WO1991000357A1 - New strain with filamentous fungi mutants, process for the production of recombinant proteins using said strain, and strains and proteins produced by said process - Google Patents

New strain with filamentous fungi mutants, process for the production of recombinant proteins using said strain, and strains and proteins produced by said process Download PDF

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Publication number
WO1991000357A1
WO1991000357A1 PCT/FR1990/000479 FR9000479W WO9100357A1 WO 1991000357 A1 WO1991000357 A1 WO 1991000357A1 FR 9000479 W FR9000479 W FR 9000479W WO 9100357 A1 WO9100357 A1 WO 9100357A1
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strain
protein
characterized
method according
sequence
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PCT/FR1990/000479
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French (fr)
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Thierry Calmels
Henri Durand
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Cayla
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    • C12R1/645Processes using microorganisms using fungi
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    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
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    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/63Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/63Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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    • C12N15/09Recombinant DNA-technology
    • C12N15/63Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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    • C12N9/14Hydrolases (3)
    • C12N9/24Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405Glucanases
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    • C12N9/24Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
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    • C12Y302/01Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01041Pullulanase (3.2.1.41)
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    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2319/00Fusion polypeptide
    • C07K2319/01Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02Fusion polypeptide containing a localisation/targetting motif containing a signal sequence

Abstract

The invention relates to a process for the production of recombinant protein using fibrous fungi, characterised by the fact that a strain of the genus Tolypocladium, into which a DNA sequence coding for said protein which has been placed under the control of elements guaranteeing the expression of said sequence in said strain, has been introduced, is cultivated on an appropriate medium and by the fact that said protein is recovered. The invention also relates to the proteins and strains obtained by putting said process into effect.

Description

MUTANT STRAIN AND MUSHROOMS FILAMEN¬ expensive, METHOD FOR PRODUCING RECOMBINANT PROTEIN USING THE SAME STRAIN AND STRAIN AND PROTEIN OBTE¬ NAKED FROM THE METHOD.

The present invention relates to a velle nou¬ strain of filamentous fungi, as well as derivatives of this strain obtained by mutation or by genetic manipulation, and a method for the production of recombinant protein, using said strains.

cules molé¬ the polypeptide synthesized by a microorganism or a cell population with recombinant proteins is meant by more than 1 * introduction into the genetic material of said microorganism or des- said cells a gene coding for EDCs considé¬ protein. The gene in question, called heterologous gene may come from another living organism or be artificially synthesized. The ability to introduce and to express heterologous genes was first highlighted and applied using bacteria, in particular Escherichia coli, as host cells. More recently, re¬ combinantes protein production by other microorganisms including yeast and fungi as well as by higher organisms cell cultures have been described.

The filamentous fungi are used cou.- monly in the fermentation industry in particular for the manufacture of certain antibiotics (penicillins, cephalosporins) and a large number of enzymes (gluco-amylases, cellulases, proteases, pectinases -).

Filamentous fungi have a cer¬ tain number of advantages over prokaryotic microorganisms (bacteria) in particular créter ex¬ capacity large amounts and a wide variety of proteins with post tradition¬ tional modifications, such as glycosylations, specific eukaryotes. Compared with eukaryotic cells supé¬ laughing, mushrooms are much easier to grow large-scale separation of mycelium and culture medium after fermentation is also very easy. The present invention relates to a recombinant protein production process using cham¬ filamentous sprockets, characterized in that cultivated on a suitable medium a strain of the genus Tolypocladium wherein has been introduced a DNA sequence encoding said protein placed under the control of elements ensuring the expression of said sequence in the said strain and in that said recovered protein.

Specifically, the strain used is a strain Tolypocladium geodes. This strain has been deposited under the No. 1-880 29 June 1989 to the final Natio¬ Culture Collection of Microorganisms - 28, rue du Docteur Roux - 75015 PARIS.

The invention also relates to mutants défi¬ cients in protease activity and / or hyperexcréteur exogenous protein from this strain.

The present invention is based in particular on the solement ï¬ a new filamentous fungal strain particularly adapted to the production of recombinant proteins. The wild strain NC14 was isolated from a soil sample rich in decaying organic matter (humus undergrowth of Graulhet region, Tarn, France). It was originally sélec¬ tioned for its high proteolytic activity, and proved to the analysis produce significant quantities of very au extracellular enzymes, chitinases and -gluca- nases in particular.

The NC14 strain was entrusted to laboratory identification of the National Museum of Natural History in Paris. It was transplanted in Petri dishes on two standard culture media used for détermina¬ tion hyphomycetes Malt-agar 2% and Potato Dextrose Agar (PDA) and incubated at 25 ° C.

Culture on malt grows slowly, only 1 cm in 3 weeks in culture and form a tight colony wet appearance. The microscopic observation mon¬ be a heterogeneous mycelium to vacuolated cytoplasm. Conidia are very rare and spore cells few and scattered. The PDA medium is more favorable. The culture has a slow growth, attaining 13-20 mm in 10 days. The frond is white, flaky surface, dense deep enough, irregular contour. We observe a lé¬ manages beige pigmentation on the back, no pigment dif- fuse in the middle. No characteristic odor.

Microscopic observation shows spore-forming cells (phialides) integral or grouped by two or three on a short side cell of the mycelium. Phialides measure of 9 to 14 .mu.m long, are surely légè- swollen at the base and then elongated to the neck which is sometimes bent. Spores (conidia) are grouped in heads; very heterogeneous shape (spherical, cylindrical, oval) with ratios generally between 3.6 to 0.9 microns long and 2.9 to 0.9 microns wide with two main classes: 3 × 1.5 (cylindrical) 1.5 x 1.8 (globular). The mycelium measure between 1.5 and 2 microns wide. No chlamydospores.

The cultural aspect, the speed of development and morphology of the spore-forming device are that this species belongs to the genus Tolypocladium (Gams) the shape, size and grouping phialides much swollen and grouped in whorls at inflatum. It also differs from Tc lindrosporum (Gams) the shape phialides but especially by the form of regular cylindrical spores in T.cylindrosporum. The NC14 strain is finally very close to the species geodes (Gams) by the method of grouping phialides described above, by their shape as well as by cultural appearance. Despite the heterogeneity of spores, very heterogeneous size and shape, 40% thereof corres¬ lay characteristics of Tolypocladium geodes (Gams), that is to say 1.6 to 2.2 .mu.m.

So it is a strain of Tolypocladium geodes whose morphology of the spores is somewhat atypical.

The NC14 strain Tolypocladium geodes was the subject of a breeding program, as consis¬ to select repeatedly mutants growing strongly proteolytic enzyme producers ticks, then chitinolytic enzymes. The méthodolo¬ mutagenesis and selection gies used are those previously described for Trichoderma reesei (Durand et al .. Enzyme Microb Technol, 1988, 10.. 341). And Penicillium Occitanis (Jain et al, submitted for publication in Enzyme Microbiol. Technology). The first selection step was carried out from a spore suspension of the strain NC14 treated with a mutagenic agent, ultraviolet light. Dilution of said suspen¬ sion of spores was used to inoculate Petri dishes containing; agar medium having the following composition:

- skimmed milk powder 10 g

- 2 g Monopotassium phosphate

- Magnesium sulfate 0.3 g

- Calcium chloride 0.2 g - Iron sulfate 0.005 g - yeast extract: 1 g

- agar: 15

- deionized water qs to 1 liter pH adjusted to 7.0 with sodium hydroxide After 5 days incubation at 27 ° C, colonies appeared on this medium are surrounded by a clear halo, due to the hydrolysis of milk proteins by proteolytic enzymes excreted by the strain. This sieve sé¬ election, which had already been used for the isolation of the NC14 strain, allowed the research mutant proteases best producers; thereof are detected by the ap¬ pearance of a hydrolysis halo of a diameter substantially greater than the average of those observed around ned colo¬ of the starting strain. One mutant, called NC18, charac- terized by a hydrolysis halo very significantly increased, was isolated. From the strain NC18, a second selection step was performed, after muta- genesis by N-methyl-N'-nitro-N-nitrosoguanidine (NTG) to the medium mentioned above to which was added the casein acid hydrolyzate (Biokar) to the concentra¬ tion of 5 g / 1. Under these conditions, pro¬ teases production by the NC18 strain is suppressed, which tra¬ Duit by a sharp decrease in the size of the hydrolysis los ha¬. A colony surrounded by a broad halo was selected; the thus isolated mutant NC21 has been a new selection step after mutagen ganese by 1 'thyl-methane sulfonate (EMS) on the medium based on powdered milk, to which ammonium sulfate the concentration of 5 g / 1 was added as a repressor of protease production. A de-repressed mutant NC28, has been isolated. From this latter strain, a mutant hyperproducer tick chitinoly- activity has been sought after mutagenesis by ni- trous acid, the basal medium described above, where the milk powder was replaced with chitin dale colloids at a concentration of 10 g / 1. A mutant hyperchiti- nolytique, NC35, selected based on the size of the halo of hydrolysis of chitin, was subjected to a selection step velle nou¬ on the same medium based on chitin, additionally containing 10 g / 1 glucose as ré¬ presser. An isolated mutant strain on this medium, NC39 was chosen. This strain has proved hyper- urinary protein: concentrations of about 10 g / 1 protein were obtained in 6 days of fermentation in a chemically defined medium (basic salts medium according Mandels and Weber, J .... Adv Chem Ser, 1969, 95: 391) containing glucose (added continuously at a rate of 20 g / 1 per day) as sole source of car¬ bone. Under the same conditions, the wild-type strain NC14 produced about 0.5 g / 1 of extracellular proteins.

This exceptional ability of the NC39 strain to excrete proteins in extremely simple conditions and a culture medium, which is one of as¬ pects of the present invention, led to consider using it for the production of heterologous proteins. However, in this context, the strong lytic activity proteolytic strains is a disadvantage. Also from the NC39 strain deficient mutants were sought pro¬ teases. After mutagenesis with NTG, and then the EMS, colonies do not produce a hydrolysis halo on based medium addition¬ milk powder born of glucose (2 g / 1) and ammonium sulfate (0, 5 g / 1) were selected in two stages. The NC46 strain ain¬ so isolated is characterized by the absence of detectable proteolytic lytic activity by the test azocasein (Tomarelli et al, J. Lab Clin Med, 1949, 34.... 428) in sur¬ swimming culture in the conditions of production of extracellular proteins mentioned above and dé¬ Crites in example II below. NC46 this strain was subjected to a new mutagenic treatment with nitrous acid and then deficient mutants amino peptidase activity were SEARCH ~ kets according to a modification of the technique described by Miller and MacKinnon (J.Bact., 1974, 120 : 355): after 4 days growth on the medium containing milk powder described above, the colonies are coated with a solution containing 0.5 mg / ml of L. Leucine β -naphtylamide and 2 mg / Fast Garnet GBC ml (Sigma). The presence of aminopeptidase activity is visualized by the appearance after a few minutes, a brown color around the colonies. The colonies producing no color are recovered and re¬ tested using the same protocol.

An isolated mutant by this technique, NC50, no longer presents the Leucine aminopeptidase activity detected in the supernatant of the cultures of strains and NC46 preceding generations.

The genealogy of the NC50 strain is summarized in Table 1.

The mutant strain NC50 has the same Capa- cited excretion quantita¬ tif view point of the protein, the hyperexcrétrice NC39 strain mentioned above. An additional advantage of the strain NC50 ré¬ side in the absence of detectable proteolytic activity in the culture supernatants, which avoids or at least minimize degradation pro¬ teins .produites, particularly proteins hetero- rologues in the fermentation medium. This is an important aspect of the present invention.

Another aspect is the identification of the ability of NC14 and derivative strains to be transformed according to a simplified protocol, original as compared to those mentioned. Table 1: Genealogy derived mutant strains of Tolypocladium geodes NC 4

Figure imgf000010_0001
previously used for other fungi such Aspergil- read nidulans (Ballance et al, Biochem BioPh s Res Commun 1983, 112:.... 284) or Trichoderma reesei (KNO THE et al .. European Patent No. EP 0244234 A2, 1987). The ori¬ nality of the Protocol lies in particular in that it does not involve the use of protoplasts, the formation and regeneration often a tedious step in the development of a transformation protocol. In the case of the NC14 strain and its derivatives, it has been found that the foreign DNA pou¬ Vait be introduced, with a high frequency, in spores treated under mild conditions with a lytic enzyme such that their resistance the osmotic pressure and germination capacity are not impaired. The development of the transformation protocol was carried out using a plasmid DNA carrying a dominant selectable marker: A gene conferring resistance to phleomycin (ARMAU et al, Patent.

French No. 2 569 723. This protocol is described in Ȭ xample I below.

According to the present invention, the elements assu¬ rant expression of said DNA sequence in the strain essentially comprise a promoter sequence of a fungal gene. Said DNA sequence encoding said protein preceded by a signal sequence assu¬ rant excretion of the protein followed by a transcription terminator sequence. A further aspect of the present inven¬ tion is constituted by the possibility to select for transformed strains in which expression of the gene of interest is amplified through a sieve sélec¬ tion based on the use of a gene component a dominant marker, placed downstream of the gene of interest, without a promoter or transcription termination signal between the two genes. This technique is illustrated in Example V below.

This gene constituting the marker encodes the re resistance to an antibiotic which is preferably a com¬ posed of the family phleomycins.

In the case of a gene encoding resistance to an antibiotic of the family of phleomycins, this gene is of genomic origin and originates from the genome of fungi actino- producer of said antibiotic. The DNA sequence encoding said protein as well as the elements ensuring the expression of said sequence are carried by a plasmid which is preferably an autonomously replicating plasmid comprising an effective autonomously replicating sequence in Tolypo¬ Cladium TARS

According to the invention, said plasmid ensures the in¬ tegration chromosomal DNA sequences involved. In summary, the present invention relates to a new strain of filamentous fungus Tolypocladium geodes NC14, and mutants derived from this strain, sélec¬ tioned for their ability to excrete large quanti¬ sides of proteins in an extremely simple culture medium, made up essentially of sugars and inorganic salts, which facilitates the extraction and purification of said protein. This unique capability can be exploited for the production of proteins recombi¬ Nantes which is a first aspect of the present invention. In a second aspect, the present invention provides a way to preserve the stability of proteins excreted in the culture medium by using mutants deficient in proteolytic activities.

According to a third aspect, the present invention provides a means to introduce heterologous genes into the strain Tolypocladium geodes NC14 and its derivatives se¬ lon a simplified protocol not involving protoplasts.

According to a fourth aspect of the present invention tion, the expression of heterologous genes by Tolypocla¬ dium geodes NC14 and its derivatives can be increased by a screening technique using a dominant marker placed downstream of the gene of interest without promoter or terminator between the two genes. The present invention also relates to proteins obtained by the implementation of the method se¬ lon the invention. These proteins encoded according to the in¬ vention, are more particularly pullulanase, human lysozyme and Sh protein, tidique nucleo Sh sequence of this protein is shown in Figure 2.

The present invention likewise relates to the transformed Tolypocladium sou¬ of tasks, obtained during the pro¬ sold production of recombinant proteins. The following examples serve to illustrate la present invention without limitation.

These examples will be described with reference to figures in which:

- Figure 1 is a diagram of plasmid pUT720 - Figure 2 shows the nucleotide sequence of the gene Sh protein preceded by the tick synthé¬ sequence used as excretion signal

- Figure 3 is a diagram of the plasmid pUT715

- Figure 4 is a diagram of plasmid pUT771 - Figure 5 shows the nucleotide sequence of the promoter of the plasmid pUT771

- Figure 6 is a diagram of the plasmid pUT760

- Figure 7 shows the nucleotide sequence syn¬ thetic encoding human lysozyme - Figure 8 is a diagram of the plasmid pUT772

Abbreviations used in these figures are considered known or will be explained in the description.

Unless otherwise indicated, the different ceded pror and products mentioned are implemented according to known techniques and / or recommended by the manufacturer, Example I: Transformation Tolypocladium geodes

The mutant strain NC50, deficient in proteolytic activities, was transformed by the plasmid pUT703 (Durand et al, Proc. Biochemistry and Genetics of Cellulose Degradation, Aubert JP Ed., Academic Press, 1988, p 136). This plasmid is characterized by the presence of a gene for resistance to antibiotics of the family of phleomycins (ARMAU et al, French Patent No. 2569723 under the control of a promoter that fongi¬. The gene promoter the glyce'raldéhyde 3-phos- phate dehydrogenase of Aspergillus nidulans (denoted promo¬ tor gpd) (Van GORCOM et al, Gene, 1986, 48. 211) and bounded downstream by the yeast CYC1 terminator (SMITH et al .., Cell, 1979, 16: 753) the resistance gene from a producer actinomycetes antibiotics of the family phleomycines; Streptoalloteichus hindus- tanus the protein encoded by this gene, called Sh protein inactivates the antibiotic. matter by formation of an equimolar complex (Gatignol et al, FEBS Letters, 1988, 230. 171).

Phleomycin and related antibiotics have high toxicity Tolypocladium geodes as for virtually all living organisms. Resistance gene thus provides an easy way to sé¬ lectionner clones transformed by their ability to grow in the presence of phleomycin.

The protocol used for transformation is as follows: spores obtained by culturing the NC50 strain on PDA medium for 7 days at 27 ° C are suspended in the following composition of MnP medium: saline base according Mandels supplemented with sucrose 100 g / 1 MES buffer 5 g / 1, pH 5.5. After filtration on sintered glass No. 2 to remove the mycelium, the suspension is centrifuged (10 minutes, 10,000 rpm) and the pellet is re¬ taken by the same medium supplemented with MnP Caylase C3 (lytic enzyme marketed by CAYLA) 10 mg / ml. After 4 hours of incubation on table stirred at 32 ° C, 100 rpm suspension is again centrifuged and the pellet is taken up in MnP medium supplemented with calcium chloride, 50 mM, in a volume such that the title of the spore suspension obtained, determined by counting mid-

Q croscopique to 1'hématimètre, is about 10 / ml. 200 .mu.l of this suspension, a solution of plasmid DNA (5 to 10 micrograms of DNA in a volume of 5 to 20 .mu.l) was added. After 10 minutes at temperature am¬ ature was added 50 .mu.l of MPC solution (10 mM MOPS pH 5.8, 60% PEG 6000 pv, CaCl 2 75 mM) and the mixture was incubated 30 minutes in ice and then added 2.5 ml of MPC. After 15 minutes at room temperature, aliquots of 0.5 ml of this mixture are introduced into tubes each containing 3 ml of MnR Soft Agar (saline medium according Mandels supplemented with sucrose 150 g / 1, glucose 2.5 g / 1 , yeast extract 2.5 g / 1 agar 6 g / 1) kept molten at 45 ° C. The content of cha¬ tube that is then poured on the surface of a petri dish containing the same medium MnR, agar 15 g / 1 and supplemented with 30 g / ml of phleomycin (marketed by CAYLA) as selection agent of clones transformed mants. They appear as small colonies after 4 days of incubation at 27 ° C.

The conversion rate obtained using the plasmid pUT703 were of the order of 2000 to 5000 transformants per microgram of DNA. The stability of the transformants was tested by transplanting the colonies on PDA medium containing 30 ug / ml phleomycin (the minimal inhibitory concentration - MIC - for the untransformed strain NC50 is about 10 ug / ml under these conditions). Approximately 90% of transplanted colonies confirmed their strength of character.

DNA hybridization experiments of sou¬ tasks transformed with a labeled probe corresponding to a portion of the phleomycin resistance gene showed that this gene was integrated into the chromosome of the transformants; or the integration sites and the number of copies varies from strain to strain. Example II: Expression and excretion of a heterologous protein. In Example I above, obtaining transformants resistant clones phleomycin indicates that the gene encoding the protein Sh is expressed in the MC50 strain Tolypocladium geodes. However, teine ​​pro¬ could not be demonstrated in surna¬ culture giants transformed strains and is therefore not likely excreted detectable amounts. In the following example, the NC50 strain was transformed using the protocol described in Example I, using the plasmid pUT720 which the card is re¬ presented in Figure 1. This plasmid is characterized by the presence in response the gpd promoter, the gene of the protein Sh upstream of which was added a synthetic signal sequence for excretion (Figure 2).

Among the resistant colonies obtained with a frequency of 3000 transformants per microgram of DNA, a hundred were picked on PDA plates containing increasing concentrations of phléo¬ mycin, 25 to 1000 mcg / ml. 3 strains resistant to more than 1000 mcg / ml of phleomycin were thus sélec¬ tioned; one of them was cultivated in laboratory fermenter under the following conditions: the spores from a PDA box incubated 7 days at 27 ° C were used to inoculate 500 ml of Mandels medium according conte¬ ing 20 g / 1 glucose and 1 g / 1 yeast extract, ré¬ party in two flasks of 1 1. After incubation for 48 hours over table stirred at 27 ° C, 200 rpm, this cul¬ ture allowed to inoculate a fermenter 14 liters of total volume (Chemap) containing 8 liters of the same medium. The fermentation lasted 6 days at 27 ° C with stirring at 500 rpm and an aeration of 0.5 vvm, pH being regulated above 5.0 with ammonia. From the second day, a sterile solution of glucose at 200 g / 1 was continuously fed at 0.8 liter per 24 hours, or about 20 grams of glucose per liter per day. After 6 days, the culture was harvested and centrifuged.

The total protein concentration, détermi¬ born by the method of RY LO (LO RY et al, J. Biol Chem, 1951, 193... 265) was found to be 9.6 g / 1. The presence of Sh protein in the culture medium was detected by chromatography

(FPLC) on MonoQ anion exchange column, Pharmacia (buffer Tris-HCl 20 mM pH 7.0 - liné¬ gradient elution area 0 - 0.6 M NaCl). In comparison with the supernatant of a culture carried out under the same conditions with the untransformed strain, there is the appearance of an additional protein peak strongly retained on the MonoQ column, eluted at 0.45 M. The area of this peak repré¬ feel about 20% of the total area of ​​the chromatogram. Analysis by SDS-PAGE, the corresponding sponding to this peak fractions revealed the presence prati¬ cally pure state of a protein of about 14 kd molecular weight 000, corresponding to the size of Sh protein (Gatignol et al, FEBS Letters, 1988, 230. 171). The presence in the protein of culture supernatant inactivating phleomycin has been confirmed by the following test: on paper disks impregnated with phleomycin solution at different concentrations (from 10 to 300 mcg / ml) different volumes of supernatant cul¬ ture (1 to 50 .mu.l) are deposited. The discs are subsequently en- layered on the surface of an agar medium (Medium No. 2 for the test antibiotics, BioMérieux) inoculated with a strain of Escherichia coli sensitive to cine phléomy¬: HB101. After overnight incubation at 37 ° C, the inhibition halos of the bacterial veil are visible au round discs containing only phleomycin. When known amounts of purified protein Sh are added, there is a reduction in the diameter of the haloes, and a total disappearance thereof in a molar ratio protein / antibiotics 1/1 or approximately 10/1 by weight. This test thus allows a semi-quantitative assay .the Sh protein concentration in an unknown solution. In the present example, Sh protein con¬ centration in the culture supernatant was found close to 2 g / 1. It was thus possible by fermentation of a transformant strain of Tolypocladium geodes NC50 by pUT720 plasmid to produce about 2 g / 1 of recombinant extracellular teine ​​pro¬ in 6 days. Example III i Isolation of a promoter sequence The plasmid pUT715, whose map is presented in Figure 3 was used as probe-vector in the search for fungal promoter sequences. The plas¬ mide was linearized with BamHI dual-AsuII cleavage (the two unique restriction sites are located in the multi-site sequence located immediately upstream of the coding part of the gene Sh). After purification by elec- troélution, the large fragment was brought into contact with chromosomal DNA of Trichoderma reesei partially digested by TaqI and MboI enzymes to generate fragments between 1 and 5 kilobases and DNA ligase. The population of hybrid plasmids thus obtained ser¬ vi to transform Escherichia coli strain DH5 ov, with selection based on resistance to ampicillin. Plasmid DNA extracted from the "pool" of mants transformed E. coli was used to transform the strain NC14

T.géodes according to the protocol described in Example I above. 50 transformants strongly resistant phléo¬ mycin (MIC> 250 micrograms / ml) were selected and divided into 10 groups of 5 clones. The DNA from each group was extracted, digested with the BstEII enzyme and subjected to ligation. With each preparation transformation of E.coli DH5 CRT was performed.

Plasmid DNA from 6 colonies of E. coli résul¬ as each transformation or 60 total transformants was analyzed separately by electrophoresis in agarose after gel microextraction. 7 plasmids of various sizes and greater than that of the pUT715 vector were chosen. Each of these plasmids extracted from the corresponding E. coli strain was used to transform sea T.géodes NC14. One of transformations led to the emergence of highly resistant clones to phléo¬ mycin (MIC> 250 micrograms / ml) with a frequency of approximately 5000 transformants per microgram of DNA. In two other changes, the frequency and level of re- transforming the resistance found much lower (some transformants / ug, MIC <100 micrograms / ml). Finally four conversions have yielded no result.

The plasmid that allowed the transformation of the most effective T.géodes called pUT771 was analyzed, it contains an insertion of a size of about 1.8 kb; mapping pUT771 is shown in Figure 4. The nucleotide sequence of the promoter portion is shown in Figure 5.

In comparative experiments, the plasmid pUT771 has always led to higher rates of processing approximately a factor of two to those obtained in the same conditions with pUT703.

Example IV: T.géodes Co-transformation with a mide plas¬ carrying the resistance marker for phleomycin and a plasmid carrying a non-selectable gene. A transformation experiment was carried out according to the protocol described in Example I above, with a mixture containing about 1 .mu.g of plasmid DNA pUT771 described in Example II and 10 .mu.g of plasmid DNA pUT760 , whose map is shown in Figure 6. the plasmid pUT760 is characterized by the presence of Klebsiella pneumoniae ATCC 15050 gene coding for the mature pullulanase (Michaelis et al, J. Bact, 1985, 164:.. 633), under dependence of the Aspergillus nidulans gpd engine pro¬ and preceded by the synthetic signal accordingly sé¬ described in example I (fi¬ gure 2).

Approximately 500 transformants selected on the basis of resistance to phleomycin were subcultured individually on the one hand on a PDA medium containing 30 .mu.g / ml of phleomycin and secondly on an agar medium containing: Glucose 5 g / 1 sulfate ammonium 1 g / 1, monopotassium phosphate 2 g / 1, mag- nesium sulfate 0.3 g / 1, calcium chloride 0.2 g / 1, yeast extract 1 g / 1, pullulan (Sigma) 10 g / 1 agar 15 g / 1. After 7 days incubation at 27 ° C, the surface of this der¬ deny medium was covered with ethyl alcohol, and the plates were placed at -20 ° C for one hour. At this temperature and in the presence of alcohol, pullulan precipitates, making the opaque medium. About 2% of co¬ lonies tested were surrounded by a clear halo more or less wide, probably due to hydrolysis of pullu¬ lane. No halo was detected around colonies of the strain NC50 unprocessed tested under the same conditions. The 10 clones transforming pretend présen¬ ter strongest pullulanase activity, that is to say those corresponding to colonies surrounded the broadest halos were recovered on the PDA-phléomy- middle cine and grown in shake flasks in the middle according Mandels supplemented with glucose 30 g / 1, yeast extract 1 g / 1 and buffered with potassium phthalate 5 g / 1. After 5 days incubation at 27 ° C, 200 rpm, the cultures were centrifuged and the pullulanase activity was assayed in the supernatants using the following protocol: 0.5 ml of a pullulan solution at 10 g / 1 in acetate buffer 0.1 M pH 5.5, 0.5 ml enzyme solution is added (optionally supernatant diluted in the same buffer). After incubation 60 minutes at 55 ° C, the glucose liberated was assayed by the method of Miller (Analytical

Che. , 1959, 31: 426). Activity is expressed in International Units (es) corresponding to the number of micro moles of glucose released per minute. In the supernatants of the 10 tested cultures, activities pullulanse 12 iu / ml to 350 IU / ml were found. No activity was detected in the supernatants of untransformed strain grown under the same conditions. Example V:

This example is intended to illustrate the possi- bility to preferentially select recom¬ binants clones T.géodes NC14 and its derivatives which are strongly producing a heterologous protein with plasmid constructs using the prin¬ ciple of the transcription polycistronic. The expression of several adjacent genes under the control of the same promoter and 1'intermédiaire the same messenger RNA, said polycistronic is a phéno¬ leads well known in bacteria. However, it is generally accepted that this phenomenon does not exist in eukaryotes. However, it was recently reported

(Kaufman et al, EMBO J., 1987, 6. 187) of an expression by animal cells in culture of the dihydroxy drofolate reductase gene allowing resistance trexate methodical, in constructions where said gene was placed immediately downstream of other non-selectable gene, the latter being preceded by a promoter. This allows indirect selection of clones strongly expressing the selectable gene not based on the level of résis¬ tance conferred by the gene placed downstream. It has been shown by the authors of the present invention that this principle could be applied to T.géodes, using for example as a selectable parqueur Sh gene for resistance to phleomycin.

A synthetic gene for human lysozyme was prepared pa * ς General assembly with 10 oligonucleotides syn¬ thesis. The sequence shown in Figure 7 includes 14 base pairs preceding the initiation codon followed by codons 18 starting with an ATG corresponding to amino acids of the human lysozyme signal peptide except for the 17th codon (replacing the glutamine a cine leu¬) (CHUNG, KESHAU and Gordon, Proc Natl Acad Sci 1988, 85:....... 6227) and 130 amino acid codons of the human lysozyme (JOLLES and JOLLES, Mol Cell Biochem, 1984, 63 , 165) and finally a stop codon followed by 6 base pairs. The DNA fragment flanked by sites res¬ triction EcoRV-BamHI was cloned into the EcoRV site of plas¬ mide pUT771 mentioned in Example II to give in one of two possible orientations, the plasmid pUT772 (Figure 8 ). The NC50 strain T.géodes was transformed by the plasmid pUT772 according to the protocol described in Ȭ xample I with the only difference that the selection of transformants was carried out with a lesser concentration of phleomycin (15 mg / ml). Colonies obtained with a frequency 50-100 times less than that provided by the pUT771 DNA used as a control were subcultured on PDA and individually examined for the calf ni¬ resistance to phleomycin ^ and verified for the production of lysozyme. Determining the minimum inhibitory concentration of phleomycin tions of mants transfor¬ by pUT772 showed that clones resistors levels were low (2 to 10 times the MIC by rap¬ port to the untransformed strain) while transfor¬ mants by ρUT771 were very high contrast (30 to 200 times the MIC). Producing lysozyme is checked indirec¬ ment by the agar cylinder method for the pro¬ duction of a lytic activity against Micrococcus lyso deikticus PDA. Of 83 strains tested from the processing by pUT772, 61 producing a detectable ac¬ tivity by the presence of a halo around inhibi¬ tion of agar cylinders. In contrast no issues colony of transformation was not pUT771 présen¬ this activity (55 tested). Clonal analysis showed that the production character was kept for 2/3 posi¬ stem tive after a stage of sporulation. PUT772-28 the NC50 strain selected from the most active strains by the test box was particularly studied. In successive crops on solid PDA medium containing increasing concentrations of phleomycin, a variant strain NC50 / pUT772-28 was selected. This variant noted NC50 / 28R4 resistant to 100 ug / ml mycin phléo¬ while the maximum concentration of growth for the parent is 20 ug / ml. Haloes tion inhibi¬ on a sailing Micrococcus lysodeikticus éga¬ LEMENT are superior to strain NC50 / 28R4 comparai¬ by its parent NC50 / pUT772-28. Both strains were grown in shake flask in the conditions described in 1'exemple IV. After five days of incubation at 27 ° C the lysozyme activity was determined by an assay turbi- dimetric from lyophilized cells of microtips coccus lysodeikticus (Shugar, Biochim Biophys, 1952, Acta 8:.. 302). Proteins from cultures supernatants were fractionated on a Pharmacia column MonoS

(50 mM phosphate buffer pH 7.2 - Elution gradient liné¬ range of 0 to 1 M NaCl). A high protein peak at 0.25 M is present in the two transformed strains and not present in the NC50 parent. For the bioassay, immunological detection with an antibody antilyso- human zyme and electrophoresed in acrylamide gel, it was verified by comparison with a sample of human authen¬ tick lysozyme that eluted at 0.25 M proteins contain lysozyme. The final concentrations in culture supernatants were estimated at 25 ug / ml and 200 mg / mL lysozyme recombinant respecti¬ tively for NC50 pUT772-28 and NC50-28R4 strains.

Claims

- CLAIMS -
1 - protein production method recombi¬ nante using filamentous fungi, characterized in that cultivated on a suitable medium a strain of the genus Tolypocladium wherein has been introduced a DNA sequence encoding said protein under elements providing control the expression of said sequence in the said strain and in that said la¬ recovered protein. 2 - Process according to claim 1, caracté¬ corseted in that the strain is a strain of Tolypocladium geodes.
3 - The method of claim 1, caracté¬ corseted in that the strain is a strain of Tolypocladium geodes deposited as No. 1-880 on 29 June 1989 at the Collection Nationale de Cultures of Microorganisms or a mutant deficient in protease activity and / or hyperexcréteur exogenous protein.
4 - Method according to one of claims 1 to 3, characterized in that the elements ensuring the expres¬ sion essentially comprise a promoter sequence of a fungal gene.
5 - Method according to one of claims 1 to
4, characterized in that the DNA sequence encoding said protein is preceded by a signal sequence assu¬ rant excretion of the protein.
6 - Method according to one of claims 1 to
5, characterized in that the DNA sequence encoding said protein is followed by a marker gene under the control of the same expression elements.
7 - Method according to claim 6, caracté¬ corseted in that said marker gene encodes for resistance to an antibiotic.
8 - The method of claim 7, characterizes in that the antibiotic is a compound of the fa- thousand of phleomycins.
9 - The method of claim 7, caracté¬ corseted in that the gene coding for resistance to an antibiotic of the family of phleomycins is not originating from genomic and genome of said antibiotic producing actinomycetes.
10 - Method according to one of claims 1 to
9, characterized in that the encoded protein is selected from pullulanase and human lysozyme. 11 - Method according to one of claims 5 to
10, characterized in that the signal sequence is the synthetic signal sé¬ accordingly set out in Figure 2.
12 - Method according to one of claims 1 to
11, characterized in that the promoter sequence is the promoter sequence shown in Figure 5.
13 - Method according to one of claims 1 to
12, characterized in that the DNA sequence encoding said protein is followed by a transcription terminator sequence. 14 - Method according to one of claims 1 to
13, characterized in that the DNA sequence encoding said protein as well as the elements ensuring the ex¬ pressure of said sequence are carried by a plasmid.
15 - Process according to Claim 14, characterizes in that said plasmid is a plasmid comprising tion réplica¬ autonomous replication sequence au¬ effective tonome at Tolypocladium: T-ARS.
16 - The method of claim 14 or 15, characterized in that said plasmid ensures 1'intégra- tion chromosomal DNA sequences involved.
17 - Protein obtained by the implementation of the method according to one of claims 1 to 16.
18 - Protein Sh characterized in that it is obtained by implementing the method according to one of claims 1 to 16. 19 - Protein Sh according to claim 18, characterized in that it comprises the amino acid sequence amino shown in Figure 2.
20 - strain Tolypocladium obtained during the implementation of the method according to one of reven¬ dications 1 to 16.
21 - strain Tolypocladium deposited under n ° 1-880 and mutants deficient in protease and / or hyperexcréteur exogenous protein activity.
PCT/FR1990/000479 1989-06-30 1990-06-28 New strain with filamentous fungi mutants, process for the production of recombinant proteins using said strain, and strains and proteins produced by said process WO1991000357A1 (en)

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US9079971B2 (en) 2007-03-30 2015-07-14 Ambrx, Inc. Modified FGF-21 polypeptides comprising non-naturally occurring amino acids
WO2015116902A1 (en) 2014-01-31 2015-08-06 Genentech, Inc. G-protein coupled receptors in hedgehog signaling
US9133495B2 (en) 2006-09-08 2015-09-15 Ambrx, Inc. Hybrid suppressor tRNA for vertebrate cells
US9156899B2 (en) 2008-09-26 2015-10-13 Eli Lilly And Company Modified animal erythropoietin polypeptides and their uses
US9175083B2 (en) 2004-06-18 2015-11-03 Ambrx, Inc. Antigen-binding polypeptides and their uses
EP3026123A1 (en) 2006-04-27 2016-06-01 Klaritos, Inc. Method and kit for predicting antibody therapy
US9434778B2 (en) 2014-10-24 2016-09-06 Bristol-Myers Squibb Company Modified FGF-21 polypeptides comprising an internal deletion and uses thereof
US9452222B2 (en) 2010-08-17 2016-09-27 Ambrx, Inc. Nucleic acids encoding modified relaxin polypeptides
US9488660B2 (en) 2005-11-16 2016-11-08 Ambrx, Inc. Methods and compositions comprising non-natural amino acids
EP3103880A1 (en) 2008-02-08 2016-12-14 Ambrx, Inc. Modified leptin polypeptides and their uses
EP3112468A1 (en) 1998-05-15 2017-01-04 Genentech, Inc. Il-17 homologous polypeptides and therapeutic uses thereof
US9567386B2 (en) 2010-08-17 2017-02-14 Ambrx, Inc. Therapeutic uses of modified relaxin polypeptides
EP3178835A1 (en) 2009-02-03 2017-06-14 Amunix Operating Inc. Extended recombinant polypeptides and compositions comprising same
EP3208612A1 (en) 2008-04-09 2017-08-23 Genentech, Inc. Compositions and methods for the treatment of immune related diseases
EP3278813A1 (en) 2009-06-08 2018-02-07 Amunix Operating Inc. Growth hormone polypeptides and methods of making and using same
EP3284825A1 (en) 2006-11-02 2018-02-21 Daniel J. Capon Methods of producing hybrid polypeptides with moving parts
EP3327026A1 (en) 2007-07-09 2018-05-30 Genentech, Inc. Prevention of disulfide bond reduction during recombinant production of polypeptides
EP3332798A1 (en) 2010-08-31 2018-06-13 Generon (Shanghai) Corporation Ltd. Use of interleukin-22 in treating viral hepatitis
WO2018152496A1 (en) 2017-02-17 2018-08-23 The Usa, As Represented By The Secretary, Dept. Of Health And Human Services Compositions and methods for the diagnosis and treatment of zika virus infection
EP3372617A2 (en) 2010-04-02 2018-09-12 Amunix Operating Inc. Binding fusion proteins, binding fusion protein-drug conjugates, xten-drug conjugates and methods of making and using same
WO2018200742A1 (en) 2017-04-25 2018-11-01 The Usa, As Represented By The Secretary, Dept. Of Health And Human Services Antibodies and methods for the diagnosis and treatment of epstein barr virus infection
WO2019018629A1 (en) 2017-07-19 2019-01-24 The Usa, As Represented By The Secretary, Dept. Of Health And Human Services Antibodies and methods for the diagnosis and treatment of hepatitis b virus infection
US10233235B2 (en) 2008-09-22 2019-03-19 The Regents Of The University Of Colorado, A Body Corporate Modulating the alternative complement pathway
US10266578B2 (en) 2017-02-08 2019-04-23 Bristol-Myers Squibb Company Modified relaxin polypeptides comprising a pharmacokinetic enhancer and uses thereof

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2033398A (en) * 1978-10-18 1980-05-21 Sandoz Ltd L compositions containing them cyclosporin derivatives their production and pharmaceutica
WO1986001537A1 (en) * 1984-08-31 1986-03-13 Centre National De La Recherche Scientifique (Cnrs Application of antibiotics of the phleomycine family as selection agent in the field of genetic engineering
EP0244234A2 (en) * 1986-04-30 1987-11-04 Alko Group Ltd. Transformation of trichoderma
EP0215594B1 (en) * 1985-08-29 1995-01-11 Genencor International, Inc. Heterologous polypeptide expressed in filamentous fungi, processes for their preparation, and vectors for their preparation

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2033398A (en) * 1978-10-18 1980-05-21 Sandoz Ltd L compositions containing them cyclosporin derivatives their production and pharmaceutica
WO1986001537A1 (en) * 1984-08-31 1986-03-13 Centre National De La Recherche Scientifique (Cnrs Application of antibiotics of the phleomycine family as selection agent in the field of genetic engineering
EP0215594B1 (en) * 1985-08-29 1995-01-11 Genencor International, Inc. Heterologous polypeptide expressed in filamentous fungi, processes for their preparation, and vectors for their preparation
EP0244234A2 (en) * 1986-04-30 1987-11-04 Alko Group Ltd. Transformation of trichoderma

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
Bio/Technology, Vol. 1, Octobre 1983, (New York, US), S. SHOEMAKER et al.: "Molecular Cloning of exo-Cellobiohydrolase I Derived from Trichoderma Reesei Strain L27", pages 691-696 voir le document en entier; en particulier figure 5 *
Biochemical and Biophysical Research Communications, Vol. 150, No. 2, 29 Janvier 1988 Academic Press, Inc., (Duluth, MN, US), K. YOSHIMURA et al.: "Human Lysozyme: Sequencing of a cDNA, and Expression and Secreation by Saccharomyces Cerevisiae", pages 794-801 voir Resume; figure 2 *
CHEMICAL ABSTRACTS, Vol. 104, No. 23, 9 Juin 1986, (Columbus, Ohio, US), S.N. AGATHOS et al.: "Physiological and Genetic Factors for Process Development of Cyclosporine Fermentations", voir page 598, Resume 205460n, & J. Ind. Microbiol. 1986, 1(1), 39-48 *
FEBS Letters, Vol. 230, No. 1,2, Mars 1988, Elsevier Science Publishers B.V., (Amsterdam, NL), A. GATIGNOL et al.: "Bleomycin Resistance Conferred by a Drug-Binding Protein", pages 171-175 voir le document en entier cite dans la demande *
Journal of Bacteriology, Vol. 164, No. 2, Novembre 198k, American Society for Microbiology, (Baltimore, US), S. MICHAELIS et al.: "Characterization and Expression of the Structural Gene for Pullulanase, a Maltose-Inducible Secreted Protein of Klebsiella Pneumoniae", pages 633-638 voir le document en entier cite dans la demande *

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0786257A1 (en) 1992-06-03 1997-07-30 Genentech, Inc. Tissue plasminogen activator glycosylation variants with improved therapeutic properties
US5728676A (en) * 1994-09-08 1998-03-17 Ciba-Geigy Corporation Use of insulin-like growth factors I and II for inhibition of inflammatory response
US5843899A (en) * 1994-09-08 1998-12-01 Ciba-Geigy Corporation Use of insulin-like growth factors I and II for inhibition of inflammatory response
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US5851984A (en) * 1996-08-16 1998-12-22 Genentech, Inc. Method of enhancing proliferation or differentiation of hematopoietic stem cells using Wnt polypeptides
US6159462A (en) * 1996-08-16 2000-12-12 Genentech, Inc. Uses of Wnt polypeptides
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US7741439B2 (en) 2000-01-13 2010-06-22 Genentech, Inc. Isolated stra6 polypeptides
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US6673580B2 (en) 2000-10-27 2004-01-06 Genentech, Inc. Identification and modification of immunodominant epitopes in polypeptides
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EP2000545A1 (en) 2001-06-20 2008-12-10 Genentech, Inc. Compositions and methods for the diagnosis and treatment of tumor
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US7888055B2 (en) 2001-08-07 2011-02-15 The University Of Chicago Polypeptide immobilization
US9074300B2 (en) 2001-08-07 2015-07-07 The University Of Chicago Polypeptide immobilization
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US7138512B2 (en) 2002-04-10 2006-11-21 Georgetown University Gene SHINC-2 and diagnostic and therapeutic uses thereof
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US7575893B2 (en) 2003-01-23 2009-08-18 Genentech, Inc. Methods for producing humanized antibodies and improving yield of antibodies or antigen binding fragments in cell culture
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US8907064B2 (en) 2004-02-02 2014-12-09 Ambrx, Inc. Modified human four helical bundle polypeptides and their uses
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WO2005093064A1 (en) 2004-03-29 2005-10-06 Galpharma Co., Ltd. Novel galectin 9 modification protein and use thereof
US7250298B2 (en) 2004-04-07 2007-07-31 The University Of Chicago Monomeric red fluorescent proteins
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US9175083B2 (en) 2004-06-18 2015-11-03 Ambrx, Inc. Antigen-binding polypeptides and their uses
WO2006069246A2 (en) 2004-12-22 2006-06-29 Ambrx, Inc. Compositions containing, methods involving, and uses of non-natural amino acids and polypeptides
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EP2511257A2 (en) 2005-03-21 2012-10-17 ViroBay, Inc. Alpha ketoamide compounds as cysteine protease inhibitors
WO2006124667A2 (en) 2005-05-12 2006-11-23 Zymogenetics, Inc. Compositions and methods for modulating immune responses
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US7749704B2 (en) 2005-11-01 2010-07-06 Mayo Foundation For Medical Education And Research Promoter polymorphisms of the BLyS gene and use in diagnostic methods
US9488660B2 (en) 2005-11-16 2016-11-08 Ambrx, Inc. Methods and compositions comprising non-natural amino acids
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EP3026123A1 (en) 2006-04-27 2016-06-01 Klaritos, Inc. Method and kit for predicting antibody therapy
EP2522678A1 (en) 2006-05-15 2012-11-14 Sea Lane Biotechnologies, LLC Neutralizing antibodies to influenza viruses
EP2826788A1 (en) 2006-06-21 2015-01-21 Musc Foundation for Research Development Targeting complement factor H for treatment of diseases by the sue of CR2-FH molecules
US9133495B2 (en) 2006-09-08 2015-09-15 Ambrx, Inc. Hybrid suppressor tRNA for vertebrate cells
WO2008030558A2 (en) 2006-09-08 2008-03-13 Ambrx, Inc. Modified human plasma polypeptide or fc scaffolds and their uses
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EP3284825A1 (en) 2006-11-02 2018-02-21 Daniel J. Capon Methods of producing hybrid polypeptides with moving parts
EP2450050A1 (en) 2006-11-29 2012-05-09 Genentech, Inc. IL-17A/F heterodimeric polypeptides and therapeutic uses thereof
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WO2008103962A2 (en) 2007-02-22 2008-08-28 Genentech, Inc. Methods for detecting inflammatory bowel disease
EP2436781A1 (en) 2007-02-22 2012-04-04 Genentech, Inc. Methods for detecting inflammatory bowel disease
US9079971B2 (en) 2007-03-30 2015-07-14 Ambrx, Inc. Modified FGF-21 polypeptides comprising non-naturally occurring amino acids
US9975936B2 (en) 2007-03-30 2018-05-22 Ambrx, Inc. Nucleic acids encoding modified FGF-21 polypeptides comprising non-naturally occurring amino acids
US9517273B2 (en) 2007-03-30 2016-12-13 Ambrx, Inc. Methods of treatment using modified FGF-21 polypeptides comprising non-naturally occurring amino acids
EP3327026A1 (en) 2007-07-09 2018-05-30 Genentech, Inc. Prevention of disulfide bond reduction during recombinant production of polypeptides
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EP3241846A1 (en) 2007-10-04 2017-11-08 ZymoGenetics, Inc. B7 family member zb7h6 and related compositions and methods
WO2009046407A2 (en) 2007-10-04 2009-04-09 Zymogenetics, Inc. B7 FAMILY MEMBER zB7H6 AND RELATED COMPOSITIONS AND METHODS
US8679749B2 (en) 2007-11-01 2014-03-25 The University Of Chicago Red fluorescent proteins with enhanced bacterial expression, increased brightness and reduced aggregation
EP2930182A1 (en) 2007-11-20 2015-10-14 Ambrx, Inc. Modified insulin polypeptides and their uses
US8946148B2 (en) 2007-11-20 2015-02-03 Ambrx, Inc. Modified insulin polypeptides and their uses
WO2009067636A2 (en) 2007-11-20 2009-05-28 Ambrx, Inc. Modified insulin polypeptides and their uses
US8003325B2 (en) 2007-11-30 2011-08-23 Mayo Foundation For Medical Education And Research Polymorphisms of the BLyS gene and use in diagnostic methods
EP2657253A2 (en) 2008-01-31 2013-10-30 Genentech, Inc. Anti-CD79b antibodies and immunoconjugates and methods of use
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US9938333B2 (en) 2008-02-08 2018-04-10 Ambrx, Inc. Modified leptin polypeptides and their uses
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US10138283B2 (en) 2008-07-23 2018-11-27 Ambrx, Inc. Modified bovine G-CSF polypeptides and their uses
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US10233235B2 (en) 2008-09-22 2019-03-19 The Regents Of The University Of Colorado, A Body Corporate Modulating the alternative complement pathway
US9644014B2 (en) 2008-09-26 2017-05-09 Ambrx, Inc. Modified animal erythropoietin polypeptides and their uses
WO2010037062A1 (en) 2008-09-26 2010-04-01 Ambrx, Inc. Non-natural amino acid replication-dependent microorganisms and vaccines
US9121025B2 (en) 2008-09-26 2015-09-01 Ambrx, Inc. Non-natural amino acid replication-dependent microorganisms and vaccines
US9121024B2 (en) 2008-09-26 2015-09-01 Ambrx, Inc. Non-natural amino acid replication-dependent microorganisms and vaccines
US9156899B2 (en) 2008-09-26 2015-10-13 Eli Lilly And Company Modified animal erythropoietin polypeptides and their uses
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EP3178835A1 (en) 2009-02-03 2017-06-14 Amunix Operating Inc. Extended recombinant polypeptides and compositions comprising same
WO2010120561A1 (en) 2009-04-01 2010-10-21 Genentech, Inc. Anti-fcrh5 antibodies and immunoconjugates and methods of use
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WO2010144508A1 (en) 2009-06-08 2010-12-16 Amunix Operating Inc. Glucose-regulating polypeptides and methods of making and using same
US8535912B2 (en) 2009-10-15 2013-09-17 Genentech, Inc. Chimeric fibroblast growth factors with altered receptor specificity
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WO2011056494A1 (en) 2009-10-26 2011-05-12 Genentech, Inc. Activin receptor-like kinase-1 antagonist and vegfr3 antagonist combinations
WO2011056497A1 (en) 2009-10-26 2011-05-12 Genentech, Inc. Activin receptor type iib compositions and methods of use
WO2011056502A1 (en) 2009-10-26 2011-05-12 Genentech, Inc. Bone morphogenetic protein receptor type ii compositions and methods of use
WO2011060246A2 (en) 2009-11-12 2011-05-19 Genentech, Inc. A method of promoting dendritic spine density
WO2011066503A2 (en) 2009-11-30 2011-06-03 Genentech, Inc. Compositions and methods for the diagnosis and treatment of tumor
EP3002297A2 (en) 2009-11-30 2016-04-06 F. Hoffmann-La Roche AG Antibodies for treating and diagnosing tumors expressing slc34a2 (tat211)
EP2805965A1 (en) 2009-12-21 2014-11-26 Ambrx, Inc. Modified porcine somatotropin polypeptides and their uses
EP2805964A1 (en) 2009-12-21 2014-11-26 Ambrx, Inc. Modified bovine somatotropin polypeptides and their uses
WO2011106297A2 (en) 2010-02-23 2011-09-01 Genentech, Inc. Compositions and methods for the diagnosis and treatment of tumor
EP3372617A2 (en) 2010-04-02 2018-09-12 Amunix Operating Inc. Binding fusion proteins, binding fusion protein-drug conjugates, xten-drug conjugates and methods of making and using same
WO2011139985A1 (en) 2010-05-03 2011-11-10 Genentech, Inc. Compositions and methods for the diagnosis and treatment of tumor
WO2011161491A1 (en) 2010-06-25 2011-12-29 Inserm (Institut National De La Sante Et De La Recherche Medicale) Methods and pharmaceutical compositions for the treatment of respiratory tract infections
US10253083B2 (en) 2010-08-17 2019-04-09 Ambrx, Inc. Therapeutic uses of modified relaxin polypeptides
US9567386B2 (en) 2010-08-17 2017-02-14 Ambrx, Inc. Therapeutic uses of modified relaxin polypeptides
US9962450B2 (en) 2010-08-17 2018-05-08 Ambrx, Inc. Method of treating heart failure with modified relaxin polypeptides
US9452222B2 (en) 2010-08-17 2016-09-27 Ambrx, Inc. Nucleic acids encoding modified relaxin polypeptides
EP3332798A1 (en) 2010-08-31 2018-06-13 Generon (Shanghai) Corporation Ltd. Use of interleukin-22 in treating viral hepatitis
WO2012036884A2 (en) 2010-09-15 2012-03-22 Aligna Technologies, Inc. Bioproduction of aromatic chemicals from lignin-derived compounds
WO2012099566A1 (en) 2010-11-17 2012-07-26 Sea Lane Biotechnologies, Llc Influenza virus neutralizing agents that mimic the binding site of an influenza neutralizing antibody
WO2013163606A1 (en) 2012-04-27 2013-10-31 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Vascular endothelial growth factor antagonists and methods for their use
WO2014144911A2 (en) 2013-03-15 2014-09-18 Capon Daniel J Hybrid immunoglobulin containing non-peptidyl linkage
WO2015116902A1 (en) 2014-01-31 2015-08-06 Genentech, Inc. G-protein coupled receptors in hedgehog signaling
US9434778B2 (en) 2014-10-24 2016-09-06 Bristol-Myers Squibb Company Modified FGF-21 polypeptides comprising an internal deletion and uses thereof
US10189883B2 (en) 2014-10-24 2019-01-29 Bristol-Myers Squibb Company Therapeutic uses of modified FGF-21 polypeptides
US9631004B2 (en) 2014-10-24 2017-04-25 Bristol-Myers Squibb Company Modified FGF-21 polypeptides comprising an internal deletion and uses thereof
US10266578B2 (en) 2017-02-08 2019-04-23 Bristol-Myers Squibb Company Modified relaxin polypeptides comprising a pharmacokinetic enhancer and uses thereof
WO2018152496A1 (en) 2017-02-17 2018-08-23 The Usa, As Represented By The Secretary, Dept. Of Health And Human Services Compositions and methods for the diagnosis and treatment of zika virus infection
WO2018200742A1 (en) 2017-04-25 2018-11-01 The Usa, As Represented By The Secretary, Dept. Of Health And Human Services Antibodies and methods for the diagnosis and treatment of epstein barr virus infection
WO2019018629A1 (en) 2017-07-19 2019-01-24 The Usa, As Represented By The Secretary, Dept. Of Health And Human Services Antibodies and methods for the diagnosis and treatment of hepatitis b virus infection

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