WO1993024631A1 - PRODUCTION OF STREPTAVIDIN FROM $i(BACILLUS SUBTILIS) - Google Patents

Abstract

Method for producing biologically active streptavidin and streptavidin fusion proteins by cloning the sav gene from Streptomyces avidinii into Bacillus subtilis and purifying the secreted streptavidin proteins from the growth medium.

Description

TITLE

PRODUCTION OF STREPTAVIDIN FROM BACILLUS SUBTILIS FIELD OF THE INVENTION

This invention relates to a method for cloning the streptavidin gene into B. subtilis and the secretion of tetrameric, biologically active streptavidin protein into the growth medium.

BACKGROUND OF THE INVENTION

Streptavidin is a tetrameric protein isolated from the actinobacterium Streptomyces avidinii and is remarkable for its ability to bind up to four molecules of d-biotin with unusually high affinity (1).

Streptavidin is a nearly neutral 60,000 dalton protein consisting of four identical subunits each having a molecular weight of 15,000 daltons (2). The ability of streptavidin to bind derivitized forms of biotin has led to its widespread use in diagnostic assays where high affinity protein-ligand interactions are important.

Some of the current applications include streptavidin coated liposomes used for drug delivery and diagnostic tests to detect human antibodies or pathogens using streptavidin linked to enzymes such as alkaline or phosphatase or horseradish peroxidase. Streptavidin is currently produced in commercial quantities by

S. avidinii . S. avidinii naturally secretes relatively low amounts of streptavidin into the growth media.

Recombinant E. coli produce streptavidin in relatively high amounts, but only intercellularly and generally in insoluble form. Streptavidin is available commercially from several manufacturers, however, the commercial reagent exhibits considerable variation in molecular weight and purity between suppliers and between lots from the same supplier. Furthermore, the reagent is expensive and its cost prohibits broader applications. The primary cause for the lack of homogeneity in the streptavidin currently produced is the presence of protease susceptible sites in the protein which exist outside of the biotin binding domain. Another factor contributing to the low purity of streptavidin is the presence of trace amounts of biotin in the growth medium from which the streptavidin is purified. This results in biotin-bound streptavidin and contributes to the molecular weight variations mentioned above.

Argarana and Meade (2) and (3) describe the cloning of the streptavidin gene from a genomic library of

Streptomyces avidinii as well as the DNA sequence of the coding region of the gene.

Meade also reports secretion of 250 mg/liter of streptavidin from S. llvidans. This process is time-consuming as the fermentation time alone is 4 to 7 days.

Cantor (4) describes the isolation of the DNA which encodes streptavidin from Streptomyces avidniii, which includes the region encoding the signal peptide and the subsequent cloning of the DNA into a bacterial host cell, typically E. coli . Additionally, Cantor describes the construction and subsequent expression in bacteria of a fused gene comprising a first DNA fragment encoding a target protein of interest (specifically human LDL receptor) fused to a DNA fragment encoding streptavidin.

Sano and Cantor (5) describe the construction of systems for expressing the cloned streptavidin gene in E. coli where the streptavidin accumulated to more than 35% of the total cell protein. Sano further describes the creation of expression vectors for streptavidin containing chimeric proteins which are also capable of expression in E. coli (6). Meade shows that

streptavidin is present in the periplasm of E. coli . However, the work of Sano and Cantor has indicated that the streptavidin signal peptide does not function in E. coli .

The methods of Sano and Cantor are useful and provide an alternative to the production of streptavidin from Streptomyyes avidinii . However, a significant problem with the production of streptavidin from E. coli is that the protein is expressed intracellularly and in insoluble form. Recovery of useful protein from this system generally requires methods for isolation of the inactive streptavidin followed by purification and refolding of the protein into the active form. These methods are time-consuming and not readily adapted for commercial production since refolding of the protein is not efficient.

B. subtilis, a gram positive bacterium, has great potential for producing commercially important proteins because it can be genetically manipulated, adapted to various nutritional and physical conditions of growth, and because it is not pathogenic or toxigenic to humans. Under the proper conditions, B. subtilis is known to synthesize and secrete specific proteins relatively free of contaminating species making the proteins easier to purify.

Chang, Nagarajan and Koracevic (7), (8), and (9) separately disclose cloning vectors capable of

replication in B. subtilis which include heterologous genes coding for proteins which can be secreted into the bacterial growth media. Proteins that may be secreted by these systems include proteases. Protein A,

prorennin, insulin, human growth hormone, interferon and urokinase.

It is generally understood that translocation of secreted proteins across bacterial membranes requires a signal peptide. While a number of studies directed to understanding the role of the signal peptide in protein secretion have been done, the mechanism of such

translocation and the exact manner by which the signal peptide influences translocation and removal of the signal peptide from the signal peptide-mature protein complex to yield secreted mature protein is not fully understood.

Vectors enabling the secretion of a number of different heterologous proteins by B. subtilis have been demonstrated (8), (9), and (10). These include vectors that are based on genes for bacterial exoenzymes such as amylase, protease, levansucrase and β-lactamases.

Palva demonstrated the secretion by B. subtilis of the heterologous proteins β-lactamase of E. coli (12) and human leukocyte interferon (13) by transforming the bacteria with a vector wherein the genes for β-lactamase of E. coli and human leukocyte interferon were operably linked to the promoter, ribosome binding site and signal sequence of the α-amylase gene from Bacillus

amyloliquefaciens. They found that only a low amount of interferon was secreted.

Secretion vectors based on levansucrase were reported by Dion (14). Dion obtained low levels of mouse interferon compared to levansucrase. The authors suggested that the low yield of interferon was due to a poorly understood incompatibility between the signal sequence of the B. subtilis levansucrase gene and the mouse interferon α2 gene.

Nagarajan (15) describes a method to design vectors for the secretion of heterologous proteins in bacteria, including B. subtilis. The method is drawn to ways of enabling combinations of promoters, ribosome binding sequences and signal peptides with sequences from desired heterologous proteins such that translocation via an effective signal peptide is achieved. However, in the case of streptavidin secretion by B. subtilis, the method disclosed will not ensure a commercially viable secretion process. It is known, for example, that the tetrameric form of streptavidin protein is needed for efficient biotin binding and prior to the instant invention it had not been demonstrated that

B. subtilis could efficiently secrete and accumulate a tetrameric protein. Most of the proteins that have been efficiently secreted to date are monomeric proteins, with the exception of E. coli alkaline phosphatase which is dimeric. Also, the production of biologically active streptavidin in the B. subtilis growth medium requires several processes to occur simultaneously and

efficiently. These include the efficient translocation of the mature protein across the membrane and removal of signal peptide from the precursor protein. This must be followed by the release of the mature protein from the membrane and oligomerization of the monomers to yield a tetrameric protein which passes into the medium. It is not clear from current knowledge whether the

oligomerization in B. subtilis occurs in the space between the cell membrane and cell wall, or if it occurs in the growth medium. Further, it is known that the Streptomyces genome contains a high GC base pair content of at least 70%, whereas the GC content in B. subtilis is known to be on the order of 42%. This would indicate that translation of a Streptomyces gene by B. subtilis may not be possible due to ca incongruous number of GC codons in the Streptomyces genes. Furthermore, it is well known that B. subtilis secretes some proteins very inefficiently, which, in the case of streptavidin, would prove lethal to the cell.

Therefore, a need remains for an expression system that is capable of expressing and secreting streptavidin protein as an active, soluble protein in commercially useful quantities which eliminates the need for additional solubilization and purification. The instant invention provides a method for efficiently expressing and secreting soluble, active streptavidin from

B. subtilis in high quantities virtually free of biotin and other contaminants.

SUMMARY OF THE INVENTION

This invention provides a method for producing tetrameric, biologically active streptavidin by

secretion from Bacillus subtilis, comprising:

a. transforming Bacillus subtilis with a gene construct comprising a sequence encoding streptavidin operably linked to a sequence encoding a signal peptide and an expression element wherein said sequence encoding a signal peptide is isolated from DNA encoding

exproteins of bacteria, and said expression element is isolated from DNA encoding gram positive bacterial proteins;

b. growing the transformed Bacillus subtilis in suitable growth medium whereby streptavidin is secreted into the growth medium; and

c. purifying the streptavidin from the growth medium.

This invention further provides a Bacillus subtilis bacterium transformed as described in (a) above.

This invention also provides a method for producing a fused gene product comprised of tetrameric,

biologically active streptavidin fused to a second desired protein, by secretion from Bacillus subtilis, comprising:

a. transforming Bacillus subtilis with a fused gene construct comprising a sequence encoding the streptavidin gene fused to a sequence encoding a second desired protein wherein said fused sequence is operably linked to a sequence encoding a signal peptide and an expression element; wherein said sequence encoding a signal peptide is isolated from DNA encoding exoproteins of bacteria and said expression element is isolated from

DNA encoding gram positive bacterial proteins;

b. growing the transformed Bacillus subtilis in suitable growth medium whereby the fused streptavidin and desired protein is secreted into the growth medium; and

c. purifying the fused streptavidin and desired protein from the growth.medium.

This invention further provides a Bacillus subtilis bacterium transformed with the fused gene construct as described in (a) above, and also the fused gene products as described above.

Also, plasmids pBE659, pBE660, pBE661, pBE662, pBE663, pBE673 and pBE655 are provided.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure la describes the construction of a plasmid pBE659 containing a hybrid gene fusion construct

consisting of sav gene fused to npr expression elements and signal peptide-from the plasmids pBE651(sav) and pBE83 (npr-lvs) . A) The DNA sequence across npr signal peptide cleavage site in pBE83 is designated as SEQ ID NO: 10. B) The DNA sequence across sav mature protein sequence in pBE651 is designated as SEQ ID NO: 11. C) The DNA sequence across nprsav fusion junction in pBE659 is designated as SEQ ID NO:12.

Figure lb describes the DNA sequence of the streptavidin gene. The DNA sequence is designated as SEQ ID NO:13. The amino acid sequence is designated as SEQ ID NO:14.

Figure 2 describes the construction of plasmids containing the gene fusion constructs pBE660 (apr-sav), pBE661 (npr-sav), pBE662 (bar-sav), and pBE663 (Ivs-sav) from the plasmids pBE30 (apr-phoA), pBE90 (npr-phoA), pBE91 (bar-phoA), and pBE597 (lvs-phoA), respectively. Figure 3 is a Western Blot analysis of B. subtilis, pBE20, and pBE659 culture supernatant, respectively.

Numbers denote time in hours.

Figure 4a depicts the U.V. absorption spectra of fractions eluted from an,iminobiotin agarose column loaded with an ammonium sulfate fraction of B. subtilis growth media.

Figure 4b depicts the U.V. absorption spectra of fraction eluted from a Sephacryl S200 column loaded with the streptavidin-containing fraction from the

iminobiotin column.

Figure 4c is a Western Blot analysis of fractions from both the iminobiotin and S200 columns.

Figure 5a describes the creation of plasmid pBE93 (npr-phoA) from plasmid pBE592 (lvs-phoA) by

restriction enzyme digestion and the ligation of the npr expression element DNA from pBE93 with the mature sav gene of plasmid pBE670 by Nhe1-Pst1 digest resulting in the creation of plasmid pBE673 (npr-sav).

Figure 5b depicts a Western blot of B. subtilus strains containing pBE659 (1-159) and pBE673 (15-159).

Figure 6 describes the streptavidin-heterologous gene (PhoA) fusions: Npr_ss-Sav_15-133-PhoA;

Npr_ss-Sav_15-138-PhOA; Npr_ss-Savi_15-145-PhoA; and

Npr_ss-Sav_{15_159}-PhoA.

Figure 7 describes the EcoRV digestion and ligation of the lvs expression element and mature lvs gene from plasmid pBE311 with the mature sav gene of pBE653 (sav) to produce the plasmid pBE655 containing the gene fusion lvs-sav-lvs.

DETAILED DESCRIPTION OF THE INVENTION

In support of the disclosure of the instant invention, the following terms are intended to convey the following meanings. "Mature protein" is the final protein product without the signal peptide attached.

"Desired protein" is any protein considered a valuable product to be obtained from genetically

engineered bacteria. The term "desired protein," or

"second desired protein" is used herein to describe that protein which, in the fused gene product of the

invention, is fused to streptavidin. Also, unless otherwise indicated. Applicants intend, according to the instant invention, that the fused gene construct may be constructed so that the streptavidin is fused to either the C-terminus, or the N-terminus, of the second desired protein.

"Signal peptide" is an amino terminal pόlypeptide preceding the secreted mature protein. The signal peptide is cleaved from and is therefore not present in the mature protein. Signal peptides function by directing and translocating extracellular proteins across cell membranes. Signal peptide is also referred to as signal protein.

"Compatible restriction sites" are different restriction sites which, when cleaved, yield nucleotide ends that can be ligated without any additional

modification.

"apr" and "Apr" refer to alkaline protease gene and protein, respectively.

"bar" and "Bar" refer to ribonuclease gene and protein, respectively.

"lvs" and "Lvs" refer to levansucrase gene and protein, respectively.

"npr" and "Npr" refer to neutral protease gene and protein, respectively.

"phoA" and "PhoA" refer to E. coli alkaline phosphatase gene and protein, respectively. "sav" and "Sav" refer to streptavidin gene and protein, respectively.

The term "streptavidin" refers to the protein comprising amino acid residues 1-159 of a 2 kb BamHI fragment isolated from spreptomyces avidinii as

described in Example 1 and Figure 1b, or any sequential subset of amino acid residues thereof, or mutations or derivatives thereof, which retain the ability to bind biotin.

The term "biotin" refers to biotin, biotin

derivatives, and biotin analogs capable of binding streptavidin.

"Ap-" refers to ampicillin.

"Kan-" refers to kanamycin.

"Cm-" refers to chloramphenicol.

"Shuttle phagemid" as used herein is a vector that is normally double stranded and contains both the origins of replication for E. coli and B. subtilis and also the F1 intragenic region for the preparation of single stranded DNA.

The terms "peptide", "polypeptide" and "protein" are used interchangeably.

The terms "restriction endonuclease cleavage site" and "restriction site" are used interchangeably.

The term "expression element" refers herein to a

DNA fragment containing the necessary information for transcription and translational initiation in

B. subtilis including, for example, the promoter sequence and the ribosome binding site sequence.

The term "exoproteins of bacteria" is used to describe those proteins produced by bacteria which are able to cross the cytoplasmic bacterial membrane and are known to be naturally secreted into the bacterial growth media. The term "gram positive bacterial proteins" refers to those proteins which are known to be naturally synthesized by gram positive bacteria.

The term "biologically active", as used herein, refers to a soluble streptavidin protein molecule which is able to bind to biotin, biotin analogs, or

derivatized biotin.

Suitable methods of genetic engineering employed herein are described in the references (16), (17), and (18) and in the instructions accompanying commercially available kits for genetic engineering. Bacterial cultures and plasmids necessary to carry out this invention are commercially available and, along with their sources, are identified in the text and examples which follow.

The source of the bacteria strains, genes and the various vectors described herein are readily available to one skilled in the art. The complete nucleotide sequence for the genes for apr, npr, bar and lvs from B . amyloliquefaciens, the E. coli phoA gene and the sav gene from S. avidinii have been published (2), (19), (20), (21), and (22). In addition, these sequences are accessible in the GenBank from Nucleic Acid Data base from Los Almos, California.

The bacterial strains B. subtilis, E. coli and S. avidinii and plasmids pBE322, pTZ18R, pSK, pC194, pUB110 and phage M13KO7 are readily available from a variety of sources. For example, they can be obtained from the American Type Culture Collection, Rockville, MD, or the Bacillus Stock Center, Ohio, and are also available from other commercial suppliers. The position of the newly engineered restriction sites and sequence of the mutagenic oligonucleotide is indicated in the figures or examples and one skilled in the art may readily prepare these constructs with the available information in this art.

The anti-streptavidin antiserum can be purchased from several manufacturers. In the present study, it was purchased from Sigma, St. Louis, MO. The

streptavidin used as standard was obtained from Bethesda

Research Laboratories, MD. The various techniques used for the construction of plasmids and bacterial strains are standard methods and relevant variations are indicated in the text when necessary. These methods are found in references (11), (16), (17), and (18).

The present invention utilizes an isolated 2 kb DNA fragment which encodes streptavidin (Figure lb). The DNA was isolated according to techniques well known in the art based on published DNA sequence (2, 3). The

2 kb fragment contains the entire sav open reading frame encoding a signal peptide and the mature protein which comprises amino acid sequences 1-159 and the flanking region DNA which occurs naturally at the 3' and 5' ends of the coding region.

A recombinant cloning vehicle is described which comprises DNA encoding a suitable expression element for streptavidin expression and the DNA fragment encoding the streptavidin protein, wherein said cloning vehicle is further characterized by the presence of a first and a second restriction enzyme site, the DNA fragment encoding streptavidin being inserted into said site.

The present invention entails development of a vector which comprises expression elements including a promoter sequence controlling transcription and a ribosomal binding site sequence controlling translation; and also a sequence for a signal peptide which enables translocation of the protein through the bacterial membrane and the cleavage of the signal peptide from the mature protein. Suitable vectors will be those which are compatible with the bacterium employed. For example, for B. subtilis such suitable vectors include

E. coli-B subtilis shuttle vectors which have compatible regulatory sequences and origins of replication. They will be preferably multicopy and have a selective marker gene, for example, a gene coding for antibiotic

resistance. For example, pTZ18R is a phagemid

obtainable from Pharmacia, Piscataway, NJ 08854, which confers resistance to ampicillin in E. coli, and pC194 from BGSC which confers resistance to chloramphenicol (cm^r) in E. coli and B. subtilis.

The expression elements containing DNA sequences encoding the promoter and ribosome binding site may be from any gram positive bacterial protein, and the signal peptide may be from any single bacterial gene which encodes a secreted product. The DNA sequences encoding the promoter and ribosome binding site may also be from a different gene than that encoding the signal peptide. These DNA sequences encoding the promoter, ribosome binding site and signal peptide can be isolated by means well known to those skilled in the art and illustrative examples are documented in the literature (23). The promoters in the DNA sequences may be either

constitutive or inducible. Suitable signal peptides and expression elements may be selected from the group comprising, for example, apr, npr, lvs and bar.

The addition of a restriction endonuclease cleavage site to the 3' end of the DNA encoding the signal peptide is also easily accomplished by means well known to those skilled in the art (16). Several methods may be employed to add the restriction endonuclease cleavage site to the 3' end of the DNA encoding the signal peptide. One such method might incorporate polymerse chain reaction (PCR). The method used in the present invention is site-directed mutagenesis which is most preferred, and is described in Mutagene manual (Biorad,

1414 Harbour Way South, Richmond, CA 96804). The means to isolate DNA sequences encoding a desired protein and the addition of restriction sites on the 5' end of the mature coding sequence is also well known to those skilled in the art (16). Any restriction endonuclease site may be used but the use of a restriction site unique to the target vector is desirable. The

restriction endonuclease site on the 3' end of the DNA sequence encoding the signal peptide and that on the 5' end of the DNA sequence encoding the mature desired protein must be compatible. Suitable compatible restriction sites are well known in the art. See, for example the Restriction Fragment Compatibility Table of the New England Biolabs 1988-1989 Catalog, New England Biolabs, Inc., Beverly, MA 01915 (1988), which is herein incorporated by reference. Preferred for use herein are EcoRV or NheI. The combined DNA sequences encoding a promoter, ribosome binding site and signal peptide with a restriction site at its 3' end and the DNA sequences encoding mature polypeptides or proteins with a

compatible restriction site at its 5' end can be operably integrated by conventional techniques (16) and (17).

The recombinant cloning vehicle of the present invention has been inserted into a bacterial host cell. A suitable host cell would be derived from the genus Bacillus, the most preferred host cell would be of the species subtilis. One method to transform B. subtilis bacteria is described by Vasantha et al. (9). Standard microbiological methods well known to those skilled in the art can be used for the growth and maintenance of bacterial cultures. Several genetically engineered B. subtilis host cells containing the recombinant cloning vehicle of the present invention have been prepared by transforming the strain BE1500 or its derivatives (1510) with the plasmids, pBE659A, pBE660, pBE661, pBE662, pBE663, pBE673, pBE659 and pBE655.

BE1500 has the genotype trpC2, metB10, lys3, Δ-aprE66, Δ-npr82, Δ-sacB::ermC (24).

A method of producing streptavidin comprises cultivating a genetically engineered host cell of the present invention under suitable conditions permitting expression of the streptavidin gene and recovering the streptavidin so produced from the growth media.

The present invention also provides a fused gene which comprises a first DNA fragment encoding

streptavidin fused to a second DNA fragment encoding a target protein of interest and wherein the fused gene is capable of expressing a fused protein in vivo when the gene is inserted into a host cell.

In one embodiment of the invention, the second DNA fragment is the gene encoding B. amyloliquefaciens levansucrase (Lvs). Such a fused gene expresses a protein which consists of streptavidin at the N-terminal region of the fused protein and levansucrase at the C-terminal region of the fused protein when the fused gene is inserted into a suitable expression vector and introduced into a suitable host cell. The fused gene may be cloned into a bacterial expression vector and used to transfect a bacterial host cell with the fused gene. A preferred bacterial host cell is B. subtilis .

The invention also provides a secretion vector capable of expressing and secreting the fused gene of the present invention when said vector is introduced into a suitable host cell. The vector comprises DNA encoding the promoter, ribosomal binding site and signal sequence of the first gene followed by DNA encoding the mature protein of the first gene, fused to DNA encoding the mature protein of the second gene. Also provided is a fused protein encoded by the fused gene of the present invention wherein a desired protein of interest is fused to streptavidin. In one embodiment of the invention, the desired protein is B. amyloliquefaciens levansucrase.

For purification of the streptavidin protein product, bacteria secreting streptavidin are grown in a standard growth media such as S7 medium and are

separated from the growth media by centrifugation. The proteins in the growth media may be concentrated either by membrane filtration techniques or ammonium sulfate precipitation or both, but most preferably by 70% ammonium sulfate precipitation. The concentrated proteins are reconstituted in an appropriate. buffer compatible with binding to an iminobiotin affinity resin. The buffer for the reconstitution of the protein and the equilibration of the iminobiotin affinity resin is preferably about pH 8.5 to pH 11.0, but most

preferably about pH 11.0. The concentrated protein fraction is loaded onto the iminobiotin affinity resin where the streptavidin is bound. The column is washed with equilibration buffer and the streptavidin is eluted with an ammonium acetate buffer, most preferably 50mM ammonium acetate at about pH 4.0. Streptavidin

fractions are identified by U.V. detection and are further desalted and purified by standard gel filtration chromatography. Several gel filtration resins may be used but Sephacryl 200 is preferred. Final

determination of the presence of pure streptavidin may be made by several methods, including for example.

Western blot analysis.

The examples-illustrate the isolation of the streptavidin gene, and the engineering of suitable closing vectors containing ligated DNA encoding operable expression elements and streptavidin. The examples also describe the transformation of Bacillus host cells capable of expressing and secreting the streptavidin protein and the means whereby said streptavidin may be purified.

EXAMPLE 1

Construntion of a npr-sav Hvdrid Gene

Fusion in B. subtilis

The sav gene was isolated as a 2 kb BamHI fragment by standard methods from Streptomyces avidinii and cloned in plasmid pSK (Stratagene, 11099 North Torrey Pines Road, La Jolla, CA 92037). An EcoRV site was engineered at the start site of the mature sav gene by site-directed mutagenesis and the resulting plasmid containing the sav gene was designated pBE651. The restriction map of plasmid pBE651 and the position of

EcoRV site with reference to the mature coding region is illustrated in Figure la. Plasmid pBE83 (24) is an E. coli-B. subtilis phagemid vector containing the npr expression element and signal peptide fused to mature Lvs . B. subtilis strains containing pBE83 secrete Lvs. Colonies can be easily visualized on agar plates due to the formation of levan which is produced in the presence of sucrose contained in the agar. Plasmid pBE83 was digested with EcoRV and BamHI and ligated to the

EcoRV-BamHI digested pBE651. B. subtilis strain BE1510 was transformed with the ligated DNA and plated on LB agar + 5% sucrose + chloramphenicol (5 ug/ml). A total of approximately 800 transformants were obtained and 128 B. subtilis clones that did not produce levansucrase were identified and were screened by colony immunoassay using commercially purchased anti-streptavidin

antiserum. Four independent positive clones were obtained and designated as pBE659A, pBE659B, pBE659C and pBE659D. All further characterizations were carried out using plasmid pBE659A, which will be referred to as pBE659. This plasmid has been deposited in the

permanent culture collection of the American Type

Culture Collection, Rockville, MD, 20852 and has been designated accession number ATCC 68977. The deposit was made in accord with the Budapest Treaty Requirements for purposes of patent procedure with access provisions as set forth under 37 C.F.R. 1.14 and 35 U.S.C. 122.

Construction apr-sav. npr-sav. bar-sav and lys-sav Gene Fusions in E. coli

The following series of sav gene fusions were also constructed in E. coli and were subsequently transferred into B. subtilis. Plasmids pBE30, pBE90, pBE91 and pBE597 all contain the mature sequence of phoA fused to apr, npr, bar and lvs expression elements, respectively. These are shuttle phagemids containing origins of replication, of pBR322, pUB110 and M13K07 (24).

E. coli strains containing pBE30, pBE90, pBE91 and pBE597 secrete PhoA in E. coli . PhoA reacts with 5 bromo-4-chloro-3-indolyl phosphate (Sigma, St. Louis, MO) to provide a blue color on the indicator plates

(23). Plasmids pBE30, pBE90, pBE91, pBE597 and pBE651 were digested with EcoRV and Pst 1 and separated on 1-2% low melting agarose. The large fragment from pBE30, pBE90, pBE91, ρBE597 and the small fragment from pBE651 were cut and purified using Geneclean (P.O. Box 2284,

La Jolla, CA 92038). The small sav fragment was ligated to each of the large fragments and E. coli strain XLl (24). was transformed. The white transformants were screened by colony immunoassay using anti-sav antiserum (18). The plasmids were isolated, verified by methods well known to one skilled in the art and designated as pBE660(Apr-Sav), pBE661 (Npr-Sav), pBE662 (Bar-Sav) and pBE663 (Lvs-Sav) . B. subtilis strain BE1500 was

transformed with pBE660, pBE661, pBE662 and pBE663 and Kan^r transformants were obtained. Colony immunoassay revealed that all transformants secreted Sav. (Western blot analysis revealed that strains containing pBE660, pBE661, pBE662, and pBE663 secreted streptavidin into the growth medium.)

EXAMPLE 2

Expression of Sav i n B . subtilis

B. subtilis strain containing pBE659A(npr-sav) and pBE20 (vector) (24), was grown in Medium B +

chloramphenicol (5 ug/ml) and samples of the medium (1 ml) were withdrawn at 4, 6, 8, 10, 12 and 24 hours after the start of the culture (20). Samples were centrifuged in the presence of the 2mM phenyl

methylsulfonyl fluoride (PMSF) for 5 min and the supernatant was respun and processed for Western analysis. Samples from pBE659 (npr-sav) vector and commercially purchased streptavidin were separated on a 10 to 20% SDS-PAGE gel (Daiichi min gel, Integrated Separation Systems, MA 01136) followed by protein electroblotting onto a nitrocellulose filter and analyzed using anti-streptavidin antibody (Sigma,

St. Louis, MO). Western blot analysis demonstrated that a protein band corresponding to mature-Sav (npr-sav) was present in pBE659 (npr-sav) at 4 hours which increased in intensity with time and was present even after 24 hours (Figure 3). This band was absent in the strain that contained only the pBE20 vector. Thus, it is

demonstrated that B. subtilis can secrete soluble Sav into the growth medium. It was noted that in this instance extracellular streptavidin accumulated to approximately 20 to 30 mg/liter after about 12 hours.

EXAMPLE 3

Purification of Streptavidin from the

B . subtilis Culture Supernatant

The method of purification of streptavidin was based on the published methods described in references (25), (26), and (27). B. subtilis strain BE1510

containing plasmid pBE659 was grown in 250 ml of the following medium: 0.6% Casaminoacids in 1X Castenholz medium + 1% glycerol + 0.01% yeast extract + 25 mM potassium phosphate buffer pH 7.0, 50 ug per ml of tryptophan, methionine and lysine and Cm (5 ug/ml) for 8 hours at 37°C [10X Castenholz basal stock contains per liter of distilled water nitriloacetic acid lg;

CaSo₄.2H₂O, 0.6 g; MgSO₄.7H₂O, 1.0; NaCl, 0.8 g; KNO₃, 1.03 g; NaNO₃, 6.89 g; Na₂HPO₄, 1.11 g; FeCl₃ solution 0.28 g/liter, 10 ml; Nitsch's Trace elements 10 ml;

(Nitsch's Trace element Solution per liter of distilled water H₂SO₄, 0.5 ml; MnSo₄.H₂O, 2.2 g; ZnSo₄7H₂O, 0.5 g; H₃BO₃, 0.5 g; CuSO₄, 0.016 g; Na₂MoO₄2H₂O, 0.025 g and CoCl₂.6H₂O, 0.046 g)].

The pH of the medium was checked and maintained around pH 7.0 by the addition of sodium hydroxide. The bacteria were harvested after 8 hours and the growth medium was separated from the bacteria by centrifugation at 6,000 g for 20 min at 4°C in the presence of protease inhibitor (2 mM phenyl methyl sulfonyl fluoride).

Ammonium sulfate was added to 70% to the supernatant and left stirring at 4°C overnight. The ammonium sulfate precipitate containing the partially purified

streptavidin was collected by centrifugation at 6,000 g for 30 min and dissolved in 7 ml of 0.05M sodium

bicarbonate buffer pH 11 + 0.5M NaCl and loaded onto a iminobiotin agarose column (5 ml). Iminobiotin agarose (Sigma) was prepared prior to sample loading by washing with 0.05M sodium bicarbonate buffer pH 11 + 0.5 M NaCl. After sample loading, the column was washed with 10 ml of 0.05 M sodium bicarbonate buffer pH 11 + 0.5 M NaCl and fractions were collected. The column was then eluted with 4 ml of 0.05 M ammonium acetate elution buffer pH 4.0 and fractions were collected. Absorbance at 280 nm was measured for the various fractions as shown in Figure 4. The peak fraction after the pH shift on the iminobiotin column was subjected to gel

filtration on a Sephacryl 200 column. The elution profile was consistent with an apparent molecular weight of approximately 60 kd suggesting that the tetrameric form of streptavidin had been purified. Samples from a few fractions as indicated in Figure 4 were analyzed by

Western blot analysis. The results obtained in the Western blot were consistent with the properties of a tetrameric Sav, i.e., elution after shift in pH from iminobiotin column and a molecular weight of

approximately 60 kd.

B. subtilis strain BE1510 containing pBE659 was grown in synthetic S7 medium and Sav was isolated using the above method or a batch method in which the

iminobiotin agarose was added to the ammonium sulfate fraction followed by loading onto a column. Thus, Sav could be isolated from different media using methods that could be scaled up for downstream processing.

EXAMPLE \

Role of N-termjnal Residues

1 to 14 in the Secretion of Streptavidin Construction of a gene fusion consisting of the npr signal peptide coding region fused to the 15th codon of mature streptavidin is described. The following steps were involved in the gene fusion construction:

a) engineering the B. amyloliquefaciens npr secretion vector; b) engineering the sav mature sequence and c) fusion of the modified sav sequence to the modified npr vector.

(a) Engineering the npr Secretion Vector

The promoter and signal peptide coding region of npr gene was amplified from plasmid pBE80 (24) using primers VN16 5'-ATGCATGGTACCGATCTAACATTTTCCCC-3' (SEQ ID NO:1) and VN47 5'-GACGTATATGATATCCGCGCTAGCACCCGGCAGACTGAT-3'

(SEQ ID NO:2) using the PCR method according to the manufacturer's instructions (Perkin Elmer Cetus , 761 Main Ave., Norwalk, CT 06859, Gene Amp PCR kit). The amplified fragment was treated with Klenow fragment to fill in any ragged ends and digested with Kpn and EcoRV, and was then purified using Geneclean kit (P.O. Box

2284, La Jolla, CA 92038). Plasmid ρBE592 was the source of the vector containing E. coli alkaline

phosphatase and is similar to plasmid pBE597 (24) except that it contains a seven amino acid deletion in the signal peptide. E, coli alkaline phosphatase. activity is indicative of export of the protein and colonies that secrete phosphatase appear blue on indicator plates (LB agar + 5 bromo-4-chloro-3-indolyl phosphate) (23).

E. coli strain containing plasmid pBE592 is white on LB agar + 5 bromo-4-chloro-3-indolyl phosphate because phosphatase is not secreted. The PCR amplified pBE80 fragment (Kpn-EcoRV) was ligated to pBE592 digested with Kpn-EcoRV and E. coll was transformed with the ligated DNA and plated on a LB plate + 100 ug/ml ampicillln + 50 ug/ml 5 bromo-4-chloro-3-indolyl phosphate (Sigma). Blue colonies were isolated, verified by restriction analysis, and one such plasmid was designated by pBE93.

(b) Engineering of sav Gene

Single stranded DNA was isolated from pBE651 and site directed mutagenesis was performed using two oligonucleotides VN44 and VN51. Oligonucleotide VN44 (GTC TCG GCC GCC GAG GCT AGC GCC GCC GGC ATC ACC GGD) (SEQ ID NO: 3) codes for a Nhe 1 site which precedes codon 15 of Sav. VN51 (GAC ACC TTC ACC AAG GTG TAG GT C GAC. AAG CCG TCC GCC) (SEQ ID NO: 4) codes for a

translational terminator and a Sal 1 site downstream of codon 133 of mature sav. The transformants were

screened primarily for the presence of Nhe 1 site followed by screening for the Sal 1 site. pBE670 contained the newly engineered Nhe 1 site; however, Sal I digestion resulted in a partial digestion

suggesting that it had both the parent (without a Sal site) and the mutant plasmids in the cell.

(c) Fusion of the Modified sav Sequence to the Modified npr Veotor

Plasmid pBE93 was digested with Nhe and Pst and ligated to Nhe 1-Pst digested pBE670, and E. coli was then transformed and screened for streptavidin

production by colony immunoassay. One of the positive clones was designated as plasmid pBE673. Restriction analysis of pBE673 revealed that it did not contain the Sal site at the 3' end and thus it encoded for a Sav protein consisting of residues 15 to 159. B. subtilis BE1500 was transformed with pBE673 and Kan R

transformants were screened by colony immunoassay for streptavidin production. B. subtilis strain

BE1500(pBE673) and BE1510(pBE659) were grown in medium A + kanamycin (10 ug/ml) or chloramphenicol (5 ug/ml). The extracellular streptavidin was analyzed by Western blot analysis (Figure 5b). The mobility of streptavidin produced by pBE673 was faster than that of pBE659 due to the deletion of residues 1 to 14 of mature streptavidin. B. subtilis strain containing pBE673 was able to secrete streptavidin efficiently into the growth medium

suggesting that the hybrid fusion junction of the npr signal peptide fused to the 15th residue of streptavidin was efficiently recognized by B. subtilis.

The DNA sequence across the fusion junction in pBE673 is shown below. npr-sav(15-159)

Sequence at the N-terminus of sav

Signal (npr) Nhe I +15 (sav)

ATC AGT CTG CCG GGT GCT AGC GCC GCC GGC ATC

(SEQ ID NO:5)

EXAMPLE 5

Vectors to Make C-terminal Fusions This example describes the construction of a set of vectors that can be used to make a variety of C-terminal fusions. The rationale for the construction of these vectors is at least two-fold. Firstly, the role of the C-terminal tail in the oligomerization of streptavidin in the growth medium can be studied. Secondly, one can identify the most stable bifunctional molecule that can maintain both the biotin binding feature of the Sav and contain an additional enzymatic activity such as levansucrase, alkaline phosphatase, β-lactamase, protein A and luciferase. Fusion proteins are normally rapidly clipped at the fusion junction in the

B. subtilis growth medium and thus by creating different fusion junctions, the most stable protein can be

identified.

The restriction enzyme site that was engineered was Msc 1 because it generates a blunt end and is unique in the vectors. Msc 1 site was created by a six base

(TGG CCA) insertion between mature-sav codons 132 and 133, 138 and 139, 145 and 146 and 159 and terminator codon by site-directed mutagenesis. The newly created Msc 1 site can be used to create translation fusions to any heterologous protein.

VN66 to create Msc 1 site at between codons 133 and 134. ACC TTC ACC AAG GTG TGG CCA AAG CCG TCC GCC

(SEQ ID NO: 6) VN67 to create Msc 1 site between codons 138 and 139. CCG TCC GCC GCC TGG CCA TCC ATC GAC GCG GCG

(SEQ ID NO:7)

VN68 to create Msc 1 site between codons 145 and 146. GAC GCG GCG AAA AAG TGG CCA GCC GGC GTC AAC AAC

(SEQ ID NO:8) VN62 to create Msc 1 site at the end of sav(159).

GAC GCC GTT CAG CAG TGG CCA TAG TCG CGT CCC GGC

(SEQ ID NO: 9)

Plasmids with the Msc 1 site were identified by

restriction analysis and designated as pBE626 (VN62), PBE627 (VN66), pBE628 (VN67), and pBE629 (VN68), respectively. E. coli strains containing pBE626, pBE627, pBE628, and pBE629 were positive for

streptavidin production as determined by colony

immunoassay. These vectors can be used to fuse any reporter protein (levansucrase, β-lactamase, PhoA,protein A or luciferase). Thus, the most stable protein that has both the biotin binding activity and the reporter protein can be obtained. Schematic

representation of the relevant hybrid fusions are shown in Figure 6.

EXAMPLE 6

Secretion of Streptavidin as a Fusion Protein

This example describes the construction of a hybrid fusion protein containing streptavidin and levansucrase using a different signal peptide. Single stranded DNA from plasmid pBE651 (Figure 1a) was used to create an EcoRV site between codons 159 and the translational terminator of sav resulting in plasmid pBE653. Thus the sav gene can be isolated as an EcoRV fragment from plasmid pBE653. Plasmid pBE311 codes for levansucrase and contains an EcoRV site between the second and third codon of mature levansucrase (24). Plasmid pBE311 was digested with EcoRV and ligated to the EcoRV digested pBE653. In order to identify clones coding for the fusion protein, B. subtilis transformants were screened by colony immunoassay using anti-streptavidin antiserum. pBE653 contains sav gene but sav is not expressed.

pBE311 does not contain sav gene and therefore only the recombinants will express the fusion protein. Plasmids containing the sav fragment fused to levanscurase were designated as pBE655. B. subtilis strains containing pBE655 were grown and labelled with ³⁵S-methionine and the medium was analyzed by gel electrophoresis for the presence of streptavidin and levansucrase. Upon

analysis, proteins corresponding to the fusion protein (Sav-Lvs) and mature levansucrase (Lvs) were found to be present in the growth medium. B. subtilis strains pBE655 and pBE311 were grown in medium A (18), and the extracellular levansucrase activity was measured (20). Levansucrase activity in the two independent

transformants from pBE655A and pBE655B were found to be 39% and 86% higher, respectively, than those of pBE311.

REFERENCES

1. Weber, P. C., Ohlendorf, D. H., Wendolski, J. J., and Salemme, F. R., (1989) Science 243:85-88.

2. Argarana, C. E., Kuntz, I D., Birken, S., Axel R., and Cantor, C. R., (1986) Nucleic Acids

Research 14:1871-1882.

3. Meade, H., Garwin, J. PCT Patent Application WO86/02077.

4. Cantor C., Axel, R., and Argarana, C., (1989) U.S. Patent 4,839,293. Claims 1-25 withdrawn due to

Inteference Decision No. 102,477.

5. Sano, T., and Cantor, C. R., (1990) Proc.

Natl. Acad. Sci. 87:142-146.

6. Sano, T., and Cantor, C. R., (1991)

Biochemical and Biophysical Research Communications 176(2) :571-577.

7. Chang, S., (1987) U.S. Patent 4,711,843.

8. Nagarajan, V., Rhodes, C. S., and

Banner, C. D. B., (1989) U.S. Patent 4,801,537.

9. Kovacevic, S., Miller, J. R., and

Hsuiung, H. M., (1988) U.S. Patent 4,783,405.

10. Stephens et a1., U.S. Patent 4,769,327.

11. Biotechnology Handbook 2, Bacillus,

C. R. Hardwood, Ecf., Plenum Press, New York (1989).

12. Palva et al., (1982), Proc. Natl. Acad. Sci., USA 79, 5582-5586.

13. Palva et al., (1983), Gene 22, 229-235.

14. Dion et al., (1989), Biochime, 71, 747-7555.

15. Nagarajan, V., et al., U.S. Patent Application Serial No. 07/803,843 and PCT Application US91/05909.

16. Sambrook, J., Fritsch, E. F., Maniatis, T., (1989) Molecular Cloning Volumes 1, 2 and 3, Cold Spring

Harbor Laboratory Press, New York. 17. Goeddel, D. V., (1990) Methods in Enzymology

185; Gene Expression Technology Academic Press, New York.

18. Nagarajan, V., (1990) Methods in Enzymology 185 214-223.

19. Vasantha, N., Thompson, L. D., Rhodes, C, Banner, C, Nagle, J., and Filpula, D., (1984) J.

Bacteriol. 159 811-189.

20. Tang, Lr., Lenstra, R., Borchert, T. V., and Nagarajan, V., (1990) Gene 96:89-93.

21. Chang, C. N., Kuang, W. J., Chen, E. Y., (1986) Gene 44, 121-126.

22. Paddon, C. J. and Hartley, R. W., (1985) Gene 40:231-239.

23. Harwood, C. R., and Cutting, S. N., (1990) Molecular Biological Methods for Bacillus, John Wiley and Sons, New York.

24. Nagarajan, v., Albertson, H., Chen, N., and Ribbe, J., (1992) Modular Expression and Secretion Vectors for Bacillus subtilis, Gene, in press.

25. Hofmann, K., Wood, S. W., Brinton, C. C,MontibeHer, J. A., and Finn, F. M., (1980) Proc. Natl. Acad. Sci. 77(8)4666-4668.

26. Heney, G., and Orr, G. A., (1981) Analytical Biochemistry 114:92-96.

27. Cuatrecasas, P., and Wilchek, M., (1968), Biochemical and Biophysical Research Communications 33(2)235-239.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: NAGARAJAN, VASANTHA

(ii) TITLE OF INVENTION: PRODUCTION OF STREPTAVIDIN

FROM BACILLUS SUBTILLIS

(iii) NUMBER OF SEQUENCES: 14

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: DU PONT COMPANY

(B) STREET: BARLEY MILL PLAZA 36

(C) CITY: WILMINGTON

(D) STATE: DELAWARE

(E) COUNTRY : USA

(F) ZIP: 19880-0036

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentln Release #1.0, Version #1.25 (vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE:

(C) CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: GEIGER, KATHLEEN W

(C) REFERENCE/DOCKET NUMBER: CR 9029

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: 302-892-2118

(B) TELEFAX: 302-892-7949

(2) INFORMATION FOR SEQ ID NO:1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 29 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS : single (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:

ATGCATGGTA CCGATCTAAC ATTTTCCCC

(2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 39 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS : single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

GACGTATATG ATATCCGCGC TAGCACCCGG CAGACTGAT

(2) INFORMATION FOR SEQ ID NO:3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 39 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:

GTCTCGGCCG CCGAGGCTAG CGCCGCCGGC ATCACCGGC

(2) INFORMATION FOR SEQ ID NO: 4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 39 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:

GACACCTTCA CCAAGGTGTA GGTCGACAAG CCGTCCGCC

(2) INFORMATION FOR SEQ ID, NO: 5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 33 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:

ATCAGTCTGC CGGGTGCTAG CGCCGCCGGC ATC

(2) INFORMATION FOR SEQ ID NO: 6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 33 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:

ACCTTCACCA AGGTGTGGCC AAAGCCGTCC GCC

(2) INFORMATION FOR SEQ ID NO: 7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 33 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: CCGTCCGCCG CCTGGCCATC CATCGACGCG GCG

(2 ) INFORMATION FOR SEQ ID NO : 8 :

(i ) SEQUENCE CHARACTERISTICS :

(A) LENGTH: 36 base pairs

(B) TYPE : nucleic acid

(C) STRANDEDNESS : single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:

GACGCGGCGA AAAAGTGGCC AGCCGGCGTC AACAAC

(2) INFORMATION FOR SEQ ID NO: 9:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 36 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:

GACGCCGTTC AGCAGTGGCC ATAGTCGCGT CCCGGC

(2) INFORMATION FOR SEQ ID NO: 10:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii ) MOLECULE TYPE : DNA (genomic)

(xi ) SEQUENCE DESCRIPTION : SEQ ID NO : 10 :

GTTCAGGCCG CTGAGGATAT C (2) INFORMATION FOR SEQ ID NO: 11:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:

GCTTCGGCAG ATATCTCC

(2) INFORMATION FOR SEQ ID NO: 12:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:

GTTCAGGCCG CTGAGGATAT CTCC

(2) INFORMATION FOR SEQ ID NO: 13:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 552 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:

ATGCGCAAGA TCGTCGTTGC AGCCATCGCC GTTTCCCTGA CCACGGTCTC GATTACGGCC 60

AGCGCTTCGG CAGACCCCTC CAAGGACTCG AAGGCCCAGG TCTCGGCCGC CGAGGCCGGC 120 ATCACCGGCA CCTGGTACAA CCAGCTCGGC TCGACCTTCA TCGTGACCGC GGGCGCCGAC 180

GGCGCCCTGA CCGGAACCTA CGAGTCGGCC GTCGGCAACG CCGAGAGCCG CTACGTCCTG 240

ACCGGTCGTT ACGACAGCGC CCCGGCCACC GACGGCAGCG GCACCGCCCT CGGTTGGACG 300

GTGGCCTGGA AGAATAACTA CCGCAACGCC CACTCCGCGA CCACGTGGAG CGGCCAGTAC 360

GTCGGCGGCG CCGAGGCGAG GATCAACACC CAGTGGCTGC TGACCTCCGG CACCACCGAG 420

GCCAACGCCT GGAAGTCCAC GCTGGTCGGC CACGACACCT TCACCAAGGT GAAGCCGTCC 480

GCCGCCTCCA TCGACGCGGC GAAGAAGGCC GGCGTCAACA ACGGCAACCC GCTCGACGCC 540

GTTCAGCAGT AG 552

( 2 ) INFORMATION FOR SEQ ID NO : 14 :

( i ) SEQUENCE CHARACTERISTICS :

(A) LENGTH : 183 amino acids

(B) TYPE : amino acid

(C ) STRANDEDNESS : unknown

(D) TOPOLOGY : unknown

( ii ) MOLECULE TYPE : protein

(xi ) SEQUENCE DESCRIPTION : SEQ ID NO : 14 :

Met Arg Lys Ile Val Val Ala Ala Ile Ala Val Ser Leu Thr Thr Val 1 5 10 15

Ser Ile Thr Ala Ser Ala Ser Ala Asp Pro Ser Lys Asp Ser Lys Ala

20 25 30

Gln Val Ser Ala Ala Glu Ala Gly Ile Thr Gly Thr Trp Tyr Asn Gln

35 40 45 Leu Gly Ser Thr Phe Ile Val Thr Ala Gly Ala Asp Gly Ala Leu Thr

50 55 60

Gly Thr Tyr Glu Ser Ala Val Gly Asn Ala Glu Ser Arg Tyr Val Leu 65 70 75 80

Thr Gly Arg Tyr Asp Ser Ala Pro Ala Thr Asp Gly Ser Gly Thr Ala

85 90 95

Leu Gly Trp Thr Val Ala Trp Lys Asn Asn Tyr Arg Asn Ala His Ser

100 105 110

Ala Thr Thr Trp Ser Gly Gin Tyr Val Gly Gly Ala Glu Ala Arg Ile

115 120 125

Asn Thr Gln Trp Leu Leu Thr Ser Gly Thr Thr Glu Ala Asn Ala Trp 130 135 140

Lys Ser Thr Leu Val Gly His Asp Thr Phe Thr Lys Val Lys Pro Ser 145 150 155 160

Ala Ala Ser Ile Asp Ala Ala Lys Lys Ala Gly Val Asn Asn Gly Asn

165 170 175

Pro Leu Asp Ala Val Gln Gln

180

Claims

What is claimed is:

1. A method for producing tetrameric,

biologically active streptavidin by secretion from

Bacillus subtilis, comprising:

a. transforming Bacillus subtilis with a gene construct comprising a sequence encoding streptavidin operably linked to a sequence encoding a signal peptide and an expression element, wherein said sequence

encoding a signal peptide is isolated from DNA encoding an exoprotein of bacteria and said expression element is isolated from DNA encoding a gram positive bacterial protein;

b. growing the transformed Bacillus subtilis in suitable growth medium whereby streptavidin is secreted into the growth medium; and

c. purifying the streptavidin from the growth medium.

2. The method of Claim 1 wherein said sequence encoding a signal peptide and said expression element are each independently selected from the group

consisting of apr, npr, lvs and bar derived from

Bacillus amyloliquefaciens .

3. The method of Claim 1 wherein said sequence encoding a signal peptide and said expression element are derived from npr from Bacillus amyloliquefaciens .

4. The method of Claim 1 wherein said sequence encoding a signal peptide and said expression element are derived from lvs from Bacillus amyloliquefaciens.

5. The method of Claim 1 wherein said expression element is comprised of a promoter sequence and a ribosomal binding site sequence.

6. The method of Claim 5 wherein said promoter sequence is inducible.

7. A transformed Bacillus subtilis bacterium capable of secreting tetrameric, biologically active streptavidin having a gene construct comprising a sequence encoding streptavidin operably linked to a sequence encoding a signal peptide and an expression element, wherein said sequence encoding a signal peptide is isolated from DNA encoding an exoprotein of bacteria and said expression element is isolated from DNA encoding a gram positive bacterial protein.

8. A bacterium of Claim 7 wherein said sequence encoding a signal peptide and said expression element are each independently selected from the group

consisting of apr, npr, lvs and bar genes derived from Bacillus amyloliquefaciens.

9. A method for producing a fused gene product comprised of tetrameric, biologically active

streptavidin fused to a second desired protein, by secretion from Bacillus subtilis, comprising:

a. transforming Bacillus subtilis with a fused gene construct comprising a sequence encoding streptavidin fused to a sequence encoding a second desired protein, wherein said fused sequence is operably linked to a sequence encoding a signal peptide and an expression element, and wherein said sequence encoding a signal peptide is isolated from DNA encoding an

exoprotein of bacteria and said expression element is isolated from DNA encoding a gram positive bacterial protein;

b. growing the transformed Bacillus subtilis in suitable growth medium whereby the fused streptavidin and desired protein are secreted into the growth medium; and

c. purifying the fused streptavidin and desired protein from the growth medium.

10. The method of Claim 9 wherein said sequence encoding a signal peptide and said expression element are each independently selected from the group

consisting of apr, npr, lvs and bar genes from Bacillus amyloliquefaciens.

11. The method of Claim 9 wherein said fused gene product is comprised of tetrameric, biologically active streptavidin fused to the C-terminus of the second desired protein.

12. The method of Claim 9 wherein said fused gene product is comprised of tetrameric, biologically active streptavidin fused to the N-terminus of the second desired protein.

13. The method of Claim 9 wherein said second target protein is selected from the group consisting of levansucrase, alkaline phosphatase, β-lactamase, luciferase, and Protein A.

14. The method of Claim 12 wherein the fused gene product is comprised of streptavidin at the N-terminal region of the fused protein and levansucrase at the C-terminal region of the fused protein; and wherein said fused gene construct is comprised of the expression element and sequence encoding the signal peptide

isolated from the levansucrase gene of Bacillus

amyloliquefaciens, fused to a sequence encoding mature streptavidin, fused to a sequence encoding mature levansucrase from Bacillus amyloliquefaciens.

15. A fused gene product produced by the method of Claim 11 or 12.

16. A transformed Bacillus subtilis bacterium capable of secreting tetrameric, biologically active streptavidin fused to a second desired protein; said bacterium having a fused gene construct comprising a sequence encoding streptavidin fused to a sequence encoding a second desired protein; wherein said fused sequence is operably linked to a sequence encoding a signal peptide and an expression element, and wherein said sequence encoding a signal peptide is isolated from DNA encoding an exoprotein of bacteria and said

expression element is isolated from DNA encoding a gram positive bacterial protein.

17. The bacterium of Claim 15 wherein said fused protein is comprised of tetrameric, biologically active streptavidin fused to the C-terminus of the second desired protein.

18. The bacterium of Claim 15 wherein said fused protein is comprised of tetrameric, biologically active streptavidin fused to the N-terminus of the second desired protein.

19. The bacterium of Claim 16 wherein said sequence encoding a signal peptide and said expression element are each independently selected from the group consisting of apr, npr, lvs and bar genes from Bacillus amyloliquefaciens.

20. The bacterium of Claim 16 wherein said second target protein is selected from the group consisting of levansucrase, alkaline phosphatase, β-lactamase, luciferase, and Protein A.

21. Plasmids pBE659, ρBE660, pBE661, pBE662, pBE663, pBE673, and pBE655.