# Patent

## Title
Expression systems and associated methods

## Abstract

A method of producing synthetic spider silk, including: transforming Escherichia coli with an expression vector; fermenting the transformed E. coli in a culture medium; inducing spider silk protein expression in the cultured E. coli; extracting the spider silk; and purifying the spider silk. Related vectors and genetically modified cells are also disclosed.

## Background
RELATED APPLICATIONS
This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Patent Application No. 62/061,574, filed Oct. 8, 2014, and titled “EXPRESSION SYSTEMS AND ASSOCIATED METHODS,” which is incorporated, in its entirety, by this reference.
The following applications and patents are related and are hereby incorporated by reference in their entirety: U.S. Provisional Application Nos. 61/917,259 and 61/977,552; U.S. application Ser. Nos. 14/042,183; 14/459,244; and U.S. Pat. Nos. 7,521,228; 7,723,109; 7,288,391; 7,157,615; 5,989,894; 5,728,810; 5,733,771; 5,756,677; and 5,994,099.


GOVERNMENT SPONSORED RESEARCH
This invention was made, at least in part, with government support under contract number W911NF-14-1-0267, awarded by the Department of Defense, and contract number IIP-1318194, awarded by the National Science Foundation. The government has certain rights in the invention.


TECHNICAL FIELD
The present disclosure relates to expression systems and associated methods for enhancing the production of recombinant proteins. More specifically, it relates to spider silk protein expression system and methods of the same.
BACKGROUND
Spider silk is a natural protein fiber produced by the spiders. Despite being a biopolymer, spider silk's tensile strength is comparable to that of high-grade alloy steel, but it has only about a sixth of the density of steel. Spider silk has been used by humans for many, many years. The ancient Greeks used it to stop wounds from bleeding. Optical targeting devices and fishnets are more recent applications of spider silks. The current study of spider silk opens the potential use of spider silks in strong and biodegradable materials such as wear-resistant lightweight clothing, rust-free panels on cars, and some biomedical devices.
Because spiders are hard to farm like silkworms in high densities because of their carnivorous nature, it is difficult to produce large amount of spider silks from farm-raised spiders. Genetic engineering is an alternative approach to produce large quantities of spider silk for commercial applications. The relevant genes of spiders have been cloned and inserted into several different organisms, such as E. coli, alfalfa, goat and silkworm, to make spider silks by different groups. Producing large-scale truly spider-like silk, however, remains a big challenge due to the small protein size, low yield and low water solubility of bio-synthetic spider silk.
BRIEF SUMMARY
In one embodiment, what is disclosed is a method of producing a synthetic spider silk. The method includes transforming one or more bacterial cells with an expression vector system comprising a first antibiotic resistance gene, a spider silk protein-encoding open reading frame, and a transfer RNA gene, to generate transformed bacterial cells. The method also includes fermenting the transformed bacterial cells in a culture medium. The method includes inducing spider silk protein expression in the cultured E. coli with an inducer. The method includes purifying the synthetic spider silk protein.
In another embodiment, described herein is a DNA vector system for expressing spider silk proteins in bacteria. The vector system includes a first antibiotic resistance gene, a spider silk protein-encoding open reading frame, and a transfer RNA gene.
In one aspect, a method of producing a synthetic spider silk is disclosed. The method includes transforming one or more bacterial cells with an expression vector system comprising a first antibiotic resistance gene, a spider silk protein-encoding open reading frame, and a transfer RNA gene, to generate transformed bacterial cells; fermenting the transformed bacterial cells in a culture medium; inducing spider silk protein expression in the cultured E. coli with an inducer, and purifying the synthetic spider silk protein.
In one embodiment, the expression vector system consists of a single vector. In one embodiment, the expression vector system includes more than one type of vector, where the first antibiotic resistance gene and the spider silk protein-encoding open reading frame residing on a first vector, and the transfer RNA gene and a second resistance gene to a second antibiotic residing on a second vector.
In one embodiment, the spider silk protein-encoding open reading frame encodes at least one of flagelliform silk, MaSp 1, MaSp2, MiSp, or a combination thereof.
In one embodiment, the protein-encoding open reading frame includes sequence coding for a C-terminal tail.
In one embodiment, the step of inducing spider silk protein expression includes holding the fermentation temperature within a temperature range of from about 20° C. to about 25° C.
In one embodiment, the inducer is selected from the group consisting of isopropyl β-D-1-thiogalactopyranoside, lactose, maltose, rhamnose, and another sugar-based induction system.
In one embodiment, the inducer is a heat shock step. In one embodiment, the step of inducing spider silk protein expression includes simultaneously adding the inducer and an antibiotic to which the first antibiotic resistance gene provides resistance.
In one embodiment, the antibiotic is an aminoglycoside antibiotic. In one embodiment, the aminoglycoside antibiotic is selected from the group consisting of: a kanamycin, a neomycin, streptomycin, amikacin, tobramycin, dibekacin, gentamycin, sisomicin, and netilmicin. In one embodiment, the aminoglycoside antibiotic is kanamycin.
In one embodiment, the method includes monitoring a level of glucose in the culture medium and supplementing the culture medium with glucose and an antibiotic when the glucose level falls below a predetermined threshold amount.
In one embodiment, the method includes monitoring a pH of the culture medium and adjusting the pH of the culture medium with a nitrogen-containing base when the pH falls below a predetermined threshold amount. In one embodiment, the nitrogen-containing base is ammonium hydroxide.
In another aspect, a DNA vector system for expressing spider silk proteins in bacteria is disclosed. The system includes a first antibiotic resistance gene, a spider silk protein-encoding open reading frame, and a transfer RNA gene.
In some embodiments, the spider silk protein-encoding open reading frame encodes for at least one of flagelliform silk, MaSp 1, MaSp2, MiSp, or a combination thereof.
In some embodiments, the transfer RNA gene encodes for a glycine-bearing transfer RNA.
In some embodiments, all of (i) the resistance gene to the first antibiotic, (ii) the spider silk protein-encoding open reading frame, and (iii) a transfer RNA gene reside on a single DNA vector.
In some embodiments, the resistance gene to the first antibiotic and the spider silk protein-encoding open reading frame reside on a first vector, and the transfer RNA gene resides on a second vector with a resistance gene to a second antibiotic.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a vector map in accordance with an embodiment of the present disclosure;
FIG. 2 is a growth curve of E. coli BL21 transformed with a vector constructed in accordance with the principles of the present disclosure;
FIG. 3 is an image of an electrophoretic gel showing relative levels of expression between a normal pET19K expression vector and a vector system in accordance with the principles of the present disclosure;
FIG. 4 is an image of an electrophoretic gel showing relative levels of expression between induced cultures using a vector system in accordance with the principles of the present disclosure at different induction temperatures;
FIG. 5 is an image of an electrophoretic gel showing relative levels of expression between induced cultures using a vector system in accordance with the principles of the present disclosure illustrating that lactose can drive expression;
FIG. 6 is an image of an agarose gel illustrating the effect of providing an aminoglycoside compound to an induced culture on plasmid retention;
FIG. 7 is an analysis of the expression of protein and quantification of spider silk proteins produced thereby;
FIG. 8 is a nucleotide sequence corresponding to SEQ ID NO: 1 of the present disclosure;
FIG. 9 is a nucleotide sequence corresponding to SEQ ID NO: 2 of the present disclosure;
FIG. 10 is a nucleotide sequence corresponding to SEQ ID NO: 3 of the present disclosure;
FIG. 11 is a nucleotide sequence corresponding to SEQ ID NO: 4 of the present disclosure;
FIG. 12 is a nucleotide sequence corresponding to SEQ ID NO: 5 of the present disclosure;
FIG. 13 is nucleotide sequences corresponding to SEQ ID NO: 6 of the present disclosure;
FIG. 14 is a nucleotide sequence corresponding to SEQ ID NO: 7 of the present disclosure;
FIG. 15 is an amino acid sequence corresponding to SEQ ID NO: 8 of the present disclosure;
FIG. 16 is an amino acid sequence corresponding to SEQ ID NO: 9 of the present disclosure;
FIG. 17 is an amino acid sequence corresponding to SEQ ID NO: 10 of the present disclosure;
FIG. 18 is an amino acid sequence corresponding to SEQ ID NO: 11 of the present disclosure;
FIG. 19 is an amino acid sequence corresponding to SEQ ID NO: 12 of the present disclosure;
FIG. 20 is an amino acid sequence corresponding to SEQ ID NO: 13 of the present disclosure;
FIG. 21 is an amino acid sequence corresponding to SEQ ID NO: 14 of the present disclosure;
FIG. 22 is an amino acid sequence corresponding to SEQ ID NO: 15 of the present disclosure;
FIG. 23 is an amino acid sequence corresponding to SEQ ID NO: 16 of the present disclosure;
FIG. 24 is an amino acid sequence corresponding to SEQ ID NO: 17 of the present disclosure;
FIG. 25 is an amino acid sequence corresponding to SEQ ID NO: 18 of the present disclosure; and
FIG. 26 is an amino acid sequence corresponding to SEQ ID NO: 19 of the present disclosure.



DETAILED DESCRIPTION
The present disclosure relates to apparatuses, systems, and associated methods for the expression of spider silk proteins in E. coli. In the following description, numerous specific details are provided for a thorough understanding of specific preferred embodiments. Those skilled in the art, however, will recognize that embodiments can be practiced without one or more of the specific details, or with other methods, components, materials, etc. In some cases, well-known structures, materials, or operations are not shown or described in detail in order to avoid obscuring aspects of the preferred embodiments. Furthermore, the described features, structures, or characteristics may be combined in any suitable manner in a variety of alternative embodiments. Thus, the following more detailed description of the embodiments of the present invention, as illustrated in some aspects in the drawings, is not intended to limit the scope of the invention, but is merely representative of the various embodiments of the invention.
In this specification and the claims that follow, singular forms such as “a,” “an,” and “the” include plural forms unless the content clearly dictates otherwise. All ranges disclosed herein include, unless specifically indicated, all endpoints and intermediate values. In addition, “optional” or “optionally” refer, for example, to instances in which subsequently described circumstance may or may not occur, and include instances in which the circumstance occurs and instances in which the circumstance does not occur. The terms “one or more” and “at least one” refer, for example, to instances in which one of the subsequently described circumstances occurs, and to instances in which more than one of the subsequently described circumstances occurs.
The present disclosure covers methods, compositions, reagents, and kits for the expression of spider silk proteins. Particularly, described herein are DNA vectors and protein expression protocols for bio-synthetic production of spider silk protein by bacteria, in one embodiment E. coli. A plasmid vector system is also described that drives greatly increased expression of a spider silk protein gene to a level which exceeds other expression systems by unexpectedly high amounts, in one embodiment based on those from the golden orb weaving spider Nephila clavipes. Media conditions, fermentation parameters, and specific protein induction steps are disclosed herein. The result is a fermentation process which can express spider silk proteins in E. coli at a level at or above 0.5 g/L.
E. coli strains suitable for transformation by DNA vectors and expression of protein constructs in accordance with principles of the present invention may include BL21 StarTM (DE3) chemically competent E. coli (Life Technologies, Carlsbad, Calif., USA).
Expression vector systems can be generated by modification of a commercially available protein expression vector containing a bacterial origin of replication, such as a pET-family vector, particularly pET19b vector. Expression in pET family vectors is generally driven by the T7 promoter, but other promoters may be employed too. The expression vector may consist of a single vector, or may be made up of multiple DNA vectors, that are either transformed simultaneously into the E. coli cells, or transformed in stepwise fashion as needed.
In the one vector system, the vector may be a pET19b vector in which the ampicillin resistance gene has been replaced with an aminoglycoside resistance gene, specifically a kanamycin resistance gene, even more specifically a kanamycin resistance gene from a pET26b vector. As will be explained below, use of an aminoglycoside antibacterial compound provides a result with surprisingly high expression of spider silk proteins in fermentation expression systems.
In order to increase expression levels of recombinant spider silk proteins that are rich in glycine or proline, genes for expression of higher levels of transfer RNA (tRNA), which become charged with these amino acids, are incorporated into the vector system. These tRNAs can have anticodons to any combination of the glycine codons GGU, GGC, GGA, and GGG, and proline codons CCU, CCA, CCC, CCG. The glycine-bearing and/or proline-bearing tRNA genes may reside on the same DNA molecule as the reading frame for expression of the spider silk construct, or may be provided on a second vector. If provided on a second vector, the tRNA genes are co-resident with a second, different antibiotic resistance gene.
The vector system may also incorporate a gene that results in the expression of serine hydroxyl methyl transferase (SHMT), which converts the amino acid serine to glycine. The SHMT expression gene may reside on any vector of a vector system. A vector including SHMT and tRNA expression genes is termed pET-SX. In one embodiment, pET-SX provides an aminoglycoside resistance gene. In one embodiment, the vector system provides only the SHMT gene. In another embodiment, the vector system provides tRNA genes but not the SHMT gene. In another embodiment, the vector system provides both the SHMT gene and tRNA expression genes.
In one embodiment, a two-vector system may be employed, with the spider silk expression gene on a first vector, and tRNA expression genes on a second vector. Such an arrangement allows for transformation with smaller vectors, which increases ease of handling and transformation, and may be accompanied by an increase in protein expression. In one embodiment, the first vector may provide resistance to an aminoglycoside compound, including but not limited to a kanamycin, a neomycin, streptomycin, amikacin, tobramycin, dibekacin, gentamycin, sisomicin, and netilmicin, while the second vector has a resistance gene against another antibiotic, such as chloramphenicol.
Silk modules to be overexpressed are cloned into at least one vector of the vector system. In one embodiment, a 1.1 kbp monomer gene may be synthesized, for instance by GeneArt® (Life Technologies, Carlsbad, Calif.), to contain the consensus motif of naturally occurring highly repetitive spider silk-like sequences from the golden orb weaving spider Nephila clavipes. Since the protein sequences of the orb weaving spiders are highly conserved the technology described here will work for all of them. The complete monomer sequence (FIYS, SEQ ID NO: 12) includes the coding sequences for flagelliform silk-like putative elastic motif, GPGGSGPGGY (FlY) (SEQ ID NO: 37) combined with the major ampullate 2 silk-like putative strength motif, linker-polyalanine—GGPSGPGSA8 (S) (SEQ ID NO: 38). Multimers (FlYS)n were generated by recombinant DNA technology using a compatible but non-regenerable cloning strategy, that is, by cloning using overhangs which may or may not be generated by restriction digestion, in such a way that the restriction sites are destroyed after ligation. Cloning of the trimer (FIYS)3 ̃3,300 bp was confirmed by partially sequencing using vector specific primers. Other constructs that were cloned for expression in this way are MaSp 1, MaSp2, FlAS and MiSp. The (FIYS)3 construct cloned into pET-SX based on pET19 and having an aminoglycoside resistance gene (or kanamycin resistance gene) is referred to as pET19K-SX-FlYS3.
With regard to the sequences of constructs, a variety of different motifs are contemplated. In some cases, one silk motif, as in for example FlYS1, encoded by SEQ ID NO. 5, will be employed. Other constructs may include two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, or more silk motif repeats. Although natural spider silk proteins may be larger (for instance, in the 250-500 kilodalton range), smaller constructs may be employed as they are a more manageable size and may have better expression than wild type constructs.
Certain spider silk protein sequences contain not only the repeating motifs (such as FlY and FlYS described above) but also a non-repeating C-terminal tail. The C-terminal tail is encoded by SEQ ID NO: 1 and increases the size of a construct but may also increase ease of spinning silk fibers after the protein has been purified. It is envisioned that any spider silk sequence, including those described above, falls within the scope of this invention, with or without the presence of a C-terminal tail in the construct.
A variety of silk proteins can be used in the vectors described herein. Such proteins may include sequences conventionally known for silk proteins (see for example, U.S. Pat. No. 7,288,391, incorporated herein by reference in its entirety).
Constructs whose coding sequences may be incorporated into vectors of the present disclosure, and which may be expressed by a fermentation process as described herein, include those identified in U.S. patent application No. 61/707,571; Ser. No. 14/042,183; PCT/US2013/062722; 61/865,487; and 61/917,259 that are incorporated herein by reference in their entirety. Constructs suitable for inclusion in vectors and production by a fermentation protocol include but are not limited to: major ampullate, minor ampullate, flagelliform, tubuliform, aggregate, aciniform and pyriform proteins. These proteins may be any type of biofilament proteins such as those produced by a variety of arachnids including, for example, Nephila clavipes, Araneus ssp. and A. diadematus. Also suitable for use in the invention are proteins produced by insects such as Bombyx mori. Dragline silk produced by the major ampullate gland of Nephila clavipes occurs naturally as a mixture of at least two proteins, designated as MaSpI and MaSpII. Similarly, dragline silk produced by A. diadematus is also composed of a mixture of two proteins, designated ADF-3 and ADF-4.
The spider silk proteins may be monomeric proteins, fragments thereof, or dimers, trimers, tetramers or other multimers of a monomeric protein. The proteins are encoded by nucleic acids, which can be joined to a variety of expression control elements, including tissue-specific animal or plant promotors, enhancers, secretory signal sequences and terminators. These expression control sequences, in addition to being adaptable to the expression of a variety of gene products, afford a level of control over the timing and extent of production.
Spider silk proteins are designated according to the gland or organ of the spider in which they are produced. Spider silks known to exist include major ampullate (MaSp1, SEQ ID NO. 13, and MaSp2. SEQ ID NO: 14), minor ampullate (MiSp, SEQ ID NO: 15), flagelliform (Flag, SEQ ID NO: 17), tubuliform (SEQ ID NO: 16), aggregate, aciniform (SEQ ID NO: 19), and pyriform (SEQ ID NO: 18) spider silk proteins. Peptides including eight repeats of MaSp1 (SEQ ID NO: 10), MaSp2 (SEQ ID NO: 11), and MiSp (SEQ ID NO: 9), are artificial sequences which can also be used.
Spider silk proteins derived from each organ are generally distinguishable from those derived from other synthetic organs by virtue of their physical and chemical properties. For example, major ampullate silk, or dragline silk, is extremely tough. Minor ampullate silk, used in web construction, has high tensile strength. An orb-web's capture spiral, in part composed of flagelliform silk, is elastic and can triple in length before breaking. Tubuliform silk is used in the outer layers of egg-sacs, whereas aciniform silk is involved in wrapping prey and pyriform silk is laid down as the attachment disk.
Sequencing of spider silk proteins has revealed that these proteins are dominated by iterations of four simple amino acid motifs: (1) polyalanine (Alan); (2) alternating glycine and alanine (GlyAla)n; (3) GlyGlyXaa; and (4) GlyProGly(Xaa)n, where Xaa represents a small subset of amino acids, including Ala, Tyr, Leu and Gin (for example, in the case of the GlyProGlyXaaXaa motif, GlyProGlyGlnGln is the major form). Spider silk proteins may also contain spacers or linker regions comprising charged groups or other motifs, which separate the iterated peptide motifs into clusters or modules.
In some embodiments, suitable spider silk proteins that can be used include recombinantly produced MaSp1 (also known as MaSpI), and MaSp2 (also known as MaSpII) proteins, including repeats of each of these which contain 8 units thereof and are encoded by DNA of SEQ ID NO: 3 and SEQ ID NO: 4, respectively; minor ampullate spider silk proteins; flagelliform silks; and spider silk proteins described in any of U.S. Pat. Nos. 5,989,894; 5,728,810; 5,756,677; 5,733,771; 5,994,099; 7,057,023; and U.S. provisional patent application No. 60/315,529 (all of which are incorporated herein by reference).
In other embodiments, a vector system in accordance with the principles of the present invention may contain a sequence that encodes for silk peptides A-H as listed hereafter. A vector system is said to contain a sequence that encodes for a protein or peptide sequence when it contains DNA made up of a series of codons which, when transcribed and translated, will result in a polypeptide of that sequence. Any codon representing a particular amino acid may be used. The peptide sequences are listed in Table 1.








TABLE 1 





PEPTIDE
AMINO ACID SEQUENCE







A
(GPGGX)n; X = any of A, S, Q, or Y; 



n = 1 to 65 inclusive


 


B
(GGZ)m; Z = any of A, Q, Y, T, or V; 



n = 1 to 35 inclusive


 


C
Ah; h = 2-16 inclusive; S can substitute 



for A for up to 25% of the total sequence


 


D
(GB)j; B = any of A, S, Q, P, with A 



preferred; j = 1-10 inclusive


 


E
GPXGPGX; X = A or S


 


F
(QQSSVAQS)p; p = 1-60 inclusive 



(SEQ ID NO: 35)


 


G
(RPLPARRPLPAPLPAPRPIPAPLPRPVPI)a;  



a = 1-60 inclusive (SEQ ID NO: 36)


 


H
THEDLDITIDGADOPITISEELTISGAGGS 



(SEQ ID NO: 34)









For peptides A. B, and D, the amino acids at the position where substitution is to be made can all be the same amino acid for each repeat, or can vary independently from one another.
The sequences can be arranged in a large number of ways. Example sequences using the peptides of the table above include, but are not limited to, ABC, ABCD, ABCE, ABCDE, A, B, BC, BCD, BD, BDC, E, ABE, AB, F, G, H, and GF. As mentioned previously, use of any combination of DNA sequence which encodes for a peptide sequence as detailed above in a vector system falls within the scope of the present invention.
In other embodiments, synthetic sequences based on MaSp1 and MaSp2 can be encoded by a vector. These include the sequences of Table 2:








TABLE 2 







SEQ ID NO: 20
GGAGQGGYGGQGAGQGGYGGLGSQGAGRGGLGGQG



AGAAAAAAAA


 


SEQ ID NO: 21
SGPGQQGPGGYGPGQQGPGGYGPGQQGPSGPGSAA



AAAAAA


 


SEQ ID NO: 22
GGAGQGGYGGLGSQGAGRGGLGGQ


 


SEQ ID NO: 23
GPGGYGPGQQGPSGPGSAAAAGPSGPGSAAAA


 


SEQ ID NO: 24
GPGGYGPGQQGPGSQGPGSGGQQGPGGQGPYGPSAA



AAAAAA


 


SEQ ID NO: 25
PGGGGAGQGGYGGLGSQGAGRGGLGGQGAGAAAAAA



GPGGYGPGQQGPSGPGSAAAAAAAA









In other embodiments, synthetic sequences based on flagelliform and MaSp2 can be encoded by a vector. These include the sequences of Table 3:








TABLE 3







SEQ ID NO: 26
GPGGAGPGGAGPGGAGPGGA


 


SEQ ID NO: 27
GPGGAGPGGAGPGGAGPGGAGPSGPGSAAAAAAAA


 


SEQ ID NO: 28
GPGGYGPGGSGPGGYGPGGSGPSGPGSAAAAAAAA


 


SEQ ID NO: 29
GGAGGSGGAGGSGGVGGSGGT


 


SEQ ID NO: 30
GGAGGSGGAGGSGGVGGSGGTGPGGSGPGGYGPGG



SGPGGYGPGGSGPGGYGPGGSGPGGYGPGGSGPGG



YGPGGSGPGGYGPGGSGPGGYGPGGSGPGGY


 


SEQ ID NO: 31
GGAGGSGGAGGSGGVGGSGGTTIEDLDITIDGADG



PITISEELTISGAGGS


 


SEQ ID NO: 32
GGAGGSGGAGGSGGVGGSGEITGPGGSGPGGYGPG



GSGPGGYGPGGSGPGGYGPGGSGPGGYGPGGSGPG



GYGPGGSGPGGYGPGGSGPGGYGPGGSGPGGYTTI



EDLDITIDGADGPITISEELTISGAGGS


 


SEQ ID NO: 33
GGAGGSGGAGGSGGVGGSGGTGPGGSGPGGYGPG



GSGPGGYGPGGSGPGGYGPGGSGPGGYGPGGSGP



GGYGPGGSGPGGYGPGGSGPGGYGPGGSGPGGYT



TIEDLDITIDGADGPITISEELTISGAGGSGPGG



PAAAAA









In the cases of SEQ ID NO: 27 and SEQ ID NO: 28, the sequences GPGGAGPGGAGPGGAGPGGA (SEQ ID NO: 26) and GPGGYGPGGSGPGGYGPGGS (SEQ ID NO: 39), respectively, may occur once, twice, three times, or four times end-to-end.
The sequences of the spider silk proteins may have amino acid inserts or terminal additions, so long as the protein retains the desired physical characteristics. Likewise, some of the amino acid sequences may be deleted from the protein so long as the protein retains the desired physical characteristics. Amino acid substitutions may also be made in the sequences, so long as the protein possesses or retains the desired physical characteristics.
In one embodiment, protein production can be achieved using bioreactors. For instance, fed-batch culture growths can be conducted in New Brunswick Scientific (Endfield, Conn., USA) Bioflo 115 (3 L), Bioflo 310 (5 L) or Bioflo 610 (100 L) controller bioreactors with BioCommand software.
Culture medium for growth of bacterial culture inocula can be composed of, per liter: 2 g KH2PO4, 4 g K2HPO4.3H2O, 5 g (NH4)2HPO4, 5 g Yeast Extract, 5 g Hy Express System II (YE-HY ES II, SheffieldTM Bio-Science, Norwich, N.Y.), 25 g glucose, 0.5 g MgSO4.7H2O, 2.5 mg Thiamine and 5 ml trace metal solution A (1.25 mL 6N H2SO4, 0.5 g NaCl, 0.1 g ZnSO4.7H2O, 0.4 g MnCl2.4H2O, 0.48 g FeCl3.6H2O, 0.04 g CuSO4.5H2O, 0.058 g H3BO3, 0.05 g NaMoO4.2H2O in distilled water to a final volume of 100 mL). All culture media may contain kanamycin (50 or 100 μg/mL) and/or chloramphenicol (60 or 120 μg/mL.)
Seed medium can be prepared in a liter bottle containing, for example, 5 g Yeast Extract, 15 g Hy Express System II, 16 g glycerol, 25 g glucose, 3.3 g (NH4)2SO4, 6.8 g KH2PO4, 7.1 g Na2HPO4, 1.5 mL trace element B solution (0.3 mL 6N H2SO4, 0.25 g CuSO4.5H2, 2.4 g MsSO4, 0.30 g NaMoO4. 2H2O, 2.5 g Ni(NO3)2, 1.5 g ZnSO4 in 100 mL distilled water) and 1.5 mL trace element C solution (0.5 g NaCl, 0.475 g FeCl3.6H2O, 0.075 g CoCl2.6H2O, 0.050 g H3BO3, 0.29 g CaCl2.2H2O in 100 mL distilled water).
Glucose feeding solution may include, per liter: 500 g glucose, 200 ml 5% Hy Express System II (SheffieldTM Bio-Science, Norwich, N.Y.), 10 g MgSO4.7H2O, 40 mg Thiamine and 5 ml trace metal solution A (1.25 mL 6N H2SO4, 0.5 g NaCl, 0.1 g ZnSO4.7H2O, 0.4 g MnCl2.4H2O, 0.48 g FeCl3.6H2O, 0.04 g CuSO4.5H2O, 0.058 g H3BO3, 0.05 g NaMoO4.2H2O in distilled water to a final volume of 100 mL). In one embodiment, whenever the fermentation culture is supplemented with additional glucose feeding solution, an aminoglycoside compound, such as for example kanamycin at about 1 to about 300 micrograms per milliliter, more preferably about 25 to about 100 micrograms per milliliter, is added as well. The addition of this compound increases expression of protein.
Starter cultures may be grown from colonies in 2 mL of YE-HY ES II at 37° C. for 6 hours and later cultivated for another 6 hours on a rotary shaker at 220 rpm, until the OD600 is  ̃3-5 before addition to a fermenter. Fed-batch cultures can be carried out at 37° C. in the fermenters. The inoculum cultures are added with 5-10% (v/v) inoculation size. The culture media may contain kanamycin (50 or 100 μg/mL).
In some cases, two seed cultures may be used. For the first seed culture, BL21(DE3) bacterial colonies harboring pET19-SXT (SEQ ID NO: 7); pET19kT (SEQ ID NO: 6) and p4GPP; or pET19kTPP-4GPP may be inoculated into 10 ml of YE-Hy ES II media with, taking into account appropriate antibiotic resistances, about 100 micrograms per milliliter of kanamycin, about 60 micrograms per milliliter of chloramphenicol, or both, and cultured at about 37° C. for about 5 hours. In a second seed culture step, about 10 milliliters of the first seed culture are inoculated into about 100 ml of YE-Hy ES II media, and cultured at about 37° C. for about 5 hours, to a final OD600 about between about 2 and about 3.
Significantly, culture pH is monitored and can be controlled at 6.9 to 7.0 by the addition of 20% (vol/vol) ammonia solution. The use of an ammonia solution served to improve protein yields in the final purification by up to 10-fold. This represents an unexpectedly large increase of yield. Without wishing to be bound by any particular theory, adding a nitrogen source during fermentation increased the amount of recombinant spider silk constructs recovered.
The dissolved oxygen concentration is controlled at 45% of air saturation by automatically increasing the agitation speed up to 450 to 900 rpm (depending on the fermenters) and by changing the pure oxygen percentage. Levels of glucose are monitored (15-30 g/L) using diabetic glucose test strips throughout the run. In one embodiment, when the glucose level is below a predetermined threshold of for example 15 g/L, a computerized operator adjusts the exponential feed-line to supplement the bacteria. Such supplementation can be accompanied by the addition of an aminoglycoside compound, such as kanamycin at about 1 to about 300 micrograms per milliliter, more preferably about 25 to about 100 micrograms per milliliter.
Gene expression was induced at an optical density of OD600 ̃80 by reducing the temperature to 25° C. and adding an inducer, such as 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG). Other inducers include maltose, lactose, rhamnose, and other sugars. In some cases, the inducer can be a spike in heat in order to elicit a heat shock response. When the inducer is added, the culture may be supplemented with an aminoglycoside compound, particularly kanamycinin in a quantity of 200 g/mL. In some embodiments, the inducer and antibiotic may be added simultaneously. The term “simultaneously” or “simultaneous” as used herein means at or around the same time. Fermentation continued for an additional about 2 to about 16 hours, preferably about 4 to about 16 hours, before harvesting by centrifugation. Surprisingly, addition of the aminoglycoside compound increased protein yield greatly, in some cases up to 75%. As seen in FIG. 6, it is believed that the increase is realized owing to the maintenance of greater numbers of copies of the expression vectors of the vector system.
With the fermentation protocol disclosed herein, E. coli transformed with an expression vector as described can be grown to high cell density (OD600 of about 120, FIG. 2). E. coli with pET19K-SX-FLYS3 vector produced more spider silk protein than E. coli with pET19K-FLYS3, as shown in FIG. 3. Further, a two-vector (pET19kTPP/p4GPP) system shows increased expression of FLYS6 compared to a single-vector (pET-SXT) system (see Example 5 and FIG. 9.)
In the pET19K-FLYS3 expression trial, there was a leaky protein expression before IPTG induction, but after two hours induction, spider silk protein expression reached a much higher level. Induction at lower temperature (20-25° C.) produced more spider silk protein of a higher proportion of full-length protein than at higher temperature (32° C.) (FIG. 4). Lactose can be used to induce spider silk protein expression, but less spider silk protein is produced than with IPTG induction (FIG. 5). Addition of an aminoglycoside compound, such as kanamycin, at and after induction, assists in preventing loss of plasmid, and thus increasing expression, at high ODs (FIG. 6). After extraction and purification, at least 0.5 g/L pure spider silk protein from E. coli fermentation can be produced (FIG. 7).
The following examples are illustrative only and are not intended to limit the disclosure in any way.
EXAMPLES
Example 1
Vector system including two vectors. Two two-vector systems were developed for expression of spider silk proteins in bacteria. Each vector system made use of the vector p4GPP, which contains the gene cassettes glyT, proL, proM, and glyVXY. These cassettes provide additional tRNAs which become charged with glycine and proline. The first two-vector system also included pET19kT. pET19kT encodes a non-repetitive sequence of one hundred amino acids at the C-terminus of a protein expressed when its expression gene has been inserted at the multiple cloning site. The second two-vector system includes p4GPP and pET19kTPP. pET19kTPP was generated by replacing the serine and glycine residues after the 10× His-tag with two valine and six proline residues to make the His-tag more accessible.
Example 2
Vector system including a single vector. pET19-SXT from pET19k by incorporating the C-terminal, non-repetitive sequence and the gene that results in the expression of serine hydroxyl methyltransferase (SHMT) (which converts the amino acid serine to glycine) along with sequences that produce additional tRNAs for glycine and proline, as in EXAMPLE 1.
Example 3
Cloning of silk modules into vectors. The 1.1 kbp monomer gene was synthesized in pMA-RQ vector by GeneArt® (Life Technologies, Carlsbad, Calif.) to contain the consensus motif of naturally occurring highly repetitive spider silk-like sequences from the golden orb weaving spider Nephila clavipes. The complete monomer sequence (FlAS or FlYS) is comprised of the coding sequences for flagelliform, silk-like putative elastic motif, GPGGX1GPGGX2 (X1/X2=A/A for FlAS, X1/X2=Y/S for FIYS) combined with the major ampullate 2 silk-like putative strength motif, linker-polyalanine—GGPSGPGSA8 (SEQ ID NO: 38). Multimers of (FlAS)n or (FlYS)n were generated by recombinant DNA technology using a compatible but non-regenerable cloning strategy. The restriction sites used were 5′-XmaI and 3′-BspEI in combination with a unique restriction site (PvuI) on the pMA-RQ vector. The PvuI-XmaI and PvuI-BspEI fragments, each containing one copy of the cloned monomer sequence, were ligated together, thus, effectively regenerating a full plasmid while doubling the size of the monomer insert in the process. The regenerated plasmid containing the silk insert was cloned into bacteria and used as a template in the next cloning step. After repeated rounds of cloning, this strategy increased the size of the silk-like insert to the desired number of motif repeats. For FlAS or FlYS, the motif was repeated 2, 3, 4, and 8 times. Once the desired size of insert was achieved through the outlined steps above, the synthetic spider silk gene is released from the recombinant pMA-RQ vector by restriction digestion in 5′ with NdeI and in 3′ by BamHI. The purified insert as cloned in frame into the expression vector (pET19kT and pET19-SXT) at the NdeI/BamHI sites for expression of the protein. Cloning of the multimer (FLAS)n or (FlYS)n was confirmed by partially sequencing using vector specific primers. The same cloning strategy was also followed for MaSp 1, MaSp2 and MiSp, including MiSp8 which is encoded by SEQ ID NO: 2.
Example 4
Expression of spider silk constructs of different sizes in BL21(DE3) are shown in FIG. 8. FlYS4 and FlAS6 constructs were expressed in a pET19kT/4GPP two-vector system and a time course study was undertaken. Lane 1 of each depicted gel is a protein ladder with molecular weights indicated; lane 2 is a purified FLYS3 construct for reference; in lanes 3-10, odd numbered lanes are supernatant fractions and even numbered lanes are pellet fractions. Lanes 3-4 are uninduced cultures; lanes 5-6 are 1 hour after IPTG induction; lanes 7-8 are 3 hours after IPTG induction; lanes 9-10 are 4 hours after IPTG induction. Cultures were normalized to OD600=10 and run on an electrophoretic gel.
Example 5
Comparison of expression of FLYS6 construct in one- and two-vector systems. FIG. 9A depicts expression of FLYS6 in a two-vector (pET19kTPP/p4GPP) system, and FIG. 9B depicts expression of FLYS6 in a single-vector (pET-SXT) system. Lane 1 of each depicted gel is a protein ladder with molecular weights indicated; lane 2 is a purified FLYS3 construct for reference. Lanes 3 are uninduced cultures; lanes 4 are 1 hour after IPTG induction; lanes 5 are 2 hours after IPTG induction; lanes 6 are 3 hours after IPTG induction; lanes 7 are 4 hours after IPTG induction. Comparison of any of lanes 4-7 reveals increased expression of silk protein by the two-vector system relative to the single-vector system.
Example 6
Comparison of induction temperatures and protein expression levels. FIG. 10A shows induction by IPTG of FLAS4 in a two-vector system at 25° C.; FIG. 10B shows induction of the same construct in the same vector system by IPTG at 16° C. Lane 1 of each depicted gel is a protein ladder with molecular weights indicated; lane 2 is a purified FLYS3 construct for reference. Lanes 3 are uninduced cultures; lanes 4 are 1 hour after IPTG induction; lanes 5 are 2 hours after IPTG induction; lanes 6 are 3 hours after IPTG induction; lanes 7 are 4 hours after IPTG induction. Comparison of any of lanes 5-7 reveals increased expression of silk protein at the lower temperature relative to the 25 degree fermentation. Samples were normalized to OD600=about 10.
Example 7
Protein expression and estimated yields. A variety of spider silk constructs were expressed in single- and two-vector systems, and at different induction systems, and their expression systems analyzed by enzyme linked immunosorbent assay (ELISA). FLYS2 in single vector SXT induced at 25° C. and FLYS3 in double vector pET19kT/p4GPP induced at 25° C. yielded about 0.325 mg/ml; larger construct FLYS6 in single vector SXT induced at 16° C. yielded about 0.050 mg/ml protein, as did FLAS4 and FLAS6 in double vector pET19kT/p4GPP system, induced at 25° C. each. FLAS4 in double vector pET19kT/p4GPP yielded nearly 0.1 mg/ml when induction temperature was decreased to 16° C.
Expression of spider silk protein production is carried out by changing protein induction temperature and antibiotic (aminoglycoside, specifically kanamycin) concentration. The expression of spider silk protein is induced at 20°, 25°, 30°, 32° and 37° C. The role of kanamycin in the protein production was studied at 100 μg/mL, 200 μg/mL, 300 μg/mL and 400 μg/mL of kanamycin. In the study E. coli with pET19K-SX-FLYS3 was induced at high OD600. Samples were taken at 0 hour, 1 hour and 2 hours after induction. Samples taken at different time points were normalized to an OD600=5. Regarding FIG. 5, plasmids were extracted, purified and digested with restriction enzymes NdeI/BamHI to check presence of silk insert before (0 h) and after induction (1 h, 2 h).
It will be appreciated that various of the above-disclosed and other features and functions, or alternatives thereof, may be desirably combined into many other different systems or applications. Also, various presently unforeseen or unanticipated alternatives, modifications, variations or improvements therein may be subsequently made by those skilled in the art, and are also intended to be encompassed by the following claims.
In the following part of the present specification, numbered examples are listed which are directed to and which define advantageous embodiments. Said examples and embodiments belong to the present disclosure and description. The embodiments, examples, and features as listed, can separately or in groups, be combined in any manner to form embodiments belonging to the present disclosure.
Numbered examples: 1. A method of producing a synthetic spider silk, comprising: transforming one or more bacterial cells with an expression vector system comprising a first antibiotic resistance gene, a spider silk protein-encoding open reading frame, and a transfer RNA gene, to generate transformed bacterial cells; fermenting the transformed bacterial cells in a culture medium; inducing spider silk protein expression in the cultured E. coli with an inducer, and purifying the synthetic spider silk protein.
2. The method of example 1, wherein the expression vector system consists of a single vector.
3. The method of any one of examples 1-2, wherein the expression vector system comprises more than one type of vector, the first antibiotic resistance gene and the spider silk protein-encoding open reading frame residing on a first vector, and the transfer RNA gene and a second resistance gene to a second antibiotic residing on a second vector.
4. The method of any one of examples 1-3, wherein the spider silk protein-encoding open reading frame encodes at least one of flagelliform silk, MaSp 1, MaSp2, MiSp, or a combination thereof.
5. The method of any one of examples 1-4, wherein the protein-encoding open reading frame comprises sequence coding for a C-terminal tail.
6. The method of any one of examples 1-5, wherein inducing spider silk protein expression comprises holding the fermentation temperature within a temperature range of from about 20° C. to about 25° C.
7. The method of any one of examples 1-6, wherein the inducer is selected from the group consisting of isopropyl 3-D-1-thiogalactopyranoside, lactose, maltose, rhamnose, and another sugar-based induction system.
8. The method of any one of examples 1-6, wherein the inducer is a heat shock step.
9. The method of any one of examples 1-8, wherein inducing spider silk protein expression comprises simultaneously adding the inducer and an antibiotic to which the first antibiotic resistance gene provides resistance.
10. The method according to examples any one of claims 1-9 wherein the antibiotic is an aminoglycoside antibiotic.
11. The method of any one of examples 1-10, wherein the aminoglycoside antibiotic is selected from the group consisting of: a kanamycin, a neomycin, streptomycin, amikacin, tobramycin, dibekacin, gentamycin, sisomicin, and netilmicin.
12. The method of any one of examples 1-11, wherein the aminoglycoside antibiotic is kanamycin.
13. The method of any one of examples 1-12, further comprising monitoring a level of glucose in the culture medium, and supplementing the culture medium with glucose and an antibiotic when the glucose level falls below a predetermined threshold amount.
14. The method of any one of examples 1-13, further comprising monitoring a pH of the culture medium, and adjusting the pH of the culture medium with a nitrogen-containing base when the pH falls below a predetermined threshold amount.
15. The method of any one of examples 1-15, wherein the nitrogen-containing base is ammonium hydroxide.
16. A DNA vector system for expressing spider silk proteins in bacteria comprising: a first antibiotic resistance gene, a spider silk protein-encoding open reading frame, and a transfer RNA gene.
17. The vector system of example 16, wherein the spider silk protein-encoding open reading frame encodes for at least one of flagelliform silk, MaSp 1, MaSp2, MiSp, or a combination thereof.
18. The vector system of any one of examples 16-17, wherein the transfer RNA gene encodes for a glycine-bearing transfer RNA.
19. The vector system of any one of examples 16-18, wherein all of the resistance gene to the first antibiotic, the spider silk protein-encoding open reading frame, and a transfer RNA gene reside on a single DNA vector.
20. The vector system of any one of examples 16-19, wherein the resistance gene to the first antibiotic and the spider silk protein-encoding open reading frame reside on a first vector, and the transfer RNA gene resides on a second vector with a resistance gene to a second antibiotic.

## Claims

1. A method of producing a synthetic spider silk, comprising:
transforming one or more bacterial cells with an expression vector system comprising a first antibiotic resistance gene, a spider silk protein-encoding open reading frame, and a transfer RNA gene, to generate transformed bacterial cells;
fermenting the transformed bacterial cells in a culture medium;
inducing spider silk protein expression in the cultured E. coli with an inducer, wherein the inducing spider silk protein expression comprises holding the fermentation temperature within a temperature range of from about 20° C. to about 25° C.; and
purifying the synthetic spider silk protein.

2. The method of claim 1, wherein the expression vector system consists of a single vector.

3. The method of claim 1, wherein the expression vector system comprises more than one type of vector, the first antibiotic resistance gene and the spider silk protein-encoding open reading frame residing on a first vector, and the transfer RNA gene and a second resistance gene to a second antibiotic residing on a second vector.

4. The method of claim 1, wherein the spider silk protein-encoding open reading frame encodes at least one of flagelliform silk, MaSp 1, MaSp2, MiSp, or a combination thereof.

5. The method of claim 1, wherein the protein-encoding open reading frame comprises sequence coding for a C-terminal tail.

6. The method of claim 1, wherein the inducer is selected from the group consisting of isopropyl β-D-1-thiogalactopyranoside, lactose, maltose, and rhamnose.

7. The method of claim 1, wherein the inducer is a heat shock step.

8. The method of claim 1, wherein inducing spider silk protein expression comprises simultaneously adding the inducer and an antibiotic to which the first antibiotic resistance gene provides resistance.

9. The method of claim 1, wherein the antibiotic is an aminoglycoside antibiotic.

10. The method of claim 9, wherein the aminoglycoside antibiotic is selected from the group consisting of: a kanamycin, a neomycin, streptomycin, amikacin, tobramycin, dibekacin, gentamycin, sisomicin, and netilmicin.

11. The method of claim 9, wherein the aminoglycoside antibiotic is kanamycin.

12. The method of claim 1, further comprising monitoring a level of glucose in the culture medium, and supplementing the culture medium with glucose and an antibiotic when the glucose level falls below a predetermined threshold amount.

13. The method of claim 1, further comprising monitoring a pH of the culture medium, and adjusting the pH of the culture medium with a nitrogen-containing base when the pH falls below a predetermined threshold amount.

14. The method of claim 13, wherein the nitrogen-containing base is ammonium hydroxide.