EP0343231A1 - PRODUCTION OF ANIMAL LYSOZYME c VIA SECRETION FROM PICHIA PASTORIS AND COMPOSITION THEREFOR - Google Patents
PRODUCTION OF ANIMAL LYSOZYME c VIA SECRETION FROM PICHIA PASTORIS AND COMPOSITION THEREFORInfo
- Publication number
- EP0343231A1 EP0343231A1 EP89900706A EP89900706A EP0343231A1 EP 0343231 A1 EP0343231 A1 EP 0343231A1 EP 89900706 A EP89900706 A EP 89900706A EP 89900706 A EP89900706 A EP 89900706A EP 0343231 A1 EP0343231 A1 EP 0343231A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- lysozyme
- pastoris
- culture
- segment
- cells
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
- C07H15/02—Acyclic radicals, not substituted by cyclic structures
- C07H15/12—Acyclic radicals, not substituted by cyclic structures attached to a nitrogen atom of the saccharide radical
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2462—Lysozyme (3.2.1.17)
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
- C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
- C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
- C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
Definitions
- 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.
- lysozymes which are also known as 1,4- ⁇ -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.
- lysozymes 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- ⁇ -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.
- lysozymes 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.
- 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.
- g-type enzymes act oniy as hydrolases.
- 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
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- yeasts are eukaryotes, their intracellular environment tends to be more suitable than that of prokaryotes for the correct folding of eukaryotic proteins.
- 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.
- 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.
- lysozyme c's e.g., lysozyme c's
- 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
- the secreted mature protein has essentially the same immunologic properties and specific activity as the naturally-occurring animal lysozyme c.
- 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.
- 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.
- 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 ⁇ BL3) .
- 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.
- 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.
- 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
- 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.
- the invention encompasses a culture of P. pastoris cells transformed with a DNA according to the invention.
- 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.
- culture means a propagation of cells in a medium conducive to their growth, and all subcultures thereof.
- 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.
- 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.
- 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:
- 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.
- 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.
- 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.
- 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.
- 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.
- the genes providing complementation for auxotrophic mutations are the P. pastoris HIS4 gene (for transformation of His4 ⁇ strains of P.
- the S. cerevisiae HIS4 gene for transformation of His4 ⁇ strain of P. pastoris
- the P. pastoris ARG4 arginosu ⁇ cinate lyase gene
- the S. cerevisiae ARG4 gene for transformation of Arg4 ⁇ strains of P. pastoris
- 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.
- 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.
- P. pastoris 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.
- AOX1 gene the major alcohol oxidase gene
- 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.
- 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.
- 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.
- translation-start triplet a translation-start-site-encoding triplet
- translation stop triplet translation-stop-signal-encoding triplet
- DNA segment with a sequence for a bovine pre-lysozyme c2
- a sequence for a bovine pre-lysozyme c2 is the approximately 460 bp EcoRI site-terminated segment constructed as described in Example 2 below.
- 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.
- DNA segments with sequences encoding pre-lysozyme c's other than bovine lysozyme c2 can be isolated.
- 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.
- 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,
- ⁇ j c 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
- 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
- bovine pre-lysozyme c2 signal segment of bovine pre-lysozyme c2 is fused to the amino-terminus of mature human milk lysozyme.
- 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.
- 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, wi ⁇ ch, 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.
- 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.
- 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.
- 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.
- ClaI-(BamHI/BglII) fragment of pSL12A which includes the Clal-site terminated bovine pre-lysozyme c2 expression cassette and the P. pastoris HIS4 gene
- ClaI-(BamHI/HpaI) fragment of pBLll which includes the Clal-site-terminated bovine pre-lysozyme c2 expression cassette and the S. cerevisiae ARG4 gene
- ClaI-(BamHI/BglII) fragment of pHLZ103 which includes the Clal-site terminated human pre-lysozyme c expression cassette and the P. pastoris HIS4 gene
- 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.
- 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.
- ARS autonomous replication sequence
- 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.
- 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.
- 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.
- 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.
- 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.
- Sad an isoschizomer of SstI
- Bglll a derivative of pAO804
- the present invention allows an animal lysozyme c to be secreted with unexpected efficiency from P. pastoris cells into the culture media.
- 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.
- 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.
- Western blot analysis of the secreted lysozyme c reveals a single immunoreactive species of the same molecular weight as the naturally-occurring, mature enzyme.
- 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.
- the animal lysozyme c's provided by the present invention can be used as known in the art for such lysozymes generally.
- 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) .
- 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.
- 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.
- 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.
- 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.
- ⁇ gtlO and E. coli strain C600HF1 were obtained from Clonetech Labs, Inc., 4055 Fabian Way, Palo Alto, California 94303.
- the packaging-kit used to package ⁇ gtlO 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.
- 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.
- RNA 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.
- 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 ⁇ g/ml proteinase K (Boehringer Mannheim Biochemicals, Inc.). The mixture was immediately shaken well and then allowed to incubate at room temperature for 15 minutes.
- proteinase K buffer .14 M NaCl, .05 M Tris/7.4, .01 M EDTA/8.0, 1% sodium dodecyl sulfate (SDS)
- 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.
- 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.
- RNA was denatured in 2mM CH 3 HgOH (Alpha Products, Danver, Massachusetts) for 5 minutes at room temperature.
- this RNA was subjected to a cDNA synthesis reaction and a cDNA library was generated from total polyA-mRNA and constructed into ⁇ gtlO (Gubler et al., Gene 25:263 (1983); Lapeyer et al., Gene 37:215 (1985)).
- MMLV reverse transcriptase was used to make a cDNA from the mRNA.
- 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.
- 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 c 2 , 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 NaH 2 P0 4 -H 2 0, .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.
- the filters were prehybridized in 6 x SSPE, 5 x Denhardt's, 25% formamide, 0.2% (w/v) SDS and 200 ⁇ g/ml herring sperm DNA for 2 hours at 42 ⁇ C. They were then hybridized for 3 hours in the same buffer at 42 ⁇ C and washed several times in 1 x SSPE at 45 ⁇ C.
- plaque-purified clones which hybridized to the oligonucleotide was designated ⁇ BL3, and its cDNA insert size was determined, after EcoRI restriction enzyme digestion of phage "mini-preps"
- 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:
- 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 ⁇ ⁇ k3 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.
- the cDNA insert encodes sixteen amino acids amino-terminal to the a ino-ter inus of the mature protein.
- 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.
- 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.
- 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.
- 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.
- lysozyme c2 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 ⁇ BL3 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.
- the lysozyme c2 with histidine at position 98 has essentially the same properties as the lysozyme c2 isolated from bovine abomasum tissue.
- 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.
- 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 ⁇ BL3 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.
- 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
- terminal segment 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.
- HIS4 histidinol dehydrogenase
- 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.
- 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) .
- pAO804 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.
- pre-lysozyme c2 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.
- 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.
- AOX1 major alcohol oxidase gene
- DAS dihydroxyacetone synthase
- Such promoters provide high level transcription, even in single copy, and are tightly regulated, thus permitting the expression period to be limited.
- 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.
- the AOXl promoter is repressed and alcohol oxidase mRNA is not made and alcohol oxidase is not expressed.
- 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.
- 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.
- pSL12A 5 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.
- Pichia pastoris strain GS115 ((His4 ⁇ ); Cregg
- 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
- 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
- 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.
- 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.
- 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.
- the cells were washed once in 10 ml of sterile H 2 0, 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 ⁇ g of Haelll-digested E. coli carrier DNA, 2 ⁇ g 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 ⁇ g of vector DNA can be used to transform the cells. 0.7 ml of 40% polyethylene glycol (PEG 3350 , 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.
- PEG 3350 polyethylene glycol
- LiCl lithium chloride + TE buffer
- Transformant colonies were recovered from the surface of the agar plates by adding 5 ml of sterile H 2 0 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.
- 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 ⁇ C 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 ⁇ C with shaking was then continued and samples were harvested after 1 and 4 days of growth in methanol.
- bovine lysozyme c2 from GS115 strain A37
- a two-stage high cell-density batch fermentation scheme was employed.
- 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.
- 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 H 3 P0 4 , 4.2 mM CaS0 4 -2H 2 0, 65 mM K 2 S0 4 , and 38 mM MgS0 4 ; trace salts at final concentrations of 0.4 ⁇ M CuS0 4 -5H 2 0, 2.5 ⁇ M KI, 9.0 ⁇ M MnS0 4 -H 2 0, 4.0 ⁇ M Na 2 Mo0 4 -2H 2 0, 1.5 ⁇ M H3BO3, 35 ⁇ M ZnS0 4 -7H 2 0, and 89 ⁇ M FeCl 3 -6H 2 0, 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.
- RIA radioimmunoassay
- 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.
- 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.
- 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) .
- 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) .
- PMSF phenylmethyl sulfonyl fluoride
- 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.
- 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 ⁇ C.
- Antisera raised in rabbits against bovine lysozyme c2 were prepared by standard protocols.
- 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 125 I-protein A dilution was composed of 1 x PBS (140 mM sodium chloride, 3 mM potassium chloride, 10 mM disodium phosphate, 2 mM monopotassium phosphate, pH 7.2), 0.25% gelatin (Bio-Rad, Inc., Richmond, 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.
- the iodinated preparation was tested by precipitation in 10% trichloroacetic acid (TCA) to estimate the proportion of intact peptide.
- TCA trichloroacetic acid
- 125 I-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
- Fraction V 0.1% Triton X-100, pH 7.4
- Pansorbin' R ' Calbiochem, San Diego, California
- 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 125 I-lysozyme.
- Micrococcus luteus cells were obtained from Sigma Chemical Company, St. Louis, Missouri.
- spectrophoto etric assay and halo assay 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.
- halo assay 10 ⁇ l 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 ⁇ g.
- 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.
- 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.
- 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
- 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 ⁇ bondapak 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.
- 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.
- 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.
- P. pastoris cells of strain PPF1 (His4 ⁇ Arg4 ⁇ " ) , were transformed with an uncut plasmid, designated pBLll, in the manner described in Example 3.
- 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.
- a fermenter run (run #274) with strain CSBL3 resulted in a specific productivity of 33 ⁇ g/g wet cell/hour at a dilution rate of O.O ⁇ h "1 and a specific productivity of 28 ⁇ g/g wet cell/hour at a dilution rate of O.O ⁇ h "1 .
- Authentic and recombinant lysozymes c were found to have comparable biological activity when tested in parallel using the same assay.
- 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.
- 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.
- 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)).
- 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.
- Casta ⁇ n 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.
- 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.
- HLZ-100 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
- 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.
- M13mpl8 was digested with Hindlll and Sail and was phosphatase-treated.
- 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.
- 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:
- 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
- a plasmid was identified, by Sanger dideoxy sequencing using the universal primer and two primers, of sequences
- the plasmid was designated pHLZ102.
- Plasmid (RF-form M13mpl8) pHLZ102 was isolated using a CsCl gradient.
- 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.
- Plasmid pHLZ103 was purified on a CsCl gradient.
- 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) .
- Sac 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.
- 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 ⁇ l, 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
- a YTM trace salts solution (5.0 ml/1 sulfuric acid, 65.0 g/1 ferrous sulfate-7H 2 0, 6.0 g/1 copper sulfate-5H 2 0, 20.0 g/1 zinc sulfate-7H 2 0, 5 3.0 g/1 manganese sulfate-H 2 0, 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.
- 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.
- the glycerol feed was decreased to 3.6 ml/h (36 ml/h at 10-liters) and a methanol feed containing 12 ml/1 YTM 4 trace salts was initiated at 1.1 ml/h for the 2-liter fermentor or 11 ml/h for the 10-liter fermentor.
- 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.
- 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.
- the methanol feed containing 12 ml/1 YTM 4 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.
- the o methanol feed was turned back on for another 5-minute interval.
- methanol was added to the fermentor to maintain a residual methanol concentration between 0.2 % and 0.8 %.
- the YTM 4 trace salts were not used but, instead, 40 ml of l ⁇ trace salts (5 ml/1 sulfuric acid, 4.8 g/1 ferric chloride-2 H 2 0, 2.0 g/1 zinc sulfate-H 2 0, 0.02 g/1 boric acid, 0.2 g/1 sodium molybdate, 0.3 g/1 manganese sulfate-H 2 0, 0.08 g/1 potassium iodide, 0.06 g/1 cupric sulfate-5 H 2 0) 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.
- 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'
- Plasmid pBR322 RIBG is the same as pBR322 Ri except that pBR322 RIBG has the sequence
- 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.
- 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.
- 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.
- 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.
- pAO802 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 pBSAGI ⁇ l 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:
- -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.
- 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.
- 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.
- 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.
- fragments known in the art are fragments with the S. cerevisiae ARG4 gene, the P. pastoris ARG4 gene and the S. cerevisiae HIS4 gene.
- pAO ⁇ ll which is the analog of pAO804 with a S. cerevisiae ARG4 gene in place of the P.
- 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
- 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
- the orientation of the ARG4 gene in pAO ⁇ ll 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.
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US11594087A | 1987-11-02 | 1987-11-02 | |
US115940 | 1987-11-02 |
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EP19890900706 Withdrawn EP0343231A4 (en) | 1987-11-02 | 1988-11-02 | Production of animal lysozyme c via secretion from pichia pastoris and composition therefor |
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JP (1) | JPH02501976A (en) |
KR (1) | KR890701606A (en) |
AU (1) | AU2825489A (en) |
CA (1) | CA1340063C (en) |
DK (1) | DK327589D0 (en) |
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WO1990003431A1 (en) * | 1988-09-26 | 1990-04-05 | The Salk Institute Biotechnology/Industrial Associates, Inc. | Mixed feed recombinant yeast fermentation |
US5122465A (en) * | 1989-06-12 | 1992-06-16 | Phillips Petroleum Company | Strains of pichia pastoris created by interlocus recombination |
US5612198A (en) * | 1990-09-04 | 1997-03-18 | The Salk Institute | Production of insulin-like growth factor-1 in methylotrophic yeast cells |
CA2105064A1 (en) * | 1991-04-01 | 1992-10-02 | Martin Anthony Gleeson | Genes which influence pichia proteolytic activity, and uses therefor |
US5850025A (en) * | 1991-09-19 | 1998-12-15 | Sibia Neurosciences, Inc. | Protection of plants against plant pathogens |
US5422108A (en) * | 1991-09-19 | 1995-06-06 | Smart Plants International Inc. | Protection of plants against plant pathogens |
CN109810963B (en) * | 2017-11-22 | 2021-09-28 | 上海复华兴生物技术有限公司 | DNA sequence of bovine intestine-derived lysozyme protein and production process and application thereof |
CN110903991A (en) * | 2019-11-13 | 2020-03-24 | 浙江新银象生物工程有限公司 | Recombinant pichia pastoris engineering bacteria containing high-copy-number humanized lysozyme gene and application thereof |
CN112029783B (en) * | 2020-09-15 | 2022-05-27 | 西南民族大学 | Method for heterogeneously expressing ruminant stomach lysozyme by using pichia pastoris |
CN115873733A (en) * | 2022-07-20 | 2023-03-31 | 青岛蔚蓝生物集团有限公司 | Pichia pastoris strain for high yield of lysozyme and application thereof |
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1988
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- 1988-11-02 JP JP1500523A patent/JPH02501976A/en active Pending
- 1988-11-02 WO PCT/US1988/003907 patent/WO1989004320A1/en not_active Application Discontinuation
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1989
- 1989-06-30 FI FI893207A patent/FI893207A0/en not_active Application Discontinuation
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Non-Patent Citations (6)
Title |
---|
Bio/Technology, Vol. 7, No. 2, February 1989, pages 160-164, New York, US; M.E. DIGAN et al.: "Continuous via secretion from the yeast, pichia pastoris", the whole article. * |
Developments in Industrial Microbiology, Proceedings of the 44th General Meeting SIM, August 10-14, 1987, Baltimore, MD, US, Vol. 29, pages 59-65, Society for Industrial Microbiology, Washington, D.C. US; M.E. DIGAN et al.: "Secretion of heterologous proteins from the methylotrophic yeast, pichia pastoris", the whole article. * |
Gene, Vol. 43, No. 3, 1986, pages 273-279, Elsevier Science Publishers B.V., Amsterdam, NL; Y. JIGAMI et al.: "Expression of synthetic human-lysozyme gene in saccharomyces cerevisiae: use of a synthetic chicken-lysozyme signal sequence for secretion and processing". * |
Molecular and Cellular Biochemistry, Vol. 63, 1984, pages 165-189, Martinus Nijhoff Publishers, Boston, The Hague, NL; P. JOLLES et al.: "What's new in lysozyme research", the whole article. * |
See also references of WO8904320A1 * |
The Journal of Biological Chemistry, Vol. 259, No. 18, September 25, 1984, pages 11617-11625, The American Society of Biological Chemists Inc., Washington, D.C, US; P. JOLLES et al.: "Stomach lysozymes of ruminants". * |
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WO1989004320A1 (en) | 1989-05-18 |
CA1340063C (en) | 1998-09-29 |
FI893207A0 (en) | 1989-06-30 |
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EP0343231A4 (en) | 1990-09-05 |
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