.

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, binding to nitrocellulose filters that had been incubated with different dilutions of the antisera, using a "slot blot" apparatus (Schleicher and Schuell, Inc., Keene, New Hampshire), and using a standard immunoblot protocol (Towbin et al., J. Proc. Natl. Acad. Sci. U.S.A. 76:4350 (1979)). The buffer used for filter blocking and washing, and the antibody and ¹²⁵I-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, California) , 0.05% Tween-20 (Sigma Chemical Co., St. Louis, Missouri) and 0.02% sodium azide (Sigma Chemical Co., St. Louis, Missouri). The sera from one rabbit, designated #156, was chosen due to the substantially lower cross reactivity of this sera with bovine serum albumin. Using a 1:2500 dilution of the sera from rabbit #156, 2.5 ng of the lysozyme standard was easily detectable on a slot blot, and 25 ng gave a very strong signal.

Radioiodination of lysozyme, for use as tracer in RIAs, was carried out using the IODOBEAD™ method. Briefly, one iodobead (Pierce Chemical Co., Rockville, Illinois) was added to a reaction mixture containing 1 mCi Na¹²⁵I (New England Nuclear, Boston,

Massachusetts) , 50 μg purified lysozyme c2 in 50 μl, and 10 μl of 0.5 M sodium phosphate buffer, pH 7.3, and incubated for 30 minutes on ice. Subsequently, the contents of the reaction vial 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 the position of ¹²⁵I-lysozyme was determined. The iodinated preparation was tested by precipitation in 10% trichloroacetic acid (TCA) to estimate the proportion of intact peptide. Typically, ¹²⁵I-lysozyme preparations used in RIAs were greater than 98% TCA-precipitable. The RIAs, by which the concentration of bovine lysozyme c2 in P. pastoris culture samples was determined, were carried out in a standard protocol involving incubation of varying amounts of standard or unknown samples with 1:25000 final dilution of rabbit anti-lysozyme antibody, 10000-20000 counts of

¹²⁵I-lysozyme in a final volume of 500 μl of assay buffer (50 mM 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). Subsequent to incubation overnight at 4*C, 100 μl of 1:40 dilution of Pansorbin'^R' (Calbiochem, San Diego, California) suspension of S. aureus cells coated with Protein A was added and incubated for 15 minutes at room temperature. The tubes were centrifuged for 60 minutes at 2360 x g after addition of two ml of ice-cold wash buffer (0.9% NaCl, 5 mM EDTA, and 0.1% Triton X-100) . The supernatant was decanted and the pellets were counted for ¹²⁵I-lysozyme. Under these conditions, approximately 50% of total ^{1 5}I-lysozyme was recovered in the pellet in the reference tube and 10% in the nonspecific binding tube, i.e., in the presence of excess unlabeled lysozyme. The sensitivity range for the assay was 0.2-20 ng with an ED₅₀ of approximately 2 ng. The unknown samples were assayed at three dilutions each, in duplicate.

EXAMPLE 7

BIOASSAYS FOR SECRETED BOVINE LYSOZYME C2

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

Two types of bioassays (spectrophoto etric assay and halo assay) were employed to analyze the bioactivity of the secreted bovine lysozyme c2 (Grosswicz et al., Meth. Biochem. Analys. 29:435 (1983)). Each assay measures the ability of lysozyme to lyse Micrococcus luteus cells. Activity assays were performed at pH 5.0, rather than at pH 7.0, which is standard for egg white lysozyme, due to the differing pH optima of the enzymes. The halo assay is an in vitro assay which 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 holes punched in an agarose plate composed of 1% agarose and 1.2 mg/ l dried M. luteus cells in 0.1 M phosphate buffer, pH 5.0. The samples can be crude media, lysozyme purified from media, or lysozyme standard. The diameters of the resulting halos were measured after a 16-hour incubation at room temperature and quantitated by comparison to a semilog plot of halo diameter versus amount derived by using the lysozyme standard (bovine lysozyme c2 purified from bovine abomasum tissue) . The plot was linear from 100 ng to 10 μg. The lower limit of sensitivity of the assay is approximately 50 ng and, using this assay, the minimum detectable concentration was 5 mg/L of purified bovine lysozyme c2.

In the second assay, a spectrophotometric assay which measures in vitro lysis of dried M. luteus cells, different amounts of purified lysozyme are 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 monitored by recording the decrease in absorbency 450 nm every fifteen seconds for two minutes. The slope of the line was calculated by linear regression and quantitated by comparing this slope to a line on which slopes versus concentration of the purified standard were plotted. The minimum detectable concentration of the spectrophotometric assay was 5 mg/L.

EXAMPLE 8

TRANSFORMATION OF GS115 CELLS WITH pSL12A; STRAIN Ll

An Aoxl⁺, His⁺ strain, designated Ll, which is a single-copy integrant of bovine lysozyme c2 expressed from the AOXl promoter in Aoxl⁺ cells, resulted after G5115 cells were transformed, as described in Example 3, with pSL12A, cut with Sail (which cuts in the P. pastoris HIS4 gene region) to integrate at the HIS4 locus. Cells were grown in shake flasks for 4-5 days in

100 mM sodium phosphate buffered minimal media containing 0.2% glycerol and 1.0% methanol to a density of between 4 and 5 OD₆₀o units/ml. The final level of bovine lysozyme c2 accumulated from the cells was between 0.24 and 0.56 mg/1 (as defined by RIA).

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

Dilution rate experiments performed with Ll in continuous fermentations, where growth rate could be controlled, showed that expression levels were growth-rate dependent. Data from run #255, a methariol-limited continuous fermentation conducted in a 1 liter fermenter with a 33% MeOH feed, in which the dilution rate was varied between O.lh and 0.04h~¹ (td=6.9 and 17.25 hours per generation, respectively) demonstrated that dilution rate has a marked effect on the specific productivity (amount of product/cell/hour) of bovine lysozyme c2 from strain Ll. Three samples taken after three fermenter volumes at each dilution rate were assayed to determine steady state values. The maximum specific productivity of 20 μg/g wet cells/hour, which was seen at a dilution rate of O.OδSh^""1, or a generation time of 10.6 hours, is significantly greater than the specific productivity of 7 μg/g wet cells/hour of the Aoxl^" strain, A37. In the course of the dilution-rate experiment, a marked decline in bovine lysozyme c2 concentration in the fermenter was observed after 600h of continuous culture. During the first 550 hours, bovine lysozyme c2 production varies as a function of dilution rate. Southern blot analysis of DNA extracted from cell samples taken from the fermenter at 550, 643 and 717 hours revealed that, at the later two time points, a significant proportion of the cells in the fermenter showed an altered restriction map. The alteration was an approximately 300 bp deletion in the DNA fragment which includes the bovine lysozyme c2 0 coding sequence and the alcohol oxidase promoter. The new fragment containing the deletion was present in approximately 50% of the cells in the fermenter by 643 hours and in approximately 90% of such cells by 717 hours. 5

EXAMPLE 9

TRANSFORMATION OF GS115 WITH pSL12A; STRAIN C6

o An Aoxl⁺, His4⁺ strain, designated C6, which is a single-copy integrant of a heterologous gene expressing bovine pre-lysozyme c2 from the AOXl promoter, resulted after GS115 cells were transformed, as described in Example 3, with pSL12A cut with SstI (or Sad at the 5 same site) , in the 5' AOXl region, indicated in the Figures, upstream of the promoter (with respect to the direction of transcription from the promoter) . Certain of the transformants had the pSL12A integrated at the AOXl locus, 5' of the AOXl promoter. 0 Cells were grown in shake flasks for 4-5 days in

100 mM sodium phosphate buffered minimal media containing 0.2% glycerol and 1% methanol to a density of between 4 and 5 OD₆₀₀ units/ml. The final level of bovine lysozyme c2 accumulated from the cells in the media was 5 0.24 and 0.42 mg/L. EXAMPLE 10

LARGE-SCALE PURIFICATION PROCEDURE; STRAIN Ll

Purification procedures adaptable to large-scale production were utilized for purification of recombinant bovine lysozyme c2 secreted from strain Ll in run #253. Cells from 80 liters of run #253 fermentation broth were removed using a Ceraflow cartridge (1 micron, Amicon Corp., Danvers, Massachusetts), and the resulting filtrate (80 liters) was concentrated with a 3,000 molecular weight cutoff spiral cartridge (Amicon) . The concentrate was diafiltered with distilled water to reduce the conductivity below 4 mS. Two and one-half liters of the diafiltrate were pumped at a flow rate of 15 mg/minute onto a ZetaChrom SP-100 capsule (Western Analytical, Temecula, California) which had been previously equilibrated with 50 mM sodium acetate, pH 5.8. After loading was complete, the capsule was washed with the equilibration buffer and the bovine lysozyme c2 was eluted with a linear gradient consisting of one liter of 50 mM sodium acetate, pH 5.8, and one liter of 300 mM sodium acetate, pH 8.0. The bovine lysozyme c2 that eluted in the first third of the peak contained small amounts (not more than 15%) of contaminating proteins as estimated by SDS-gel electrophoresis and high pressure liquid chromatography (HPLC) . These contaminants were removed by repeating the binding, washing, and elution from the SP-100 capsule. HPLC analysis was accomplished using a μbondapak C-18 column (Waters Associates, Milford, Massachusetts) and an acetonitrile gradient in aqueous trifluoroacetic acid. This HPLC method also was used to quantify lysozyme during fermentation and purification procedures. Lysozyme activity was determined by the spectrophotometric assay described in Example 7. One unit is defined as a 1% change in OD₄₅₀ per minute 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 analysis using norleucine as the internal standard.

Using this three-step procedure, the bovine lysozyme c2 secreted by Pichia pastoris can be purified to near homogeneity. The overall yield of enzyme from cell-free broth is approximately 60% of the initial amount of lysozyme measured in crude media and is increased to 75% when impure fractions are re-chromatographed on the sulphopropyl disk. 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 Aoxl⁺, Arg⁺ transformant, designated CSBL11, was selected for further work.

Plasmid pBLll, which is illustrated in Figure 3, is similar to plasmid pSL12A, except that it bears an Hpal-site-terminated segment with the S. cerevisiae ARG4 gene cloned into the pBR322 BamHI site of pSL12A for selection in place of the Bglll-site-terminated fragment with the P. pastoris HIS4 gene. The S. cerevisiae ARG4 gene is functional in P. pastoris. The 5'-AOXl sequence on pBLll directed circular integration at the 5'-end of the AOXl locus in some of the transformants. Transformants were first screened for Arg⁺ phenotype. Arg⁺, Aoxl⁺ transformants were shown by Southern hybridization to have integrated the plasmid at the 5'-end of the AOXl locus. 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, AOXl promoter-driven expression cassette. The location of the pSL12A DNA in the genome of the double-copy integrants remains unknown, although it is not at the HIS4 or AOXl locus. One Aoxl⁺, His⁺, Arg⁺ transformant, designated CSBL3, was selected for further work. A fermenter run (run #274) with strain CSBL3 resulted in a specific productivity of 33 μg/g wet cell/hour at a dilution rate of O.Oδh^"1 and a specific productivity of 28 μg/g wet cell/hour at a dilution rate of O.Oδh^"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 pHLZ103

A plasmid, designated by us as pHLZlOO, was prepared by standard techniques. See European Patent Application Publication No. 0 222 366 and Castaήόn et al.. Gene 66, 223-234 (1988). Plasmid pHLZlOO consists of pUC9 (Vieira and Messing, Gene 19, 259 (1982)) with an insert, between the Sail and Hindlll sites, which comprises a segment of 435 base pairs encoding, in addition to a translational 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.

Employing the Sanger dideoxy method, the sequence of this 435-bp segment was found to be that indicated in the following Figure XII:

Figure XII

5'-ATTGTTCTGGGGCTTGTCCTCCTTTCTGTTACGGTCCAGGGCAAGGTCTTTGAAA GGTGTGAGTTGGCCAGAACTCTGAAAAGATTGGGAATGGATGGCTACAGGGGAATCAG CCTAGCAAACTGGATGTGTTTGGCCAAATGGGAGAGTGGTTACAACACACGAGCTACA AACTACAATGCTGGAGACAGAAGCACTGATTATGGGATATTTCAGATCAATAGCCGCT ACTGGTGTAATGATGGCAAAACCCCAGGAGCAGTTAATGCCTGTCATTTATCCTGCAG TGCTTTGCTGCAAGATAACATCGCTGATGCTGTAGCTTGTGCAAAGAGGGTTGTCCGT GATCCACAAGGCATTAGAGCATGGGTGGCATGGAGAAATCGTTGTCAAAACAGAGATG TCCGTCAGTATGTTCAAGGTTGTGGAGTGTAA

By simply applying the genetic code to the 3' 393 base pairs indicated in Figure XII, the amino acid sequence can be deduced for the 130-amino-acid, mature lysozyme c, for which all but the 4 N-terminal amino acids of the corresponding pre-lysozyme c are encoded by the DNA with the sequence in Figure XII. As indicated by the description below of the site-directed mutagenesis of pHLZlOl to prepare pHLZ102, the sequence of the four amino acids of the N-terminus of the pre-lysozyme c, for which a nucleotide sequence encoding the 144 carboxy- terminal amino acids and translation stop codon is given in Figure XII, is MKAL.

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

The amino acid sequences of the pre-lysozyme c of placental origin and that of cell line U-937 origin are the same, as the two differences in nucleotide sequence of the cDNAs encoding the pre-lysozyme c's do not alter amino acid sequence. Castaήόn et al.. Gene 66, 223-234 (1988) . The positions of the two differences in nucleotide sequence are indicated by underlining in the sequence in Figure XII. In the U-937 cell line-derived cDNA, there is a T in place of the C in the GTC in Figure XII encoding the Val at position -3 in the signal peptide and there is an A in place of the G in the CAG in Figure XII encoding the Gin at position -2 in the signal peptide.

In pHLZlOO, between the 5'-end of the 435-bp segment indicated in Figure XII and the 5'-GTCGAC-3' of the Sail site, there is a segment with a sequence 5'-CTGCA{C}-3', wherein the 5'-CTGCA-3' is the remnant of the PstI site of pUC9 and {C} designates a polydC stretch (of not precisely known length) which arose from the polydC-tailing of polydC-tailed, Pstl-cut pUC9 into which a polydG-tailed cDNA comprising the 435-bp segment was ligated to make pHLZlOO. Further, in pHLZlOO, between the 3'-end of the 435-bp segment indicated in Figure XII and the 5'-AAGCTT-3' of the Hindlll site, there is a segment with the sequence 5'-CTCCAGAATTTT{G}TGCAGCC-3', wherein (G) designates a polydG stretch (of not precisely known length) which arose at least in part from the polydG-tailing of the cDNA comprising the 435-bp segment prior to ligation of that cDNA into polydC-tailed pUC9 to make pHLZlOO and possibly in part from a stretch of one or more dG's at the 3'-end of that cDNA prior to its being polydG-tailed; 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 make pHLZlOO was made; and 5'-TGCAGCC-3' corresponds to (i) the 5'-GCC-3' immediately preceding the Hindlll site of pUC9 and (ii) the 5'-TGCA-3' which arose from the filling-in of the gap, between the 3'-end of polydG-tailed cDNA annealed to the 3'-end of polydC-tailed, Pstl-cut pUC9, in the preparation of pHLZlOO. 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 digested with BamHI and Hindlll following manufacturer's instructions. Correct plasmid was indicated by a pattern of about 2750 bp

(Hindlll - BamHI segment of pUC9) and about 540 bp (BamHI - Hindlll segment comprising the 435-bp segment indicated in Figure XII) .

The Sail - Hindlll site-bounded, human pre-lysozyme c insert in pHLZlOO was inserted into M13mpl8 for site-directed mutageneses as follows, in order to add the 12 nucleotides required at the 5'-end of the 435-bp segment of Figure XII to encode the first four amino acids of the pre-lysozyme c and to add EcoRI ends just prior to the ATG encoding the translational start and the first amino acid of the signal peptide and just after the TAA encoding the translational stop codon. Thus, M13mpl8 was digested with Hindlll and Sail and was phosphatase-treated. Similarly, a Hindlll/Sall digest was performed on pHLZlOO and the approximately 530-bp fragment with 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 therefrom. A Hindlll/Sall digest of the plasmid yielded fragments of about 7240 bp and 530 bp, indicative of the correct plasmid.

The 3' end of the insert in M13mpl8 was then modified to add an EcoRI site immediately after the translational stop-encoding TAA triplet. To accomplish this, site-directed mutagenesis was performed following the procedure of Zoller and Smith, Methods in Enzymology 100, 468 (1983) and using the oligonucleotide with the sequence:

5'-GCC AGT GCC AAG CTT GAA TTC TTA CAC TCC ACA ACC.

Plaques were screened with the screening oligonucleotide with the sequence:

5'-AAG CTT GAA TTC TTA CAC

and a mini-template prep was performed on the positives. E. coli JM103 cells were transformed with the 3'-end- modified template from the miniprep and plaques were again screened with the same screening oligonucleotide. Positives in this second round of screening were used to prepare template for sequencing. Sequencing was accom¬ plished by the Sanger dideoxy method using the universal primer of sequence:

5'-GTA AAA CGA CGG CCA GT.

A plasmid (RF form of M13mpl8) with the 3'-end of the pre-lysozyme-encoding segment having the desired modification was identified and designated pHLZlOl. 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 mutagenizing oligonucleotide was of sequence

5'-AAG CCC CAG AAC AAT GAG AGC CTT

CAT GAA TTC GTC GAC TCT AGA GGA

and the screening oligonucleotide had the sequence:

5'-GAG AGC CTT CAT GAA TTC.

A plasmid was identified, by Sanger dideoxy sequencing using the universal primer and two primers, of sequences

5'-CTC ACA CCT TTC AAA GAC and

5'-ATA ATC AGT GCT TCT GTC TCC AGC ATT GTA,

as having both ends of the pre-lysozyme c-encoding 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 pAO804 was then digested with EcoRI and the ends were phosphatased. 25 ng of cleaved, phosphatase-treated pAO804 and 250 ng of pre-lysozyme c-encoding fragment were ligated together in a standard ligation reaction and the ligation mixture was used to transform E. coli MC1061. Transformants were identified by ampicillin- resistance and a transformant with a plasmid with the correct structure was identified by miniprepping plasmid DNA from the transformant, digesting the miniprepped DNA with PstI and analyzing the fragment sizes of the digested plasmid DNA. A plasmid with the pre-lysozyme- encoding segment inserted in the correct orientation, relative to the direction of transcription from the AOXl promoter, would have a PstI fragment of about 2100 bp in length; a plasmid with the pre-lysozyme-encoding segment inserted in the incorrect orientation, relative to the direction of transcription from the AOXl promoter, would have a PstI fragment of about 1960 bp in length. A plas¬ mid with the correct structure was found and designated pHLZ103.

EXAMPLE 13

HUMAN LYSOZYME-EXPRESSING P. PASTORIS STRAINS

Plasmid pHLZ103 was purified on a CsCl gradient.

5 μg of Sacl-digested pHLZ103 was transformed into P. pastoris strain GS115 (ATCC #20864) using the spheroplast method of transformation (Cregg et al., Mol. Cell Biol. 5, 3376-3385 1985) . (Sad is an isoschizomer of Sstl.) His^4" Mut⁺ cells were identified and the DNA of several transformants was characterized by Southern blot hybridization analysis. Thus, the DNAs were digested 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 a transformant the linearized pHLZ103 plasmid had integrated by addition into the AOXl locus 5' of the AOXl coding sequence, leaving the transformant Mut^4". One such transformant was selected for further work and designated G+HLZ103S. To develop Mut^4"' human lysozyme-expressing strains, plasmid pHLZ103 was digested with Bglll and 5 μg of fragment was transformed into GS115 cells by the spheroplast method. His⁺ cells were identified and screened for their Mut phenotype.

Screening for Mut phenotype was accomplished by plating His^4" transformants on minimal glycerol (2%) master plates to obtain colonies originating from single cells. After two days incubation at 30*C, the masters were replica-plated to minimal glycerol plates and plates containing no carbon source to which methanol was added in vapor phase. This was done by adding a drop, approx. 200 μl, of methanol to the underside of the top of the cover of the petrie dish holding the plate. The plates were incubated at 30*C for two days with additional methanol added in the vapor phase every day. Colonies showing visible growth were scored at Mut^4" and those with no visible growth were scored as Mut^4"/-. Mut^4"' transformants were characterized by

Southern blot hybridization analysis. Thus, DNA of the transformants was digested with EcoRI and probed with plasmid pAO803 DNA. Fragments of 2000 bp and 5100 bp (with loss of the wild type 5700 bp fragment) indicated integration of the expression cassette by disruption at the AOXl locus. One such Mut^4"/^"" transformant was selected for further work and designated G-HLZ103S.

EXAMPLE 14

ADDITIONAL PROTOCOLS FOR CULTURING LYSOZYME-SECRETING P. PASTORIS STRAINS

The procedures described in this Example have been employed advantageously with bovine lysozyme c2- secreting P. pastoris strains A37 (Mut^4*' ) and Ll (Mut^4*) . The procedures can be used also to make human lysozyme with P. pastoris strains G-HLZ103S and G+HLZ103S. The various strains have been carried on yeast nitrogen base (YNB) without amino acids (Difco Laboratories, Inc., Detroit, Michigan, USA) + 2 % glucose agar plates with monthly transfers. Inocula for the protocols described in this Example were grown overnight 0 at 30 ^βC with shaking at 200 rpm in YNB without amino acids + 2 % glucose + phosphate buffer (pH 6.0).

1. Fermentor Start-up and General Operation

5 The 2-liter fermentors (L.H. Fermentation,

Hayward, California, USA; or Biolafitte, LSL Biolafitte, Princeton, New Jersey, USA) were autoclaved at a 700 ml volume containing 225 ml of 10X basal salts (42 ml/1 85% phosphoric acid, 1.8 g/1 calcium sulfate-2H₂0, 28.6 g/1 o potassium sulfate, 23.4 g/1 magnesium sulfate-7H₂0, 6.5 g/1 potassium hydroxide) and 30 g glycerol. After sterilization, 3 ml of a YTM trace salts solution (5.0 ml/1 sulfuric acid, 65.0 g/1 ferrous sulfate-7H₂0, 6.0 g/1 copper sulfate-5H₂0, 20.0 g/1 zinc sulfate-7H₂0, 5 3.0 g/1 manganese sulfate-H₂0, 0.1 g/1 biotin) was added and the pH adjusted to 5.0 with the addition of concentrated ammonium hydroxide. The fermentors were then inoculated with a 10-50 ml volume of inoculum. Upon exhaustion of the initial glycerol charge, a glycerol 0 feed was started as described below in section 2, 3 or 4 of this Example. Throughout a fermentation, the pH was controlled at 5.0 with the addition of 20% ammonium hydroxide solution containing 0.1% Struktol J673 antifoam (Struktol Co., Stow, Ohio, USA) and excessive foaming was sensed by a foam probe and controlled by addition of

2% Struktol J673 antifoam. The dissolved oxygen of the fermentation was maintained above 20% of air saturation by increasing the air flow rate up to 3 liter/minute and agitation speed up to 1500 rpm during the fermentation. Ten-liter fermentations (in a 14-liter Biolafitte fer entor) were started at a 7.0 liter volume containing 1.9 liters 10X basal salts and 300 g glycerol for the Mut^" mixed-feed, methanol-non-limiting, fed-batch protocol, or a 5.0 liter volume containing 2.4 liters of 10X basal salts and 360 g of glycerol for the 0 Mut^4" methanol fed-batch protocol, or a 8 liter volume containing 3.2 liters of 10X basal salts and 480 g glycerol for the Mut^" methanol fed-batch protocol. After sterilization, 29 ml of the YTM₄ trace salts solution was added and the pH was adjusted and 5 subsequently controlled at 5.0 with the addition of ammonia gas throughout the fermentation. Excessive foaming was controlled with the addition of 10% Struktol J673 antifoam. The fermentor was inoculated with an inoculum with a volume of 200-500 ml. Upon exhaustion of o the initial glycerol charge, a feed was started as outlined in section 2, 3 or 4 of this Example. The dissolved oxygen was maintained above 20% by increasing the air flow rate up to 40 liter/minute, the agitation up to 1000 rpm and/or the pressure of the fermentor up to 5 1.5 bar during the fermentation.

2. Mut^4"/^" Cells: Mixed-Feed, Methanol Non-limiting, Fed-Batch Fermentation

0 After the glycerol batch phase was completed

(i.e., upon exhaustion of the initial glycerol charge), a 50% (by weight) glycerol feed, containing 12 ml/1 YTM trace salts was started at 5.4 ml/h for the 2-liter fermentor or 54 ml/h for the 10-liter fermentor. After 6 hours of glycerol feeding, the glycerol feed was decreased to 3.6 ml/h (36 ml/h at 10-liters) and a methanol feed containing 12 ml/1 YTM₄ trace salts was initiated at 1.1 ml/h for the 2-liter fermentor or 11 ml/h for the 10-liter fermentor. After 5 hours, the methanol feed was adjusted to give a residual methanol concentration of 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^4" Cells: Methanol-fed-batch Fermentation 0

After exhaustion of the initial glycerol charge, a 50% glycerol feed, containing 12 ml/1 YTM trace salts, was started at 12 ml/h for the 2-liter or 200 ml/h for the 10-liter fermentor and run for a total of 7 5 hours. After 6 hours on the glycerol feed, the methanol feed, containing 12 ml/1 YTM₄ trace salts, was started at 1.1 ml/h for the 2-liter and 11 ml/h for the 10-liter fermentor for 5 minutes. When a xise in dissolved oxygen was seen after the methanol feed was shut off, the o methanol feed was turned back on for another 5-minute interval. The latter process was repeated several times until an immediate response in the dissolved oxygen was observed to the methanol feed cessation; once this occurred, the methanol feed was increased by 20% per hour 5 at 30 minute intervals. The methanol feed was increased until a feed rate of 7.6 ml/h for the 2-liter or 126 ml/h for the 10-liter fermentor was reached. The fermentation was then carried out for 40-60 hours for the 2-liter or 25-35 hours for the 10-liter fermentor. 0

4. Mut^4"/- Cells: Methanol-fed-batch Fermentation

After exhaustion of the initial glycerol charge, methanol was added to the fermentor to maintain a residual methanol concentration between 0.2 % and 0.8 %. For the 10 liter runs, the YTM₄ trace salts were not used but, instead, 40 ml of lΑ trace salts (5 ml/1 sulfuric acid, 4.8 g/1 ferric chloride-2 H₂0, 2.0 g/1 zinc sulfate-H₂0, 0.02 g/1 boric acid, 0.2 g/1 sodium molybdate, 0.3 g/1 manganese sulfate-H₂0, 0.08 g/1 potassium iodide, 0.06 g/1 cupric sulfate-5 H₂0) and 2 mg/1 biotin were injected into the fermentor every second day. Fermentation was carried out for up to about 192 hours for the 2-liter runs or up to about 144 hours for the 10-liter runs.

Additional information on protocols for culturing lysozyme c-secreting P. pastoris strains is provided in United States Patent Application Serial No. 249,446, filed September 26, 1988, which is commonly owned with the present application and is hereby incorporated herein by reference.

EXAMPLE 15

PREPARATION OF PLASMIDS pAO803, pAO804, AND pA0811

The procedures of this Example were carried out using standard techniques, as described, for example, in 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 Bglll site into the PvuII site: pBR322 was digested with EcoRI, the protruding ends were filled in with Klenow Fragment of E. coli DNA polymerase I, and the resulting DNA was recircularized using T4 ligase. The recircularized DNA was used to transform E. coli MC1061 to ampicillin-resistance and transformants were screened for having a plasmid of about 4.37 kbp in size without an EcoRI site. One such transformant was selected and cultured to yield a plasmid, designated pBR322 RI, which is pBR322 with the EcoRI site xeplaced with the sequences 5'-GAATTAATTC-3'

3'-CTTAATTAAG-5'. pBR322 RI was digested with PvuII and the linker, of sequence

5'-CAGATCTG-3' 3*-GTCTAGAC-5' was ligated to the resulting blunt ends employing T4 ligase. The resulting DNAs were recircularized, also with T4 ligase, and then digested with Bglll and again recircularized using T4 ligase to eliminate multiple Bglll sites due to ligation of more than one linker to the PvuII-cleaved pBR322 RI. The DNAs, treated to eliminate multiple Bglll sites, were used to transform E. coli MC1061 to ampicillin-resistance. Transformants were screened for a plasmid of about 4.38 kbp with a Bglll site. One such transformant was selected and cultured to yield a plasmid, designated pBR322 RIBG, for further work. Plasmid pBR322 RIBG is the same as pBR322 Ri except that pBR322 RIBG has the sequence

5'-CAGCAGATCTGCTG-3' 3'-GTCGTCTAGACGAC-5' in place of the PvuII site in pBR322 RI. pBR322 RIBG was digested with Sail and Bglll and the large -fragment (approximately 2-97 kbp) was isolated. Plasmid pBSAGI5I, which is described in European Patent Application Publication No. 0 226 752, was digested completely with Bglll and Xhol and an approximately 850 bp fragment from a region of the P. pastoris AOXl locus downstream from the AOXl gene transcription terminator (relative to the direction of transcription from the AOXl promoter) was isolated. This Bglll-Xhol fragment from pBSAGI5I and the approximately 2.97 kbp, Sall-Bglll 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 3.8kbp) with a Bglll site. This plasmid was designated pAO801. The overhanging end of the Sail site from the pBR322 RIBG fragment was ligated to the overhanging end of the Xhol site on the 850 bp pBSAGISI fragment and, in the process, both the Sail site and the Xhol site in pAO801 were eliminated. pBSAGISI was then digested with Clal and the approximately 2.0 kbp fragment was isolated. The 2.0 kbp fragment has an approximately 1.0 kbp segment which comprises the P. pastoris AOXl promoter and transcription initiation site, an approximately 700 bp segment coding the hepatitis B virus surface antigen ("HBsAg") and an approximately 300 bp segment which comprises the P. pastoris AOXl gene polyadenylation signal and site- encoding segments and transcription terminator. The HBsAg coding segment of the 2.0 kbp fragment is terminated, at the end adjacent the 1.0 kbp segment with the AOXl promoter, with an EcoRI site and, at the end adjacent the 300 bp segment with the AOXl transcription terminator, with a StuI site, and has its subsegment which codes for HBsAg oriented and positioned, with respect to the 1.0 kbp promoter-containing and 300 bp transcription terminator-containing segments, operatively for expression of the HBsAg upon transcription from the AOXl promoter. The EcoRI site joining the promoter segment to the HBsAg coding segment occurs just upstream (with respect to the direction of transcription from the AOXl promoter) from the translation initiation signal-encoding triplet of the AOXl promoter. For more details on the promoter and terminator segments of the 2.0 kbp, Clal-site-terminated fragment of pBSAGISI, see European Patent Application Publication No. 0 226 846 and Ellis et al., Mol. Cell. Biol. 5, 1111 (1985) .

Plasmid pAO801 was cut with Clal and combined for ligation using T4 ligase with the approximately 2.0 kbp Clal-site-terminated fragment from pBSAGISI. 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 5.8 kbp) which, on digestion with Clal and Bglll, yielded fragments of about 2.32 kbp (with the origin of replication and ampicillin-resistance gene from pBR322) and about 1.9 kbp, 1.48 kbp, and 100 bp. On digestion with Bglll and EcoRI, the plasmid yielded an approxi- mately 2.48 kbp fragment with the 300 bp terminator segment from the AOXl gene and the HBsAg coding segment, a fragment of about 900 bp containing the segment from upstream of the AOXl protein encoding segment of the AOXl gene in the AOXl locus, and a fragment of about 2.42 kbp containing the origin of replication and ampicillin resistance gene from pBR322 and an approximately 100 bp Clal-Bglll segment of the AOXl locus (further upstream from the AOXl-encoding segment than the first mentioned 900 bp EcoRI-Bglll segment) . Such a plasmid had the Clal fragment from pBSAGISI in the desired orientation; in the opposite undesired orientation, there would be EcoRI-Bglll fragments of about 3.3 kbp, 2.38 kbp and 900 bp.

One of the transformants harboring the desired plasmid, designated pAO802, was selected for further work and was cultured to yield that plasmid. The desired orientation of the Clal fragment from pBSAGIδl in pAO802 had the AOXl locus fragments bracketing the HBsAg- encoding segment and the approximately 800 bp Bglll-site-terminated fragment from downstream of the

AOXl gene in the AOXl locus oriented correctly to lead to the correct integration into the P. pastoris genome at the AOXl locus of linearized plasmid made by cutting at the Bglll site at the terminus of the 800 bp fragment from downstream of the AOXl gene in the AOXl locus. pAO802 was then treated to remove the HBsAg coding segment terminated with an EcoRI site and a StuI site. The plasmid was digested with StuI and a linker of sequence:

5^,-GGAATTCC-3' 3'-CCTTAAGG-5' was 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 transformants were screened for a plasmid of the expected size ( 5.1 kbp) with EcoRI-Bglll fragments of about 1.78 kbp, 900 bp, and 2.42 kbp and Bglll-Clal fragments of about 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 work and was cultured to yield pAO803.

Plasmid pAO804 was then made from pAO803 by inserting, into the BamHI site from pBR322 in pAO803, an approximately 2.75 kbp Bglll fragment from the P. pastoris genome which harbors 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 digested with BamHI and combined with the HIS4 gene-containing Bglll 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), which is cut by Sail. One such transformant was selected for further work, and the plasmid it harbors was designated pAO804. pAO804 has one Sall-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 the direction of transcription from the AOXl promoter.

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

It is noteworthy that other plasmids, similar to pAO804 but with selectable marker genes, other than the P. pastoris HIS4 gene, that are capable of providing selection to P. pastoris, can be constructed by inserting a fragment into the BamHI site of pAO803 which comprises a selectable marker gene other than the P. pastoris HIS4 gene. Among such fragments known in the art are fragments with the S. cerevisiae ARG4 gene, the P. pastoris ARG4 gene and the S. cerevisiae HIS4 gene. For example, to make a plasmid, designated pAOδll, which is the analog of pAO804 with a S. cerevisiae ARG4 gene in place of the P. pastoris HIS4 gene, and which was employed to make plasmid pBLll of the present invention, the following procedure was followed: pAO803 was digested with BamHI and the overhanging ends were filled in using Klenow Fragment of E. coli DNA polymerase I. Then, the linearized, filled-in plasmid was combined with an approximately 2.1 kbp Hpal fragment from the S. cerevisiae genome which harbors the

S. cerevisiae ARG4 gene. See Tschumper and Carbon, J. Mol. Biol. 156, 293 (1982) ; European Patent Application Publication No. 0 211 455. The Hpal fragment is also available from a plasmid, designated pYM25, which is available from the Northern Regional Research Laboratory depository of the U.S. Department of Agriculture in

Peoria, Illinois, USA, where the plasmid is on deposit in E. coli MC1061 under NRRL deposit no. B-18015. The fragments were blunt end-ligated employing T4 ligase and the ligation mixture was used to transform E. coli MC1061 0 to ampicillin resistance. Transformants were then screened for plasmids of the expected size (approximately 7.2 kbp). One such transformant was selected for further work; the plasmid it harbors was designated pA0811. Although the orientation of the ARG4 gene in pAOδll has 5 not been determined, it should be noted that there is a Bglll site in the Hpal-site-terminated fragment which is about 400-500 bases from an Hpal site at a fragment terminus and which is near the 3'-end of the ARG4 gene. The P. pastoris ARG4 gene is disclosed in o European Patent Application Publication No. 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 deposit no. 5 B-18016.

The S. cerevisiae HIS4 gene is disclosed in Donahue et al., Gene 18:47 (1982). A plasmid, designated pBPGl-1, which includes a fragment with the S. cerevisiae HIS4 gene, is described in European Patent Application 0 Publication No. 0 226 752 and is available from the Northern Regional Research Laboratory depository in Peoria, Illinois, where it is deposited in E^coli MC1061 under NRRL deposit No. B-18020.

5 DEPOSITS

Cultures of P. pastoris strains GS115 and PPF1 and E. coli K-12 strain MC1061 harboring plasmid pBSAGISI have been deposited with the American Type Culture Collection (ATCC) , 12301 Parklawn Drive, Rockville, Maryland 20852 U.S.A., on the following dates with the corresponding ATCC Accession numbers: 0

ATCC Cell Line Dates Accession No.

GS115 August 18, 1987 20864

PPF1 August 18, 1987 20865

E. COli MC1061 (pBSAGI5I) September 15, 1987 67512 5

Said deposits were made, and accepted by the ATCC, under the terms of the Budapest Treaty on the International .Recognition of the Deposits of Microorganisms for Purposes of Patent Procedures and the Regulations o promulgated under said Treaty. Samples of said deposits will be available, in accordance with said Treaty and Regulations, to industrial property offices and other persons and entities legally entitled to receive them under the patent laws and regulations of the United 5 States of America and any other nation or international organization in which the present application, or an application claiming priority of the present application, is filed. All restrictions on the public availability of samples of said deposits will be irrevocably removed, at the latest, upon issuance of a United States patent on this application.

While the various aspects of the present invention have been described herein with some particularity, those skilled in the art will recognize modifications and variations that remain within the spirit of the invention. These modifications and variations are within the scope of the invention as described and claimed herein.

Claims (60)

WHAT IS CLAIMED IS:

1. A DNA which 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 oriented and positioned, with respect to said promoter and terminator segments, operatively for transcription in P. pastoris, from the promoter of said promoter segment, of said pre-lysozyme c-encoding segment and the polyadenylation-signal-encoding and polyadenylation-site-'encoding segments of said terminator segment.

2. A DNA according to Claim 1 wherein said first P. pastoris gene and said second P. pastoris gene are the same and are the P. pastoris AOXl gene.

3. A DNA according to Claim 2 wherein the pre-lysozyme c-encoding segment encodes a pre-lysozyme c from a mammalian species.

4. A DNA according to Claim 3 wherein the species is human.

5. A DNA according to Claim 4 wherein 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' encoding the Val at position -3 of the signal peptide of the pre-lysozyme c and the triplet 5'-CAA-3' encoding the Gin at position -2 of the signal peptide of the pre-lysozyme c.

6. A DNA according to Claim 3 wherein the species is bovine.

7. A DNA according to Claim 6 wherein the bovine pre-lysozyme c-encoding segment has the sequence of the pre-lysozyme c2-encoding, Eco Rl-site-terminated segment of plasmid pBLI6C.

8. A DNA according to Claim 1 which is capable of transforming P. pastoris cells to express said pre-lysozyme c and which comprises a gene to provide a

5 selectable marker to P. pastoris cells which harbor said DNA.

9. A DNA according to Claim 7 wherein the gene providing the selectable marker is selected from the group consisting of the P. pastoris HIS4 gene, the P.

10 pastoris ARG4 gene, the S. cerevisiae ARG4 gene, and the S. cerevisiae HIS4 gene.

10. A DNA according to Claim 9 wherein said first P. pastoris gene and said second P. pastoris gene are the same and are the P. pastoris AOXl gene.

₁₅ 11. A DNA according to Claim 10 wherein the pre-lysozyme c-encoding segment encodes a pre-lysozyme c from a mammalian species.

12. A DNA according to Claim 11 wherein the species is human.

2o

13. A DNA according to Claim 12 wherein 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

25 segment of plasmid pHLZ102 altered to have the triplet 5'-GTT-3' encoding the Val at position -3 of the signal peptide of the pre-lysozyme c and the triplet 5'-CAA-3' encoding the Gin at position -2 of the signal peptide of the pre-lysozyme c.

3_Q

14. A DNA according to Claim 13 which is plasmid

PHLZ103.

15. A DNA according to Claim 11 wherein the species is bovine.

16. A DNA according to Claim 15 wherein 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 according to Claim 16 selected from the group consisting of pSL12A and pBLll.

18. A culture of P. pastoris transformed with a DNA according to Claim 8.

19. A culture according to Claim 18, which is a culture of P. pastoris transformed with a DNA according to Claim 9.

20. A culture according to Claim 19, which is a culture of P. pastoris transformed with a DNA according to Claim 10.

21. A culture according to Claim 20, which is a culture of P. pastoris transformed with a DNA according to Claim 11.

22. A culture according to Claim 21, which is a culture of P. pastoris transformed with a DNA according to Claim 12.

23. A culture according to Claim 22, which is a culture of P. pastoris transformed with a DNA according to Claim 13.

24. A culture according to Claim 23, which is a culture of P. pastoris transformed with a DNA according to Claim 14.

25. A culture according to Claim 24, said culture being of a P. pastoris strain selected from the group consisting of G-HLZ103S and G+HLZ103S.

26. A culture according to Claim 21, which is a culture of P. pastoris transformed with a DNA according to Claim 15.

27. A culture according to Claim 26, which is a culture of P. pastoris transformed with a DNA according to Claim 16.

28. A culture according to Claim 27, which is a culture of P. pastoris transformed with a DNA according to Claim 17.

29. A culture according to Claim 28, said culture being of a P. pastoris strain selected from the group consisting of A37, Ll, C6, CSBL11, and CSBL3.

30. A method of making an animal lysozyme c which comprises culturing P. pastoris cells, which have a gene which is capable of expressing the corresponding pre-ani al lysozyme c in P. pastoris, said culturing being under conditions such that said gene is transcribed in said cells.

31. The method of Claim 30 wherein the cells are of a culture, or a subculture of a culture, which has been transformed with a DNA according to Claim 8 which is capable of expressing the corresponding pre-lysozyme c in P. pastoris.

32. The method according to Claim 31 wherein tϋae cells are of a culture, or a subculture of a culture, which has been transformed with a DNA according to Claim

9 which is capable of expressing the corresponding pre-lysozyme c in P. pastoris.

33. A method according to Claim 32 wherein the cells are of a culture, or a subculture of a culture, which has been transformed with a DNA according to Claim

10 which is capable of expressing the corresponding pre-lysozyme c in P. pastoris.

34. A method according to Claim 33 wherein the lysozyme c to be made is a mammalian lysozyme c and wherein the cells are of a culture, or a subculture of a culture, which has been transformed with a DNA according to Claim 11 which is capable of expressing the corresponding pre—lysozyme c in P. pastoris.

35. A method according to Claim 34 wherein the lysozyme c to be made is a human lysozyme c and wherein the cells are of a culture, or a subculture of a culture, which has been transformed with a DNA according to Claim 12 which is capable of expressing the corresponding pre-lysozyme c in P. pastoris.

36. A method according to Claim 35 wherein the lysozyme c to be made is a human lysozyme c and wherein the cells are of a culture, or a subculture of a culture, which has been transformed with a DNA according to Claim

13 which is capable of expressing the corresponding pre-lysozyme c in P. pastoris.

37. A method according to Claim 36 wherein the lysozyme c to be made is a human lysozyme c and wherein the cells are of a culture, or a subculture of a culture, which has been transformed with a DNA according to Claim

14 which is capable of expressing the corresponding pre-lysozyme c in P. pastoris.

38. A method according to Claim 37 wherein the cells are of a strain selected from the group consisting of G-HLZ103S and G+HLZ103S.

39. A method according to Claim 34 wherein the lysozyme c to be made is a bovine lysozyme c and.wherein the cells are of a culture, or a subculture of a culture, which has been transformed with a DNA according to Claim

15 which is capable of expressing the corresponding pre-lysozyme c in P. pastoris.

40. A method according to Claim 39 wherein the bovine lysozyme c to be made is bovine lysozyme c2 and wherein the cells are of a culture, or a subculture of a culture, which has been transformed with a DNA according to Claim 16 which is capable of expressing the corresponding pre-lysozyme c2 in P. pastoris.

41. A method according to Claim 40 wherein the lysozyme c to be made is bovine lysozyme c2 and wherein the cells are of a culture, or a subculture of a culture, which has been transformed with a DNA according to Claim 17 which is capable of expressing the corresponding pre-lysozyme c2 in P. pastoris.

42. A method according to Claim 41 wherein the cells are of a strain selected from the group consisting of A37, Ll, C6, CSBL11 and CSBL3.

43. The method of Claim 30 wherein said culturing is in a medium which contains methanol as a carbon source.

44. The method of Claim 31 wherein said culturing is in a medium which contains methanol as a carbon source.

45. The method of Claim 32 wherein said culturing is in a medium which contains methanol as a carbon source.

46. The method of Claim 33 wherein said culturing is in a medium which contains methanol as a carbon source.

47. The method of Claim 34 wherein said culturing is in a medium which contains methanol as a carbon source.

48. The method of Claim 35 wherein said culturing is in a medium which contains methanol as a carbon source.

49. The method of Claim 36 wherein said culturing is in a medium which contains methanol as a carbon source.

50. The method of Claim 37 wherein said culturing is in a medium which contains methanol as a carbon source.

51. The method of Claim 38 wherein said culturing is in a medium which contains methanol as a carbon source.

52. The method of Claim 39 wherein said culturing is in a medium which contains methanol as a carbon source.

53. The method of Claim 40 wherein said culturing is in a medium which contains methanol as a carbon source.

54. The method of Claim 41 wherein said culturing is in a medium which contains methanol as a carbon source.

55. The method of Claim 42 wherein said culturing is in a medium which contains methanol as a carbon source.

56. An 18-amino acid polypeptide of sequence

MKALVILGFLFLSVAVQG.

57. A DNA which comprises a segment selected from the group consisting of a segment of 54-bp in a sequence which encodes the polypeptide of sequence o MKALVILGFLFLSVAVQG, and the segments of 441-bp in sequences which encode a polypeptide of sequence

MKALVILGFLFLSVAVQGKVFERCE-

LARTLKKLGLDGYKGVSLANWLCLT- 5 K ESSYNTKATNYNPSSESTDYGIF-

QINSKWWCNDGKTPNAVDGCHVSCS-

ELMENDIAKAVACAKXg₈IVSEQGITA-

WVAWKSHCRDHDVSSYVEGCTL wherein X_9g is K or H, and a segment of 444-bp in a 0 sequence which encodes the fusion polypeptide wherein the polypeptide of sequence MKALVILGFLFLSVAVQG is fused to the amino-terminus of mature human milk lysozyme.

58. A DNA according to Claim 57 which comprises a segment of 54-bp in a sequence which encodes the 5 polypeptide of sequence

MKALVILGFLFLSVAVQG.

59. A DNA according to Claim 58 which comprises a segment of 441 bp in a sequence which encodes the polypeptide of sequence: ₀ MKALVILGFLFLSVAVQGKVFERCE-

LARTLKKLGLDGYKGVSLANWLCLT-

KWESSYNTKATNYNPSSESTDYGIF-

QINSKWWCNDGKTPNAVDGCHVSCS-

ELMENDIAKAVACAKHIVSEQGITA- ₅ WVAWKSHCRDHDVSSYVEGCTL.

60. A DNA according to Claim 58 which comprises a segment of 444 bp in a sequence which encodes the fusion polypeptide wherein the polypeptide of sequence MKALVILGFLFLSVAVQG is fused to the amino-terminus of mature human milk lysozyme.