Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
  • C07K14/36—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Actinomyces; from Streptomyces (G)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
  • C12N15/75—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Bacillus
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
  • C07K2319/036—Fusion polypeptide containing a localisation/targetting motif targeting to the medium outside of the cell, e.g. type III secretion
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
  • C07K2319/22—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a Strep-tag

Definitions

• 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.
• 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.
• 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 naturally secretes relatively low amounts of streptavidin into the growth media.
• 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.
• streptavidin 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. coll . 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.
• a target protein of interest specifically human LDL receptor
• 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. coll (6) .
• Meade shows that streptavidin is present in the periplasm of E. coli .
• the work of Sano and Cantor has indicated that the streptavidin signal peptide does not function in E. coli .
• 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.
• 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 a ylase, protease, levansucrase and ⁇ -lactamases.
• Nagarajan 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.
• 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.
• 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 J3.
• 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 c 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.
• the instant invention provides a method for efficiently expressing and secreting soluble, active streptavidin from
• 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 stre_ptavidin 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
• 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.
• plasmids pBE659, pBE660, pBE ⁇ l, pBE662, pBE663, pBE673 and pBE655 are provided.
• Figure la describes the construction of a plas id pBE659 containing a hybrid gene fusion construct consisting of sav gene fused to npr express:, .n 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 SEC 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(Jbar-sav), and pBE663(I vs-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.
• 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 Nhel-Pstl 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) .
• FIG. 6 describes the streptavidin-heterologous gene (PhoA) fusions: Npr ss -Savi5--i 3 3-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.
• Meture protein is the final protein product without the signal peptide attached.
• Desired protein is any protein considered a valuable product to be obtained rom genetically engineered bacteria.
• 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 p ⁇ lypeptide 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 prot €in.
• “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.
• n npr n and “Npr” refer to neutral protease gene and protein, respectively.
• phoA and PhoA refer to E. coli alkaline phosphatase gene and protein, respectively.
• n sav and “Sav” refer to streptavidin gene and protein, respectively.
• streptavidin refers to the protein comprising amino acid residues 1-159 of a 2 kb Ba HI fragment isolated from spreptomyces avidinii as described in Example 1 and Figure lb, or any sequential subset of amino acid residues thereof, or mutations or derivatives thereof, which retain the ability to bind biotin.
• biotin refers to biotin, biotin derivatives, and biotin analogs capable of binding " streptavidin.
• Am- 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 FI intragenic region for the preparation of single stranded DNA.
• restriction endonuclease cleavage site and “restriction site” are used interchangeably.
• expression element refers herein to a
• B. subtilis including, for example, the promoter sequence and the ribosome binding site sequence.
• 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.
• gram positive bacterial proteins refers to those proteins which are known to be naturally synthesized by gram positive bacteria.
• biologically active refers to a soluble streptavidin protein molecule which is able to bind to biotin, biotin analogs, or derivatized biotin.
• 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).
• these sequences are accessible in the GenBank from Nucleic Acid Data base from Los Almos, California.
• GenBank Nucleic Acid Data base from Los Almos, California.
• avidinii and plasmids pBE322, pTZ18R, pSK, pC194, pUBHO and phage M13K07 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
• 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) .
• 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 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.
• 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 7 ) in E. coli and B. subtilis.
• the expression elements containing DNA sequences encoding the promoter and ribosome binding si-te 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.
• 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) .
• PCR polymerse chain reaction
• the method used in the present invention is site-directed mutagenesis which is most preferred, and is described in Mutagene manual (Biorad,
• 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.
• 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, metBlO, lys3, ⁇ -aprE66, ⁇ -npr82, ⁇ -sacB::ermC (2
• 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.
• the second DNA fragment is the gene encoding B. amyloliquefaciens levansucrase (Lvs) .
• a fused gene expresses a protein which consists of streptavidin at the N-terminal region of the fused : tein and levansucrase at the C-terminal region of ⁇ .ne 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.
• a fused protein encoded by the fused gene of the present invention wherein a desired protein of interest is fused to streptavidin.
• the desired protein is B. amyloliquefaciens levansucrase.
• 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.
• 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.
• 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.
• subtilis strain BE1510 was transformed with the ligated DNA and plated on LB agar + 5% sucrose + chloramphenicol (5 ug/ l) . 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
• 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, pUBHO and M13K07 (24) .
• PhoA reacts with 5 bromo-4-chloro-3-indolyl phosphate (Sigma,-St. Louis, MO) to provide a blue color on the indicator plates
• 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, ⁇ BE597 and the small fragment from pBE651 were cut and purified using Geneclean (P.O. Box 2284,
• 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.)
• B. subtilis strain BE1510 containing plasmid pBE659 was grown in 250 ml of the following medium: 0.6% Gasaminoacids in IX 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;
• 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.
• 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.
• Sav could be isolated from different media using methods that could be scaled up for downstream processing.
• Plasmid ⁇ BE592 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) .
• indicator plates LB agar + 5 bromo-4-chloro-3-indolyl phosphate
• 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.
• 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 &fi S ⁇ S. ShS. 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.
• 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.
• subtilis strain ⁇ SE1500(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.
• 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, ⁇ -lactamase, protein A and luciferase. Fusion proteins are normally rapidly clipped at the fusion junction in the
• B. subtilis growth medium 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.
• TGG CCA six base 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.
• 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, ⁇ -lactamase, PhoA,
• 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 la) was used to create an EcoRV site between codons 159 and the translational terminator of sav resulting in plasmid pBE653.
• 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 35 S-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.
• MOLECULE TYPE DNA (genomic)
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