Classifications

• • 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)
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
  • C07K14/39—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts
  • C07K14/395—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts from Saccharomyces
• • 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
• • 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

• This invention relates to a process of recombinant DNA technology for producing human lysozyme (HLZ) peptides in methylotrophic yeast such as Pichia pastoris.
• the invention further relates to the methylotrophic yeast transformants, DNA fragments and expression vectors used for their production and cultures containing same.
• 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 l,4-3-N- acetylmuramidases, cleave the glycosidic bond between the C-l of N-acetylmuramic acid and the C-4 of N- acetylgulcosamine 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- /S-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 (e.g., lactotransferrin (which inhibits the growth of certain microorganisms by chelating iron) , complement, antibodies, vitamins, other enzymes and various antibiotics (e.g. , tetracycline and bacitracin) , as antimicrobial agents, as preservatives for foods (e.g., cheese, sausage and marine products) , as ripening agents for cheese, and in various other applications. Since 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.
• other components e.g., lactotransferrin (which inhibits the growth of certain microorganisms by chelating iron)
• complement e.g., antibodies, vitamins, other enzymes and various antibiotics (e.g. , tetracycline and bac
• Lysozymes of the c, or "chicken", type contain 129-130 amino acids in their mature, secreted forms. Forty of these 129-130 amino acids have been found to be invariant among different species.
• Two of the several carboxyl groups of lysozyme molecules (corresponding to the Glu-35 and 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 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 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 also known. See European Patent Application Publication Nos. 0 181 634, 0 208 472, and 0 222 366.
• HLZ human lysozyme
• coli tends to produce disulfide-bonded proteins such as HLZ in their reduced for s which frequently are not stable in the presence of endogenous bacterial proteases, and which tend to aggregate into inactive complexes. See, for example, Muraki et al. , in A ⁇ ric. Biol. Chem.. 49: 2829-2831 (1985). Attempts to overcome this problem, e.g., by employing a suitable leader sequence in order to produce soluble HLZ which could be readily recovered from the cell broth, will frequently result in other inconveniences, especially during purification of the product, since the bulk of the desired protein may become associated with the cell paste.
• 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 somewhat 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 can offer clear advantages over bacteria in the production of heterologous proteins, which include their ability to secrete heterologous proteins into the culture medium. Secretion of proteins from cells is generally superior to production of proteins in the cytoplasm. Secreted products are obtained in a higher degree of initial purity; and further purification of the secreted products is made easier by the absence of cellular debris. In the case of sulfhydryl-rich proteins, there is another compelling reason for the development of eukaryotic hosts capable of secreting such proteins into the culture medium: their correct tertiary structure is produced and maintained via disulfide bonds.
• European Patent Application 255,233 describes the production of human lysozyme by cultivating a cell transformed with a DNA sequence which codes for hen egg white lysozyme signal peptide bound to the 5' end of a DNA segment coding for human lysozyme. Cultivation of S. cerevisiae transformed with this construct produced only a few mg per liter of human lysozyme, with about half of the HLZ produced being secreted into the culture medium.
• European Patent Application 251,730 similarly describes the production of human lysozyme employing a synthetic signal peptide which is said to be superior to that of hen egg white lysozyme for secretive production of human lysozyme.
• the production of only about 5 mg/L of secreted HLZ is all that is demonstrated, however.
• Other references which disclose the secreted production of HLZ by the yeast S. cerevisiae include Suzuki et al. , 14th Int. Conf. on Yeast Genetics and Molecular Biology, p.
• 83 052882 (3/7/88; priority JP 86 196226 (8/21/86) ; assigned to Sumitomo Chem. Ind. KK) ; Japanese Application No. 83 052881 (3/7/88; priority JP 86 195885 (8/20/86) ; assigned to Sumitomo Chem. Ind. KK) ; Japanese Application No. 83 052876 (3/7/88; priority JP 86 195886 (8/20/86) ; assigned to Sumitomo Chem. Ind. KK) ; Japanese Application No.
• 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.
• yeast expression system based on methylotrophic yeast, such as for example, Pichia pastoris, has been developed.
• Methylotrophic yeasts have been found to be particularly favorable for use as host systems for the large-scale production of those heterologous proteins which they are capable of secreting into the culture media at significant levels in biologically active form.
• Methylotrophic yeast e.g., P. pastoris
• P. pastoris are readily adaptable to continuous industrial-scale fermentation processing, whereby the yeasts grow to high cell densities in a defined and inexpensive fermentation medium.
• production levels usually scale up from shake-flask cultures to large fermentor 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 such yeast.
• yeasts since 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 cf cultures of other types of hosts, thereby increasing the reliability and safety of the heterologous polypeptide products.
• a key feature of expression systems based on methylotrophic yeast lies with the promoter employed to drive heterologous gene expression.
• This promoter which is derived from a methanol-responsive gene of a methylotrophic yeast, is frequently highly expressed and tightly regulated (see, e.g., U.S. Patent No. 4,855,231).
• Another key feature of expression systems based on methylotrophic yeast is the ability of expression cassettes to stably integrate into the genome of the methylotrophic yeast host, thus significantly decreasing the chance of vector loss.
• heterologous proteins e.g., hepatitis B surface antigen [Cregg et al., Bio/Technology 5 , 479 (1987)], lysozyme [see WO 89/03907, published 5/18/89; assigned to Salk Institute Biotechnology/Industrial Associates; describes bovine lysozyme secretion promoted by the native lysozyme signal sequence] and invertase [Digan et al.. Developments in Industrial Microbiology 29, 59 (1988); Tschopp et al., Bio/Technology 5 , 1305 (1987)].
• heterologous proteins e.g., hepatitis B surface antigen [Cregg et al., Bio/Technology 5 , 479 (1987)]
• lysozyme see WO 89/03907, published 5/18/89; assigned to Salk Institute Biotechnology/Industrial Associates; describes bovine lysozyme secretion promoted by
• HLZ biologically active human lysozyme
• the present invention provides a powerful method for the production of secreted HLZ peptides in methylotrophic yeast.
• the invention method can easily be scaled up from shake-flask cultures to large scale fermentors with no loss in HLZ productivity.
• the invention method can readily be scaled up without the need for making major changes in the fermentation conditions used for the large scale growth of the transformed strains relative to the conditions used for small scale growth of transformed strains.
• HLZ peptides can very efficiently be produced in, and secreted from, methylotrophic yeast, such as, for example, P. pastoris. This is accomplished by transforming a methylotrophic yeast with, and preferably integrating into the yeast genome, at least one copy of a first DNA sequence operably encoding an HLZ peptide, wherein said first DNA sequence is operably associated with a second DNA sequence encoding the S. cerevisiae alpha-mating factor (AMF) pre-pro sequence (including the proteolytic processing site: lys-arg) , and wherein both of said DNA sequences are under the regulation of a methanol responsive promoter region of a gene cf a methylotrophic yeast.
• AMF S. cerevisiae alpha-mating factor
• Methylotrophic yeast cells containing in their genome at least one copy of these DNA sequences efficiently produce and secrete biologically active HLZ peptides into the medium.
• the present invention is directed to the above aspects and all associated methods and means for accomplishing such.
• the invention includes the technology requisite to suitable growth of the methylotrophic yeast host cells, fermentation, and isolation and purification of the HLZ gene product.
• Figure 1 provides the nucleotide sequence of the human lysozyme gene employed in the examples.
• Figure 2 is a restriction map of plasmid pA0815.
• Figure 3 is a restriction map of plasmid pHLZ103.
• Figure 4 is a restriction map of plasmid pHLZ105.
• Figure 5 is a restriction map of plasmid pAHZ106.
• Figure . 6 is a restriction map of plasmid pAHZ108.
• Figure 7 is a restriction map of plasmid pMHZ109.
• a DNA fragment containing at least one copy of a first expression cassette comprising in the reading frame direction of transcription, the following DNA sequences:
• the DNA fragment according to the invention can be transformed into methylotrophic yeast cells as a linear fragment flanked by DNA sequences having sufficient homology with a target gene to effect integration of said DNA fragment therein. In this case integration takes place by addition or replacement at the site of the target gene.
• the DNA fragment can be part of a circular plasmid, which may be linearized to facilitate integration, and will integrate by addition at a site of homology between the host and the plasmid sequence.
• an expression vector containing at least one copy of a DNA fragment as described hereinabove.
• novel methylotrophic yeast cells containing in their genome at least one copy of the above described DNA fragment.
• a process for producing HLZ peptides by growing methylotrophic yeast transformants containing in their genome at least one copy of a DNA sequence operably encoding an HLZ peptide, operably associated with DNA encoding the S.
• cerevisiae AMF pre-pro secretion signal sequence (including the lys-arg proteolytic processing site) , and optionally further encoding an HLZ peptide, operably associated with DNA encoding the native HLZ secretion signal peptide, wherein both the coding sequence(s) and the signal sequence(s) are maintained under the regulation of promoter region(s) of methanol responsive gene(s) of methylotrophic yeast, under conditions allowing the expression of said DNA sequence in said transformants and secreting HLZ peptides into the culture medium. Cultures of viable methylotrophic yeast cells capable of producing HLZ peptides are also within the scope of the present invention.
• the polypeptide product produced in accordance with the present invention is secreted to the culture medium at surprisingly high concentrations; the level of HLZ peptides secretion mediated solely by the AMF pre-pro secretion signal sequence is more than ten times higher than that obtained with strains wherein secretion is mediated solely by the native lysozyme signal sequence.
• the excellent results obtained in the practice of the present invention are also due to the fact that the S.
• cerevisiae alpha-mating factor pre-pro secretion signal sequence (when employed alone, or in combination with the native human lysozyme secretion signal sequence) functions unexpectedly well to direct secretion of HLZ peptides in methylotrophic yeast.
• human lysozyme or "HLZ peptide” or simply “HLZ”, as used throughout the specification and in the claims, refers to a polypeptide product which exhibits similar, in-kind, biological activities to natural human lysozyme, as measured in recognized bioassays, and has substantially the same amino acid sequence as native KLZ.
• polypeptides deficient in one or more amino acids in the amino acid sequence reported in the literature for naturally occurring HLZ or polypeptides containing additional amino acids or polypeptides in which one or more amino acids in the amino acid sequence of natural HLZ are replaced by other amino acids are within the scope of the invention, provided that they exhibit the functional activity of HLZ, e.g., the ability to lyse cells and to produce a stimulatory effect on the phagocytic activity of polymorphonuclear leukocytes and acrophages.
• the invention is intended to embrace all the allelic variations of HLZ.
• HLZ derivatives obtained by simple modification of the amino acid sequence of the naturally occurring product, e.g, by way of site-directed mutagenesis or other standard procedures, are included within the scope of the present invention.
• Forms of HLZ produced by proteolysis of host cells that exhibit similar biological activities to mature, naturally occurring HLZ are also encompassed by the present invention.
• amino acids which occur in the various amino acid sequences referred to in the specification have their usual, three- and one-letter abbreviations, routinely used in the art, i.e.:
• HLZ peptides are produced by methylotrophic yeast cells containing in their genome at least one copy of a DNA sequence operably encoding HLZ peptides operably associated with DNA encoding the S. cerevisiae ⁇ -mating factor (AMF) pre-pro secretion signal sequence (including the proteolytic processing site: lys-arg) , and optionally further encoding an HLZ peptide, operably associated with DNA encoding the native human lysozyme secretion signal peptide, under the regulation of promoter region(s) of methanol responsive gene(s) of methylotrophic yeast.
• AMF S. cerevisiae ⁇ -mating factor
• pre-pro secretion signal sequence including the proteolytic processing site: lys-arg
• an HLZ peptide operably associated with DNA encoding the native human lysozyme secretion signal peptide, under the regulation of promoter region(s) of methanol responsive gene(s)
• HLZ peptides as used herein includes DNA sequences encoding HLZ or any other "HLZ peptide” as defined hereinabove.
• DNA sequences encoding HLZ are known in the art. They may be obtained by chemical synthesis or by transcription of messenger RNA (mRNA) corresponding to HLZ into complementary DNA (cDNA) and converting the latter into a double stranded cDNA. Chemical synthesis of a gene for HLZ is, for example, disclosed by Muralli et al. , Agric. Biol. Chem.. 50: 713-723 (1986); see also Ikehara et al. , Chem. Pharm. Bull., . 34 . : 2202 (1986) .
• the requisite DNA sequence can also be removed, for example, by restriction enzyme digest of known vectors harboring the HLZ gene. Examples of such vectors and the means for their preparation are presented in the "Background" section of this disclosure.
• the structure of a presently preferred HLZ gene used in accordance with the present invention is illustrated in FIG. 1 and is further elucidated in the examples.
• Yeast species contemplated for use in the practice of the present invention are methylotrophs, i.e., species which are able to grow on methanol (as well as other) carbon source nutriment.
• Species which have the biochemical pathways necessary for methanol utilization fall into four genera, i.e., Candida, Hansenula, Pichia, and Torulopsi ⁇ . Of these, a substantial amount is known about the molecular biology of members of the species Hansenula polymorpha and Pichia pastoris.
• the presently preferred yeast species for use in the practice of the present invention is Pichia pastoris, a known industrial yeast strain that is capable of efficiently utilizing methanol as the sole carbon and energy source.
• methanol responsive genes in methylotrophic yeast the expression of each being controlled by methanol responsive regulatory regions (also referred to as promoters) .
• methanol responsive promoters are suitable for use in the practice of the present invention.
• specific regulatory regions include the promoter for the primary alcohol oxidase gene from Pichia pastoris (AOX1) , the promoter for the secondary alcohol oxidase gene from P. pastoris (AOX2) , the promoter for the dihydroxyacetone synthase gene from P. pastoris (DAS) , the promoter for the P40 gene from P. pastoris, the promoter for the catalase gene from P. pastoris, and the like.
• the presently preferred promoter region employed to drive HLZ gene expression is derived from a methanol-regulated alcohol oxidase gene of P. pastoris.
• P. pastoris is known to contain two functional alcohol oxidase genes: alcohol oxidase I (AOX1) and alcohol oxidase II (AOX2) genes.
• AOX1 alcohol oxidase I
• AOX2 alcohol oxidase II
• the coding portions of the two AOX genes are closely homologous at both the DNA and the predicted amino acid sequence levels and share common restriction sites.
• the proteins expressed from the two genes have similar enzymatic properties but the promoter of the A0X1 gene is more efficient and more highly expressed; therefore, its use is preferred for
• the AOX1 gene including its promoter, has been isolated and thoroughly characterized; see Ellis et al., Mol. Cell. * Biol. 5 , 1111 (1985) and US 4,855,231.
• the DNA fragment used for transforming methylotrophic yeast cells contains at least one copy of a first expression cassette which, in addition to a methanol responsive promoter of a methylotrophic yeast gene and the HLZ encoding DNA sequence (HLZ gene) , contains a DNA sequence encoding, in-reading frame, the S.
• cerevisiae AMF pre-pro secretion signal sequence including a DNA sequence encoding the processing site: lys-arg (also referred to as the lys-arg encoding sequence) , and a transcription terminator functional in a methylotrophic yeast.
• the DNA fragment used for transforming methylotrophic yeast cells optionally further contains at least one copy of a second expression cassette which, in addition to a methanol responsive promoter of a methylotrophic yeast gene and the HLZ encoding DNA, also includes, in reading frame, the sequence encoding the secretion signal sequence native to HLZ.
• the S. cerevisiae alpha-mating factor is a 13-residue peptide, secreted by cells of the "alpha” mating type, that acts on cells of the opposite "a” mating type to promote efficient conjugation between the two cell types and thereby formation of "a-alpha” diploid cells [Thorner et al., The Molecular Biology the Yeast Saccharomyces, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 143 (1981)].
• the AMF pre-pro sequence is a leader sequence contained in the AMF precursor molecule, and includes the lys-arg encoding sequence which is necessary for proteolytic processing and secretion (see e.g. Brake et al., Proc. Natl. Acad.
• the AMF pre-pro sequence employed in the practice of the present invention is a 255 bp fragment obtained from plasmid pAO208, which is described in WO 89/03907, which is hereby incorporated by reference herein.
• the transcription terminator functional in a methylotrophic yeast used in accordance with the present invention has either (a) a subsegment which encodes a polyadenylation signal and polyadenylation site in the transcript, and/or (b) a subsegment which provides a transcription termination signal for transcription from the promoter used in the expression cassette.
• expression cassette refers to a DNA sequence which includes sequences functional for both the expression and the secretion processes.
• the entire transcription terminator is taken from a protein-encoding gene, which may be the same or different from the gene which is the source of the promoter.
• multiple copies of the above- described expression cassettes be contained on one DNA fragment, preferably in a head-to-tail orientation.
• the DNA fragments according to the invention optionally further comprise a selectable marker gene.
• a selectable marker gene functional in methylotrophic yeast may be employed, i.e., any gene which confers a phenotype upon methylotrophic yeast cells, thereby allowing them to be identified and selectively grown from among a vast majority of untransformed cells.
• Suitable selectable marker genes include, for example, selectable marker systems composed of an auxotrophic mutant P. pastoris host strain and a wild type biosynthetic gene which complements the host's defect.
• the S. cerevisiae or P. pastoris HIS4 gene for transformation of Arg4 ⁇ mutants
• the S. cerevisiae ARG gene or the P. pastoris ARG4 gene may be employed.
• the DNA fragment is integrated into the host genome by any of the gene replacement techniques known in the art, such as by one-step gene replacement [see e.g., Rothstein, Methods Enzymol. 101, 202 (1983); Cregg et al., Bio/Technology 5 , 479 (1987); and U.S. Patent No. 4,882,279] or by two-step gene replacement methods [see e.g., Scherer and Davis, Proc. Natl. Acad. Sci. USA, 76, 4951 (1979) ] .
• gene replacement techniques known in the art, such as by one-step gene replacement [see e.g., Rothstein, Methods Enzymol. 101, 202 (1983); Cregg et al., Bio/Technology 5 , 479 (1987); and U.S. Patent No. 4,882,279] or by two-step gene replacement methods [see e.g., Scherer and Davis, Proc. Natl. Acad. Sci. USA, 76, 4951
• the linear DNA fragment is directed to the desired locus, i.e., to the target gene to be disrupted, by means of flanking DNA sequences having sufficient homology with the target gene to effect integration of the DNA fragment therein.
• flanking DNA sequences having sufficient homology with the target gene to effect integration of the DNA fragment therein.
• One- step gene disruptions are usually successful if the DNA to be introduced has as little as 0.2 kb homology with the fragment locus of the target gene; it is however, preferable to maximize the degree of homology for efficiency.
• DNA fragment according to the invention is contained within, or is an expression vector, e.g., a circular plasmid
• one or more copies of the plasmid can be integrated at the same or different loci, by addition to the genome instead of by gene disruption.
• Linearization of the plasmid by means of a suitable restriction endonuclease facilitates integration.
• expression vector is intended to include vectors capable of expressing DNA sequences contained therein, where such sequences are in operational association with other sequences capable of effecting their expression, i.e., promoter sequences.
• expression vectors usually used in recombinant DNA technology are often in the form of "plasmids", i.e., circular, double-stranded DNA loops, which in their vector form are not bound to the chromosome.
• vector and "plasmid” are used interchangeably.
• the invention is intended to include other forms of expression vectors as well, which function equivalently.
• the segments of the expression cassette(s) are said to be "operationally associated" with one another.
• the DNA sequence encoding HLZ peptides is positioned and oriented functionally with respect to the promoter, the DNA sequence encoding the processing and secretion signal, i.e., the S. cerevisiae AMF pre- pro sequence (including the DNA sequence encoding the AMF processing-site: lys-arg) , or the native HLZ secretion signal sequence, and the transcription terminator.
• the polypeptide encoding segment is transcribed, under regulation of the promoter region, into a transcript capable of providing, upon translation, the desired polypeptide.
• the expressed HLZ product is found as a secreted entity in the culture medium.
• Appropriate reading frame positioning and orientation of the various segments of the expression cassette are within the knowledge of persons of ordinary skill in the art; further details are given in the Examples.
• the DNA fragment provided by the present invention may include sequences allowing for its replication and selection in bacteria, especially E. coli. In this way, large quantities of the DNA fragment can be produced by replication in bacteria.
• methylotrophic yeast such as, for example, Pichia pastoris
• the expression cassettes are transformed into methylotrophic yeast cells either by the spheroplast technique, described by Cregg et al., in Mol. Cell. Biol. 5 , 3376 (1985) and U.S. Patent No. 4,879,231; or by the whole-cell lithium chloride yeast transformation system [Ito et al. , Agric. Biol. Chem. 48, 341 (1984)], with modification necessary for adaptation to methylotrophic yeast, such as P. pastoris [See U.S. Patent No. 4,929,555].
• the spheroplast method is preferred.
• Transformed strains which are of the desired phenotype and genotype, are grown in fermentors.
• a three-stage, high cell-density, fed-batch fermentation system is normally the preferred fermentation protocol employed.
• expression hosts are cultured in defined minimal medium with an excess of a non- inducing carbon source (e.g., glycerol) .
• a non- inducing carbon source e.g., glycerol
• methanol fed-batch mode methanol alone
• mixed-feed fed-batch mode a limiting amount of a non-inducing carbon source plus and methanol
• the heterologous protein expression system used for HLZ production utilizes the promoter derived from the methanol-regulated AOX1 gene of P. pastoris, which is very efficiently expressed and tightly regulated.
• This gene can be the source of the transcription terminator as well.
• the presently preferred expression cassette comprises, operationally associated with one another, the P. pastoris AOXl promoter, DNA encoding the S. cerevisiae AMF pre-pro sequence (including the DNA sequence encoding the AMF processing site: lys-arg) , a DNA sequence encoding mature HLZ, and a transcription terminator derived from the P. pastoris AOXl gene.
• two or more of such expression cassettes are contained on one DNA fragment, in head-to-tail orientation, to yield multiple expression cassettes on a single contiguous DNA fragment.
• the presently preferred host cells to be transformed with multiple expression cassettes are P. pastoris cells having at least one mutation that can be complemented with a marker gene present on a transforming DNA fragment.
• His4 " (GS115) or Arg4 " (GS190) auxotrophic mutant P. pastoris strains are employed.
• the fragment containing one or more expression cassette(s) is inserted into a plasmid containing a marker gene complementing the host's defect.
• pBR322-based plasmids e.g., pA0815
• Insertion of one or more copies of an AMF- based HLZ expression/secretion cassette into parent plasmid pA0815 produces plasmids pAHZ106 and pAHZ108; while insertion of one copy of an AMF-based expression cassette and one copy of a native HLZ signal sequence based expression cassette produces plasmid pMHZ109.
• the transforming DNA comprising the expression cassette(s) is preferably integrated into the host genome by a one-step gene replacement technique.
• the expression vector is digested with an appropriate enzyme to yield a linear DNA fragment with ends homologous to the AOXl locus by means of the flanking homologous sequences.
• the AOXl gene is replaced with the expression cassette(s), thus decreasing the strains' ability to utilize methanol.
• a slow growth rate on methanol is maintained by expression of the AOX2 gene product.
• the transformants in which the expression cassette has integrated into the AOXl locus by site- directed recombination can be identified by first screening for the presence of the complementing gene. This is preferably accomplished by growing the cells in media lacking the complementing gene product and identifying those cells which are able to grow by nature of expression of the complementing gene. Next, the selected cells are screened for their Mut phenotype by growing them in the presence of methanol and monitoring their growth rate.
• the fragment comprising one or more expression cassette(s) preferably is integrated into the host genome by transformation of the host with a linearized plasmid comprising the expression cassette(s) .
• the integration is by addition at a locus or loci having homology with one or more sequences present on the transformation vector. Positive transformants are characterized by
• Methylotrophic yeast transformants which are identified to have the desired genotype and phenotype are grown in fermentors. It is presently preferred to use the three-step production process described above.
• the level of HLZ secreted into the media can be determined by Western blot analysis of the media in parallel with an HLZ standard, using anti-HLZ antisera; by radioimmunoassay (RIA) ; by enzyme-inhibitor assay; or by HPLC after suitable pretreatment of the medium.
• Human lysozyme is purified to apparent homogeneity by ion exchange chromatography.
• the purification scheme typically employed comprises the following steps. First, fermentor broth is ultrafiltered, the filtrate then loaded directly onto a cationic ion exchange column. The loaded column is then washed sequentially with a series of buffers of increasing pH and varying conductivity (to remove endogenous P. pastoris proteins) . Finally the HLZ product is eluted with a high conductivity buffer, dialyzed and lyophilized.
• the purification scheme employed for the purification of AMF-based, secreted HLZ must be capable of separating authentic HLZ from the incorrectly processed forms. It has been found that this can readily be accomplished by making several modifications to the purification scheme described above. First, it has been found that the ultrafiltration step can be eliminated, and cell broth loaded directly onto the cationic ion exchange column. This alone provides a substantial time savings with respect to the processing time required to prepare purified HLZ.
• the ratio of ion exchange material to fermentation broth is at least doubled, relative to the amount of resin ordinarily employed.
• loading levels in the range of about 10 up to 20 ml of fermentation broth per gram of ion exchange resin are employed. After thorough washing of the loaded resin with a sufficient quantity of solvent having a pH and conductivity effective to cause elution of endogenous P. pastoris proteins, and thereafter the correctly processed HLZ is selectively eluted with a solvent gradient so as to separate the authentic human lysozyme from the incorrectly processed forms.
• the thorough washing is typically carried out in a stepwise fashion wherein (1) 1-2 volumes (relative to the volume of ion exchange resin) of buffered media having a conductivity in the range of about 15-25 mMho, and a pH which is higher than the pH of the fermentation broth (e.g., in the range of about 3.5-5.5) ; followed by (2) 2-4 volumes of buffered media having a conductivity which is lower than that of the initial wash (e.g., in the range of about 10-15 mMhos and a pH which is higher than that of the initial wash (e.g., in the range of about 5.5-7.0) .
• the gradient employed for selective elution of authentic HLZ from the washed cationic ion exchange resin comprises at least 6 volumes (relative to the volume of ion exchange resin employed) of a buffered media having a pH in the range of about 7.7 up to 8.2; wherein a linear gradient is employed starting at a conductivity of about 5-25 mMhos, and increasing to a final conductivity in the range of about 40-60 mMhos.
• P. pastoris is described herein as a model system for the use of methylotrophic yeast hosts.
• Other useful methylotrophic yeasts can be taken from four genera, namely Candida, Hansenula, Pichia and Torulopsis. Equivalent species from them may be used as hosts herein primarily based upon their demonstrated characterization of being supportable for growth and exploitation on methanol as a single carbon nutriment source. See, for example, Gleeson et al., Yeast 4 . , l (1988) .
• Plasmid pHLZlOO (described in WO 89/03907) , a pUC8 vector containing an almost full length cDNA clone for human lysozyme inserted into the Pstl site, was used to transform E. coli strain MC1061.
• Transformants were screened by examination of BamHI-Hindlll restriction enzyme-digested DNA for the presence of an insert of approximately 570 bp containing an internal Pstl site. A colony with the expected restriction pattern was used to prepare plasmid DNA.
• the 570 bp fragment was cloned into the Hindlll-Sall sites of mpl8, yielding plasmid pHLZlOl.
• Site-directed mutagenesis [Zoller and Smith Meth. Enzymol. 100:468 (1983)] was used to insert an EcoRI site immediately following the translation termination codon.
• the mutagenizing oligonucleotide was of the following sequence:
• the mutagenized clone was sequenced to verify the desired addition of an EcoRI site, and then used for a second site-directed mutagenesis to replace the four amino-terminal amino acids and to insert an EcoRI site immediately preceding the initiation codon.
• the mutagenizing oligonucleotide was of the following sequence:
• the fully mutagenized plasmid was called pHLZ102.
• the lysozyme gene was isolated on an EcoRI fragment (500 ng) and separately inserted into the unique EcoRI site of the Pichia pastoris vector pAO804 and pA0815 (25-50 ng) .
• the construction of pAO804 is described in Example 8.
• Plasmid pA0815 differs from pAO804 in a single restriction site, BamHI (the Hindlll/Clal/Haelll site of pAO804 is changed to a BamHI site, providing plasmid pA0815) .
• a restriction map of pA0815 is provided in Figure 2. The ligation mixture was transformed into MC1061 cells and amp R colonies were selected.
• the resulting expression vectors contain the gene for human lysozyme under the control of the Pichia pastoris AOXl promoter and regulatory regions as well as the AOXl transcription termination and polyadenylation signals.
• the vectors include the Pichia pastoris HIS4 gene used for selection in His " hosts, and additional 3' AOXl sequences used to direct integration into the host genome. These plasmids are shown in Figures 3 and 4, respectively.
• the entire lysozyme gene and approximately 20-25 bases each of the promoter and termination regions were sequenced in pHLZ103 to verify that no changes had occurred during cloning.
• the ⁇ MF pre-pro sequence consists of 89 amino acids which function to direct peptides fused to it through the secretory pathway.
• the ⁇ MF pre-pro region contains an 83-amino acid signal sequence preceding three processing sites, lys-arg and (glu- ala) 2 , which are susceptible to the proteolytic action of two specific proteases. Cleavage of the fusion protein at the junction of the ⁇ MF pre-pro and peptide sequences by secretory pathway-localized proteases allows the peptide to exit the cell.
• DNA were isolated from pHLZ102 (see Example 1) on a Sail-Hindlll fragment.
• This Sail-Hindlll fragment (1400 ng) was then inserted into an Ml3 vector (100 ng) which contained, in the EcoRI-Smal sites, a 275 bp EcoRI-Hindlll fragment from pAO208 encoding the ⁇ MF pre-pro region, including the three processing sites.
• Plasmid pAO208 is described in Example 6.
• the ligation was transformed into JM103 cells and DNA from plaques were analyzed. Correct plasmid exhibited 450 and 7500 bp sized bands upon digestion with HindiII-Sa l.
• Site- directed mutagenesis of the resulting plasmid was used to delete the M13 polylinker, the nucleotides encoding the (glu-ala) 2 processing sites, and the DNA encoding the native signal sequence, thereby fusing the sequence encoding the 83-amino acid pre-pro region and lys-arg processing site directly to the first codon of mature human lysozyme.
• the oligonucleotide used to perform this mutagenesis was of the following sequence:
• the fusion gene consisting of the DNA sequences encoding the ⁇ MF pre-pro region ending in the lys-arg processing site and the mature human lysozyme gene was isolated as an EcoRI fragment (500 ng) and inserted into EcoRI-digested Pichia pastoris vector pA0815 (50 ng) .
• the ligation was transformed into MC1061 cells and amp R colonies were selected. Correct plasmid exhibited 1825, 530, and 6130 bp sized bands upon digestion with Pstl.
• the resulting single-copy expression vector, pAHZl06 ( Figure 5) , contains one copy of the ⁇ MF pre-pro-hu an lysozyme fusion gene under the transcriptional control of the Pichia pastoris AOXl promoter and regulatory regions, as well as the AOXl transcription termination and polyadenylation signals.
• the vector also includes the Pichia pastoris HIS4 gene for selection in His " hosts and additional 3 ' AOXl sequences.
• the entire ⁇ MF pre-pro-human lysozyme fusion gene and approximately 20 nucleotides each of the promoter and termination regions of pAHZ106 were sequenced to verify that the sequences were not altered during the cloning process.
• the expression cassette consisting of the AOXl promoter, ⁇ MF pre-pro-human lysozyme fusion gene, and AOXl transcription termination region was isolated from pAHZ106 on a Bglll-BamHI fragment (250 ng) and inserted back into the unique BamHI site of pAHZ106 (25 nq) .
• the liqation was transformed into MC1061 cells and ampR colonies were selected. Correct plasmid exhibited 2400, 3700, and 4235 bp sized bands upon digestion with Bglll-BamHI.
• the resulting vector, pAHZ108 ( Figure 6) , contains two copies of the expression cassette as tandem-repeat units.
• iii Two-copy vector containing the native and ⁇ MF pre-pro signal sequences: pMHZ109
• Vector pMHZ109 which contains one copy of the native signal sequence-human lysozyme expression cassette and one copy of the ⁇ MF pre-pro-human lysozyme expression cassette, is shown in Figure 7.
• the expression cassette consisting of the AOXl promoter, ⁇ MF pre-pro-human lysozyme fusion gene, and AOXl transcription termination region was isolated from pAHZlOe on a BamHI-BgJ- . II fragment (250 ng) and inserted into the unique BamHI site of pHLZ105 (25 ng) .
• the ligation was transformed into MC1061 cells and amp R colonies were selected. Correct plasmid exhibited
• Mut + strains of P. pastoris were used to develop Mut + strains of P. pastoris.
• the Mut phenotype refers to methanol utilization.
• Mut + strains utilize methanol in a wild- type fashion by nature of insertion of the expression cassette into the genome by addition rather than by disruption.
• Mut + strains were developed by integration of the entire expression vector into either the AOXl or HIS4 locus by additive homologous recombination. For site-directed addition of the single copy vector into the AOXl locus, the single copy vectors were digested with Sad, which linearizes the vector within the AOXl promoter region.
• the multicopy vectors were digested with StuI, which linearizes the plasmid within the HIS4 region.
• undigested plasmid was allowed to integrate randomly into the AOXl locus at either the 5 ' or 3 ' regions found in the plasmid or into the HIS locus.
• the coding region of the AOXl gene was undisturbed.
• plasmid pHLZ103 was digested with Bglll. This liberates an expression cassette comprised of the AOXl promoter region, human lysozyme gene, AOXl transcription termination signals, HIS4 gene for selection, and AOXl 3' region. Both ends of this expression cassette contain long sequences which are homologous to the 5' and 3' ends of the AOXl locus.
• the expression cassette is integrated into the AOXl locus by a homologous recombination event which results in the substitution of the Bglll-ended expression cassette for the AOXl structural gene. Positive transformants were selected first by their His + phenotype and then by their Mut ' phenotype, i.e. slow growth on methanol.
• Transformants resulting from integration of the Bglll fragment of pHLZ103 were initially screened for histidine prototropy and were then analyzed for growth on methanol-containing plates. Approximately 30% were slow growers, indicative of disruption of the AOXl gene. Eight of these Mut " transformants were analyzed by the Southern blots described above.
• Example 3 Growth of human lysozyme-expressing strains in one and ten liter fermentations
• the fermentor was autoclaved with 1000 ml of medium containing 500 ml of 10X basal salts, 5% glycerol, and the remainder deionized water. After sterilization, 4 ml of PTM 1 trace salts were added, and the pH was adjusted to 5.0 with concentrated NH ⁇ OH. The pH of the medium was maintained at 5 by addition of 50% NH 4 OH containing 0.1% Struktol J-673 antifoam. Inocula were prepared from buffered YNB containing 2% glycerol. The fermentor was inoculated with 10-50 ml of the cultured cells which had grown to an OD ⁇ g of 1- ⁇ , and the batch growth regimen was continued for l ⁇ -24 hours.
• a glycerol feed (50% glycerol plus 12 ml/L PTM 1 salts) was initiated at 5-20 ml/hour and continued until 200 ml of glycerol feed had been added. Subsequently, the glycerol feed was terminated, and a methanol feed (100% methanol plus 12 ml/L PTM 1 salts) was started at an initial rate of approximately 2 ml/hour. After 2 hours, the methanol feed rate was increased in increments of 10% every 30 minutes until a final rate of 5-5.5 ml/hour was attained. The vessel was harvested 70-100 hours following methanol induction. Results are summarized below:
• the cell growth of the four strains were similar and the fermentations reached final wet cell weights of approximately 350-420 g/L. This level of cell density is typical for Pichia pastoris strains grown under the standard Mut + conditions.
• the similar amount of growth of the strains containing one and two copies of the ⁇ MF pre-pro-human lysozyme fusion gene expression cassette indicates that the presence of additional copies of the cassette does not adversely affect cell growth.
• the levels of human lysozyme secreted by these strains during one-liter fermentations were measured by both enzyme activity assay (Example 5.c) and RIA (Example 5.d) .
• the human lysozyme expression level data for the fermentations are included in the Table set forth above.
• Strain G+HLZ105S3 which contains one copy of the native signal sequence-human lysozyme qene expression cassette, secreted only 17 mg of human lysozyme/L after 94 hours of induction by methanol.
• Inocula were prepared from selective plates and grown overnight at 30°C in buffered YNB containing 2% glycerol to an OD ⁇ Q of 1-8. An aliquot of 5-50 ml of the overnight culture was added to a 2-liter capacity fermentor, and the repressed growth phase continued in 5X basal salts containing 5 ml/L of PTM. salts at 30 ° C .
• the pH was maintained at 5.5 by the addition of 50% (v/v) ammonium hydroxide, and foaming was controlled by the addition of 5% (v/v) Struktol antifoam. Dissolved oxygen was maintained above 20% by increased aeration and agitation as needed.
• This batch growth phase continued for 18-24 hours until the glycerol was exhausted.
• a 50% (w/v) glycerol feed (containing 12 ml/L of PTM, trace salts) was initiated at a rate of 5-20 ml/hour.
• a methanol feed (100% plus 12 ml/L of PTM 1 trace salts) was initiated at a rate of 1 ml/hour. The rate was increased over the course of the fermentation to maintain a residual methanol concentration of less than 0.5%.
• the vessel was harvested 70-150 hours following methanol induction.
• a 15-liter capacity fermentor was autoclaved with 6.5 liters of a solution containing 5.5 liters of 10X basal salts and 525 g of glycerol. After sterilization, the pH was adjusted to 5.0 with NH 3 gas, and 30 ml of PTM 1 trace salts were added. The fermentor was inoculated with 500 ml of an overnight culture grown in buffered YNB containing 2% glycerol. The pH was maintained at 5.0 by the addition of NH 3 gas, and the temperature was maintained at 30°C. A 5% solution of Struktol J-673 was added as necessary to control foaming. Dissolved oxygen was maintained above 20% saturation by increased agitation and aeration as needed.
• a glycerol feed (50% glycerol plus 12 ml/L of PTM 1 salts) was initiated at 50-200 ml/hour and continued until a volume of 700 ml had been added.
• a methanol feed (100% methanol plus 12 ml/L PTM 1 salts) was initiated at 7.5- 10 ml/hour. After two hours at this rate, the methanol feed was increased by 10% increments every 30 minutes to a final rate of 60 ml/hour which was maintained for the remainder of the fermentation.
• Strain G+AHZ108S20 containing two ⁇ MF pre- pro-human lysozyme fusion gene cassettes, was also grown in a 10-liter fermentation conducted using an increased methanol feed during the induction phase.
• the first two phases of Run 704 were conducted according to the standard conditions described above. Following the glycerol fed-batch phase, the methanol feed was initiated at 20 ml/hour. After 3 hours, the feed rate was increased by 20% increments every 15 minutes to a final rate of 60 ml/hour.
• the cell yield (final cell density of 459 g/L) was similar to the density achieved in the previous 10-liter fermentation of this strain (Run 695) ; and, although slightly less, the yield of human lysozyme (550 mg/L) was similar. Therefore, it appears that either protocol can be used for 10-liter fermentations of this strain.
• Run 667 G-HLZ103S5 (1 copy) Mut " strain G-HLZ103S5 was grown in a 10- liter fermentation according to a standard Mut " protocol. The initial two phases of Run 667 were conducted as described for the standard Mut * 10-liter fermentation protocol. In the induction phase, the methanol feed was introduced at 7.5-10 ml/hour and increased after two hours by 10% increments every 30 minutes until a final rate of 30 ml/hour was achieved. At this rate of methanol addition, the residual methanol level in the fermentor was maintained between 0.1 and 0.5%.
• strain G-HLZ103S5 grew to a final cell density of 428 g wet weight/L, which is typical of other Mut " recombinant strains grown in 10-liter fermentations.
• the level of human lysozyme produced in Run 667 was approximately 20 mg/L.
• the purification scheme consisted of four basic steps. First, fermentor broth was filtered through a 100,000 molecular-weight cut-off spiral cartridge. Second, the filtrate, which contained the human lysozyme, was loaded directly onto a radial flow sulphopropyl cartridge. In the third step, the cartridge was washed sequentially with a series of buffers with increasing pH and varying conductivity to remove endogenous Pichia pastoris proteins. Finally, the human lysozyme was eluted from the cartridge with a buffer of high conductivity, dialyzed, and lyophilized.
• the filtrate from the spiral ultrafiltration cartridge was pumped directly onto the sulphopropyl cartridge at 5 ml/minute.
• the cartridge was then washed with a series of six buffers of increasing pH and conductivity as follows: 1. 1 liter of 50 mM sodium acetate, pH 5.1, containing sufficient NaCl to bring the conductivity to 24.7 mMho/cm 2 ,
• the human lysozyme was eluted from the sulphopropyl cartridge with one liter of 50 mM sodium phosphate, pH 8.0, containing sufficient NaCl to bring the conductivity to approximately 100 mMho/cm 2 . Fractions of approximately 30 ml were collected, and the absorbance of the fractions was measured at 280 nM. The peak fractions containing material with absorbance at 280 nM were pooled and dialyzed in Spectrapor tubing with a molecular-weight cut-off of 6000-8000 against two liters of MilliQ-purified water. The dialysis was changed every four hours for two days. The sample was then frozen and lyophilized.
• Two sulphopropyl cartridges were used in series to accommodate the increased level of human lysozyme in the fermentation broth. Secondly, elution of the human lysozyme from the sulphopropyl cartridges was achieved with a gradient system developed to separate the major forms of the protein produced by the new strains. In addition, other modifications were introduced to simplify the overall purification procedure. ' Filtration of the cell-free broth through the 100,000 molecular-weight cut-off spiral cartridge was eliminated, and the number of washes of the ZetaPrep cartridges was reduced.
• This modified scheme was used to purify the human lysozyme produced in ten-liter fermentation Run 704 of strain G+AHZ108S20.
• the contents of the fermentor were harvested after 70 hours on methanol and centrifuged at 6500 X g for 30 minutes.
• the cell-free broth (6100 ml) was pumped at 5 ml/minute directly ontc the two ZetaPrep Modular radial flow sulphopropyl cartridges connected in series.
• the cartridges were washed with 0.8 liters of 50 mM sodium acetate, pH 5.0, containing sufficient NaCl to bring the conductivity to 20 mMho/cm 2 , followed by 1.4 liters of 50 mM sodium phosphate, pH 7.0, with sufficient NaCl to bring the conductivity to 13.8 mMho/cm 2 .
• the human lysozyme was eluted from the cartridges with a four-liter linear gradient of 50 mM sodium phosphate, pH 8.0, with increasing conductivity from 10 to 50 mMho/cm 2 . Fractions of approximately 10 ml were collected and the absorbance of the fractions was measured at 280 nM. Aliquots of fractions containing material with absorbance at 280 nM were analyzed by tricine SDS-PAGE to monitor the elution of the two forms of lysozyme produced by strain
• This pool contained approximately 1 gram of human lysozyme which appeared to be correctly processed and at least 95% pure according to analysis by SDS-PAGE, immunoblot, amino acid analysis, and/or protein sequencing (see Example 5.e-h) .
• the cell-free broth contained approximately 2. ⁇ g of human lysozyme immunoreactive material, as determined by RIA, of which approximately 1.9 g (66%) was estimated to be correctly processed. As shown in Table 3, approximately 1.7 grams (or 61%) of the 2.8 grams of immunoreactive starting material was isolated. However, the 2.8 grams of starting material contained both correctly and incorrectly processed human lysozyme, approximately 1.9 and 0.9 grams, respectively. Thus, the approximately 60% recovery noted in Table 3 represents a recovery of approximately 89% of the correctly processed human lysozyme.
• the amounts of human lysozyme secreted from recombinant strains of Pichia pastoris were quantitated by RIA and lysis assays of cell-free broth obtained during growth of the strains in fermentors.
• Lysozyme from two sources was used as standard in RIA, immunoblot, and enzyme activity assays. Lysozyme from the urine of human leukemia patients was purchased from Green Cross Corporation (distributed by Alpha Therapeutic Corporation, Los Angeles, CA) , and lysozyme isolated from human milk was obtained from Sigma Chemical Company (St. Louis, MO) .
• Polyclonal rabbit antisera to human lysozyme purchased from Dako-Immunoglobulins (Santa Barbara, CA) , were used for the RIA and immunoblot analyses.
• Samples from fermentor cultures of human lysozyme-expressing strains of Pichia pastoris were centrifuged at 6500 X g for 5 minutes to separate the broth and the cells. The broth was decanted from the cell pellet and used for immunoblot, RIA, and enzyme activity assays.
• Cell extracts were prepared from 175 mg of cells grown in fermentors. The cells were washed twice in extraction buffer (10 mM sodium phosphate, pH 7.5, 0.1% Triton X-100, 0.5 M NaCl, 2 mM PMSF) , and then vortexed four times for 1 minute each with 0.5 g of 0.5 mm glass beads and 0.35 ml of extraction buffer.
• the * beads were washed with an additional 0.35 ml of extraction buffer which was combined with the lysate and centrifuged in a microfuge for 15 minutes. The supernatant was removed, and the total protein was determined by the Bradford method [Anal. Biochem. 2-2:248 (1976)].
• the remaining cell pellets were suspended in 0.7 ml of 2X sample buffer (0.125 M Tris HC1, pH 6.8, 4% SDS, 200 mM dithiothreitol (DTT) , 20% glycerol, 0.005% bromophenol blue, 20 ⁇ g/ml pyronin Y) , boiled for 10 minutes, and centrifuged in a microfuge for 15 minutes. Following centrifugation, the supernatant was removed and retained. The cell lysates and supernatants resulting from centrifugation of resuspended insoluble pellets were analyzed by immunoblot.
• lysis activity of the human lysozyme produced in Pichia pastoris was measured, with modifications, as described [Shugar et al. Biochem. Biophvs. Acta 8.:302 (1952)]. Lysozyme was added to a suspension of Micrococcus Ivsodeikticus, and the decrease in absorbance at 405 nm was measured spectrophotometrically.
• the standard conditions used for the assay were pH 7.4 in 0.033M potassium phosphate buffer containing 0.1% BSA at 25°C.
• the specific activity of the standard human lysozyme is defined as the change in OD 405 per minute per mg of lysozyme.
• the average specific activity of both the milk and urine lysozymes was found to be " 1100 OD 405 /minute/mg in the enzyme activity assay under the standard conditions.
• the concentration of human lysozyme in the fermentation broth was calculated from the specific activity obtained for the standard in the same assay.
• a radioimmunoassay was developed for the evaluation of the expression of human lysozyme.
• Iodinated human lysozyme required for the RIA was not commercially available, therefore, lysozyme isolated from the urine of human leukemia patients was iodinated by an Iodobead procedure.
• 50 ⁇ g of lysozyme was combined with one Iodobead (Pierce, Rockford, IL) and 1 Ci of [ 25 I]NaI (NEN, Boston, MA) in 50 mM NaP0 4 , pH 7.4. After incubation for 30 minutes on ice, the 125 I-lysozyme was separated from free iodine on a
• the ED 50 for the competition of the 25 I-lysozyme by unlabeled lysozyme was 2.5 ng, and the sensitivity of the assay was approximately 0.8 ng.
• the standard curves generated in the RIA of the human lysozymes purified from milk and urine are superimposable.
• the membrane was then washed extensively (four 15-minute washes) with Western buffer, incubated with approximately 3 ⁇ Ci 125 I-Protein A (New England Nuclear, Boston, MA) at room temperature for 60 minutes, washed extensively as before, air dried, and exposed to film. Under these conditions, as little as 1 ng of human lysozyme can be detected.
• Purified human lysozyme was subjected to amino acid analysis following hydrolysis [Spackman et al. (1958). Anal. Biochem. 3):1190.] A known amount of purified human lysozyme was added to a glass tube, and the tube was placed in a reaction flask containing approximately 0.5 ml of 6N HC1. After evacuation, the sample was hydrolyzed by vapor phase hydrolysis at 110°C for 24 hours. The hydrolyzed sample was then taken up in 500 ⁇ l of 0.2 M sodium acetate, pH 2.2, and 50 ⁇ l were applied to a Beckman 6300 Amino Acid Analyzer. h. Protein seguencing
• N-terminal amino acid sequence of samples of purified Pichia pastoris-produced human lysozyme was determined according to the method of Hunkapiller and Hood [Science 219:650 (1983)].
• An Applied Biosystems 470/120 Gas Phase Protein Sequencer was utilized in the analysis of " 1 nmol of purified material.
• the plasmid was subsequently digested with Hindlll and the 350 bp fragment isolated from a 10% acrylamide gel and subcloned into pUCl ⁇ (Boehringer Mannheim) digested with Hindlll and Sail.
• the ligation mix was transformed into JM103 cells (that are widely available) and amp R colonies were selected.
• the correct construction was verified by Hindlll and Sail digestion, which yielded a 350 bp fragment, and was called pA0201. 5 ⁇ g of pA0201 was digested with Hindlll, filled in using E. coli DNA Polymerase I Klenow fragment, and 0.1 ⁇ g of Bglll linkers (GAGATCTC) were added. After digestion of the excess Bglll linkers, the plasmid was reclosed and transformed into MC1061 cells. Amp R cells were selected, DNA was prepared, and the correct plasmid was verified by Bglll, Sail double digests, yielding a 350 bp fragment, and by a Hindlll digest to show loss of Hindlll site.
• This plasmid was called pAO202.
• An alpha factor-GRF fusion was isolated as a 360 bp BamHI-PstI partial digest from pYSV201.
• Plasmid pYSV201 is the EcoRI-BamHI fragment of GRF-E-3 inserted into M13mpl8 (New England Biolabs) .
• Plasmid GRF-E-3 is described in European Patent Application No. 206,783. 20 ⁇ g of pYSV201 plasmid was digested with BamHI and partially digested with Pstl. To this partial digest was added the following oligonucleotides:
• the resulting plasmid, pA0203 was cut with EcoRI and Bglll to yield a fragment of greater than 700 bp.
• the ⁇ -factor-GRF fragment codes for the (1- 40) leu 27 version of GRF and contains the processing sites lys-arg-glu-ala-glu-ala.
• the AOXl promoter was isolated as a 1900 bp EcoRI fragment from 20 ⁇ g of pAOP3 and subcloned into EcoRI-digested pA0203.
• the development of pA0P3 is disclosed in European Patent Application No. 226,646 and described hereinbelow.
• MC1061 cells were transformed with the ligation reaction, amp R colonies were selected, and DNA was prepared.
• the correct orientation contains a «376 bp Hindlll fragment, whereas the wrong orientation has an «675 bp fragment.
• One such transformant was isolated and was called pA0204.
• the parent vector for pA020 ⁇ is the HIS4, PARS2 plasmid pYJ32 (NRRL B-15891) which was modified to change the EcoRV site in the tet R gene to a Bglll site, by digesting PYJ32 with EcoRV and adding Bglll linkers to create pYJ32(+BglII) .
• This plasmid was digested with Bglll and the 1.75 Kb Bglll fragment from pA0204 containing the AOXl promoter- ⁇ mating factor- GRF-AOX1 3' expression cassette was inserted.
• the resulting vector was called pA0208.
• An EcoRI digest of pAO20 ⁇ yielded an 650 bp fragment + vector, while vector having the other orientation yielded a 1.1 Kb fragment + vector.
• Plasmid pPG2.5 [a pBR322 based plasmid containing the approximately 2.5 Kbp EcoRI-Sall fragment from plasmid pPG4.0, which plasmid contains the primary- alcohol oxidase gene (AOXl) and regulatory regions and which is available in an E. coli host from the Northern Regional Research Center of the United States Department of Agriculture in Peoria, Illinois as NRRL B-15 ⁇ 6 ⁇ ] was linearized with BamHI.
• AOXl primary- alcohol oxidase gene
• This oligonucleotide contains the AOXl promoter sequence up to, but not including, the ATG initiation codon, fused to the sequence of the EcoRI linker; 6. Positive clones were sequenced by the
• Plasmid pA0815 was constructed by mutagenizing plasmid pA0807 (which was in turn prepared as described hereinbelow) to change the HindiII/ClaI/ Hindlll sites downstream of the AOXl transcription terminator in pA0807 to a BamHI site.
• the oligonucleotide used for mutagenizing pA0807 had the following sequence:
• the mutagenized plasmid was called pA0 ⁇ 07-Bam.
• Plasmid pA0 ⁇ 04 was digested with Bglll and 25 ng of the 2400 bp fragment were ligated to 250 ng of the 5400 bp Bglll fragment from Bglll-digested pA0 ⁇ 07-Bam.
• the ligation mix was transformed into MC1061 cells and the correct construct was verified by digestion with Pst/BamHI to identify 6100 and 2100 bp sized bands.
• the correct construct was called pA0815.
• the restriction map of the expression vector pA0815 is shown in Figure 3.
• fl-ori DNA fl bacteriophage DNA (50 ⁇ g) was digested with 50 units of Rsa I and Dra I (according to manufacturer's directions) to release the «458 bp DNA fragment containing the fl origin of replication (ori) .
• the digestion mixture was extracted with an equal volume of phenol: chloroform (V/V) followed by extracting the aqueous layer with an equal volume of chloroform and finally the DNA in the aqueous phase was precipitated by adjusting the NaCl concentration to 0.2M and adding 2.5 volumes of absolute ethanol.
• the mixture was allowed to stand on ice (4°C) for 10 minutes and the DNA precipitate was collected by centrifugation for 30 minutes at 10,000 x g in a microfuge at 4°C.
• the DNA pellet was washed 2 times with 70% aqueous ethanol. The washed pellet was vacuum dried and dissolved in 25 ⁇ l of TE buffer [1.0 mM EDTA in 0.01 M (pH 7.4) Tris buffer]. This DNA was electrophoresed on 1.5% agarose gel and the gel portion containing the «458 bp fl-ori fragment was excised out and the DNA in the gel was electroeluted onto DE ⁇ l (Whatman) paper and eluted from the paper in 1M NaCl.
• the DNA solution was precipitated as detailed above and the DNA precipitate was dissolved in 25 ⁇ l of TE buffer (fl-ori fragment) .
• Cloning of fl-ori into Dra I sites of pBR322 pBR322 (2 ⁇ g) was partially digested with 2 units Dra I (according to manufacturer's instructions). The reaction was terminated by phenol:chloroform extraction followed by precipitation of DNA as detailed in step 1 above.
• the DNA pellet was dissolved in 20 ⁇ l of TE buffer. About 100 ng of this DNA was ligated with 100 ng of fl-ori fragment (step 1) in 20 ⁇ l of ligation buffer by incubating at 14'C for overnight with 1 unit of T4 DNA ligase.
• the ligation was terminated by heating to 70°C for 10 minutes and then used to transform E. coli strain JM103 [Janisch-Perron et al., Gene, vol 22, 103(1983)].
• Amp R transformants were pooled and superinfected with helper- phage R408 [Russel et al. , supra] . Single stranded phage were isolated from the media and used to reinfect JM103.
• Amp R transformants contained pBRfl-ori which contains fl-ori cloned into the Dra I sites (nucleotide positions 3232 and 3251) of pBR322.
• plasmid pA0807 pBRfl-ori (10 ⁇ g) was digested for 4 hours at 37°C with 10 units each of Pst I and Nde I.
• the digested DNA was phenol:chloroform extracted, precipitated and dissolved in 25 ⁇ l of TE buffer as detailed in step 1 above.
• This material was electrophoresed on a 1.2% agarose gel and the Nde I - Pst I fragment (approximately 0.8 kb) containing the fl-ori was isolated and dissolved in 20 ⁇ l of TE buffer as detailed in step 1 above.
• Plasmid pA0 ⁇ 04 has been described in PCT Application No. WO 69/04320. Construction of this plasmid involved the following steps:
• 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 tc 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 replaced with the sequence:
• PBR322 ⁇ RI was digested with PvuII, and the linker having the sequence:
• Plasmid pBR322 ⁇ RIBGL is the same as PBR322 ⁇ RI except that pBR322 ⁇ RIBGL has the sequence
• PBR322 ⁇ RIBGL was digested with a 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 650 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.
• the Bglll-Xhol fragment from pBSAGI5I and the approximately 2.97 kbp, Sall-Bglll fragment from pBR322 ⁇ RIBGL were combined and subjected to ligation with T4 ligase.
• the ligation mixture was used to transform E. coli MC1061 to ampicillin-resistance and transformants were screened for a plasmid of the expected size (approximately 3.8 kbp) with a Bglll site. This plasmid was designated pAO ⁇ Ol.
• the overhanging end of the Sail site from the PBR322 ⁇ RIBGL fragment was ligated to the overhanging end of the Xhol site on the 850 bp pBSAGI5I fragment and, in the process, both the Sail site and the Xhol site in pA0801 were eliminated.
• pBSAGI5I 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 encoding 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
• Plasmid pAO ⁇ Ol was cut with Clal and combined for ligation using T4 ligase with the approximately 2.C 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 approximately 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.
• pA0802 One of the transformants harboring the desired plasmid, designated pA0802, was selected for further work and was cultured to yield that plasmid.
• the desired orientation of the Clal fragment from pBSAGISI in pA0802 had 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.
• pA0 ⁇ 02 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:
• Plasmid pA0804 was then made from pA0803 by inserting, into the BamHI site from pBR322 in pA0803, an approximately 2.75 kbp Bglll fragment from the P. pastoris HIS4 gene. See, e.g., Cregg et al. , Mol. Cell. Biol. 5_, 3376 (1985) and European Patent Application Publication Nos 180, ⁇ 99 and 18 ⁇ ,677.
• pA0 ⁇ 03 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 pA0804.
• pA0 ⁇ 04 has one Sall-Clal fragment of about 1.5 kbp and another of abut 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 pA0 ⁇ 04 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 pA0804 would also be effective for use in accordance with the present invention.

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• 1991
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