Patent Description:
Serum albumin is a monomeric non-glycosylated 67kDa transport protein that is highly abundant in mammalian plasma (<NUM>-<NUM> gr/L). It folds into a canonical heart-shaped structure comprising three helical domains, with a conserved set of <NUM> disulfide bridges (<FIG>). Serum albumin is the main blood carrier for metabolites, hormones, drugs, and some cations. It has seven binding sites for fatty acids and three major binding sites for other small molecules. Crystallographic analyses reveal two major conformations for albumin (compact and myristate-bound) (<FIG>). Besides ligand binding, serum albumin has a role in regulation of plasma colloid oncotic pressure, and has some catalytic properties. (<NUM>-<NUM>).

In addition to ligand binding, serum albumin has a role in stabilizing the extracellular fluid volume.

In addition to their physiological importance, human and bovine serum albumins (HSA and BSA, respectively) have many biochemical and pharmacological applications. Already in <NUM>, they were used to minimize osmotic shock after bleeding in patients. Additional clinical uses include vaccine preparations and treatment of burn injuries, hemorrhagic shock, hypoproteinemia, and ascites resulting from liver cirrhosis. HSA and BSA are also used in a range of biochemical procedures, such as immunological (e.g. ELISA), radioimmunological and immunoenzyme assays, as a blotting reagent, and as a molecular weight standard. Albumin is also widely used in molecular biology to stabilize the reactants and to prevent their adhesion to surfaces (NEB site). Finally, HSA and BSA are widely used as cell culture medium supplements, and have an increasingly important role in the cultured meat industry. Other novel biological applications include nanodelivery of drugs, oxygen carrier, peptide fusion.

Most albumin used in research and applications is derived from animal plasma. Animal-sourced albumin, however, increases the risk of DNA and viral contamination and batch-to-batch variability. This and serious ethical problems have encouraged the development of non-animal recombinant sources of albumin. Recombinant BSA and HSA from yeast and rice are now used to replace plasma-derived albumin. These expression systems are laborious and expensive [<NPL>; <NPL>].

Bacterial expression systems are amenable to high-throughput screening and mass production. Bacterially-expressed albumin variants may also serve as superior starting points in the development of albumin mutants with desirable carrier properties for therapeutic purposes.

Bacterial expression of albumin is challenging, however, potentially due to its large size, multidomain organization and <NUM> disulfide bonds. Indeed, bacterial expression of HSA was attempted in several studies, and some success was achieved in obtaining HSA from inclusion bodies [<NPL>], and as a fusion to maltose-binding protein [<NPL>]. HSA variants that have certain properties due to specific amino acid substitutions are described in <CIT>.

To date there is no efficient bacterial expression system for albumin.

According to an aspect of some embodiments of the present invention there is provided an albumin protein variant comprising:.

wherein the albumin protein is soluble when expressed in bacteria and characterized by increased thermostability as compared to wild type human serum albumin (HSA) of SEQ ID NO: <NUM>.

According to some embodiments of the invention, the protein binds a small molecule albumin ligand in at least the same affinity as wild type HSA of SEQ ID NO: <NUM>.

According to some embodiments, the small molecule is warfarin or Ketoptofen.

According to some embodiments of the invention, the albumin protein is folded as HSA as evidenced by the ability to bind myristate.

According to some embodiments, the protein supports cell growth in culture.

According to some embodiments of the invention, the protein further comprises at least one mutation at albumin binding sites.

According to some embodiments of the invention, the at least one mutation is selected from the group consisting of K 136N, V216I, A254M, V344T, H440L, A449I, S470N A528F and V547I,.

According to some embodiments of the invention, the protein further comprises at least one mutation selected from the group consisting of Q33K, D38E, N44K, T52K, S58T, T76Q, E95D, N99H, V116E, T125K, A163K, A172E, E184A, A191E, D259K, K286R, E297D, M298K, A300E, S304P, G328A, M329R, L349I, T355D, A362K, A364E, F374E, D375E, P379K, P384T, Q397K, K402Y, V415M, S419P, P421D, V426L, S427T, A443E, M446L, P486H, I513L, K524M, T527K, K541E, D562E, K564P, K573S and A578K.

According to some embodiments of the invention, the protein is at least <NUM> % identical to SEQ ID NO: <NUM>.

According to some embodiments of the invention, the protein comprises the amino acid sequence set forth in SEQ ID NO: <NUM>, <NUM> or <NUM>.

According to some embodiments , the protein comprises a heterologous tag.

According to some embodiments , the protein is tagless.

According to some embodiments of the invention, the protein exhibits increased yield than the wild type protein (SEQ ID NO: <NUM>) when expressed in bacteria.

According to an aspect of some embodiments of the present invention there is provided an albumin protein comprising:.

wherein the albumin protein is soluble when expressed in bacteria and characterized by increased thermostability as compared to wild type bovine serum albumin (BSA) of SEQ ID NO: <NUM>.

According to some embodiments of the invention, the at least one mutation is selected from the group consisting of V240I, H241Y, A253M, A260V, V344L, V414M, V425I, K439L, M445L, P485H, A527F, V546I and V551T.

According to some embodiments of the invention, the protein further comprises at least one mutation selected from the group consisting of Q33K, E45D, T52K, A60P, E63S, A78E, S79E, E92S, S109N, D124K, A128E, N158K, G162K, T183A, L189KE226P, V228E, D258K, K285R, K294N, A296D, N300DA321D, S328R, A361K, T371R, D374E, H378K, L379H, N385E, R412K, S418P, P420D, T438Q, S442E, E443K, S426T, A500P, E503P, D517P, T526K, A559K, D561E, V569E, V576E, S577K and T580A.

According to some embodiments, the protein comprises a heterologous tag.

According to some embodiments, the protein is tagless.

According to some embodiments, the protein is immobilized to a solid support.

According to an aspect of some embodiments of the present invention there is provided a polynucleotide comprising a nucleic acid sequence encoding the protein as described herein.

According to an aspect of some embodiments of the present invention there is provided a cell comprising the polynucleotide and/or protein as described herein.

According to an aspect of some embodiments there is provided a composition comprising the protein as described herein and an active ingredient.

According to some embodiments, the active ingredient is a protein.

According to some embodiments, the active ingredient is a drug.

According to some embodiments, the active ingredient is attached to the protein.

According to some embodiments, the composition is shaped as a tube.

According to an aspect of some embodiments there is provided a method of producing albumin, the method comprising expressing in bacteria a nucleic acid sequence encoding the protein as described herein, thereby producing albumin.

According to some embodiments, the expressing is in an inducible manner.

According to some embodiments, the method further comprises isolating the protein from the bacteria or conditioned medium thereof.

According to an aspect of some embodiments there is provided a method of cell culturing, the method comprising culturing cells in the presence of an albumin protein as described herein.

According to some embodiments, the culturing is in serum-free medium or in the presence of serum up to <NUM> %.

According to some embodiments, the cells are eukaryotic cells and optionally mammalian cells.

According to some embodiments, the cells are hybridoma cells.

According to some embodiments, the cells are bovine cells, chicken cells, duck cells, fish cells or pig cells.

According to some embodiments, the cells are stem cells or progenitor cells.

According to some embodiments, the cells are differentiated cells.

According to some embodiments, the albumin protein is formulated with any of a fatty acid, a vitamin, a hormone or an ion.

According to an aspect of some embodiments there is provided a method of isolating an albumin binding molecule of interest, the method comprising contacting a sample which may comprise an albumin binding molecule with the albumin as described herein under conditions which allow complexation.

According to an aspect of some embodiments there is provided a method of facilitating a molecular biology reaction with a nucleic acid molecule, the method comprising contacting the albumin protein as described herein with the nucleic acid molecule to thereby facilitate the molecular biology reaction.

According to some embodiments, the reaction is restriction, amplification and/or sequencing.

According to an aspect of some embodiments there is provided a composition comprising a research reagent and the albumin protein as described herein.

According to some embodiments of the invention, the research reagent is selected from the group consisting of a buffer, an enzyme and a cell culture medium.

The present invention, in some embodiments thereof, relates to albumin protein variants, production thereof and uses of same.

Serum albumin is the most abundant protein in the blood serum of mammals and has essential carrier and physiological roles. Albumins are also used in research, in a wide variety of molecular and cellular experiments. Despite their importance, however, albumins are challenging for heterologous, i.e., recombinant, expression in microbial hosts. Therefore, albumins used in research and biotechnological applications are primarily derived from animal serum despite severe ethical and reproducibility concerns.

In order to overcome technological hurdles associated with recombinant production of albumin, and whilst conceiving and reducing embodiments of the invention to practice, the present inventors designed and synthesized several stable versions of human and bovine serum albumins. The most highly mutated version of human albumin, which is termed herein "Thermalbumin" (HSA3), is stable beyond the boiling point and, unusually for a large and complex protein, exhibits reversible temperature-dependent folding and unfolding. Design accuracy is verified by crystallographic analysis of a human albumin variant with <NUM> mutations (i.e., HSA <NUM>). This albumin variant exhibits fatty-acid binding properties indistinguishable from the wild type. The stable albumin designs may be used in making reliable, animal-free reagents for molecular and cell biology and enables mutagenesis, including in high-throughput format, to study and enhance albumin physiological properties.

As used herein "albumin" refers to serum albumin having the symbol "Serum_albumin". In human, the albumin is referred to herein as SEQ ID NO: <NUM>. In bovine it is referred to herein as SEQ ID NO: <NUM>.

As used herein "protein" is interchangeably used with "polypeptide".

According to teachings of the invention, alterations are made to the wild-type sequence to produce the albumin variants having at least a same function (e.g., protein/drug binding) as the wild type protein.

The term "variant", in the structural sense, means a polypeptide derived from a wild type albumin comprising an alteration, i.e., a substitution, insertion, and/or deletion, at one or more (several) positions.

A substitution means a replacement of an amino acid occupying a position with a different amino acid; a deletion means removal of an amino acid occupying a position; and an insertion means adding <NUM>-<NUM> amino acids adjacent to an amino acid occupying a position. The altered polypeptide (variant) can be obtained through human intervention by modification of the polynucleotide sequence encoding the wild type protein.

According to a specific embodiment, the alteration is a substitution.

According to a specific embodiment, the alteration comprises a plurality of substitutions e.g., <NUM>-<NUM> amino acid coordinates, e.g., <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>.

As used herein "soluble" or "solubility" refers to accumulation of the protein variant as individual molecules (monomers or dimers) in the cytosol of the bacterial cells or upon secretion and not as aggregates such as in the case of inclusion bodies. Solunility is typically tested by Coomassie staining.

As used herein "thermostability" refers to preservation of the wild type structure and chemical properties of albumin under extreme temperatures e.g., <NUM>, <NUM>, <NUM> , <NUM>, <NUM>. <NUM> or even <NUM> or above, as determined by the melting temperature. Thermostability is determined using methods which are well known in the art and include but are not limited to nanoscale differential scanning fluorimetry (nanoDSF) or differential scanning calorimetry (DCS).

According to an aspect of the invention there is provided an albumin protein variant (HSA variant) comprising:.

According to another aspect, there is provided an albumin protein variant (BSA variant) comprising:.

Thus, the protein variant (HSA) comprises an amino acid sequence which is at least <NUM> %, at least <NUM> %, at least <NUM> %, at least <NUM> %, at least <NUM> %, at least <NUM> %, at least <NUM> %, at least <NUM> %, at least <NUM> %, at least <NUM> %, at least <NUM> %, at least <NUM> %, at least <NUM> %, at least <NUM> %, at least <NUM> %, at least <NUM> %, at least <NUM> %, at least <NUM> %, at least <NUM> %, at least <NUM> % or <NUM> % identical to SEQ ID NO: <NUM>, as determined using the BestFit software of the Wisconsin sequence analysis package, utilizing the Smith and Waterman algorithm, where gap weight equals <NUM>, length weight equals <NUM>, average match equals <NUM> and average mismatch equals -<NUM>.

According to another embodiment, the protein variant comprises an amino acid sequence which is at least <NUM> %, at least <NUM> %, at least <NUM> %, at least <NUM> %, at least <NUM> %, at least <NUM> %, at least <NUM> %, at least <NUM> %, at least <NUM> %, at least <NUM> %, at least <NUM> %, at least <NUM> %, at least <NUM> %, at least <NUM> %, at least <NUM> % or <NUM> % identical to SEQ ID NO: <NUM>, as determined using the BestFit software of the Wisconsin sequence analysis package, utilizing the Smith and Waterman algorithm, where gap weight equals <NUM>, length weight equals <NUM>, average match equals <NUM> and average mismatch equals -<NUM>. Of note, the percentage of homology refers to global homology over the albumin protein sequence.

According to a specific embodiment, the protein is at least <NUM> % identical to SEQ ID NO: <NUM>.

According to a specific embodiment, the protein comprises the amino acid sequence set forth in SEQ ID NO: <NUM>, <NUM> or <NUM>.

According to alternative embodiments, the protein variant (BSA) comprises an amino acid sequence which is at least <NUM> %, at least <NUM> %, at least <NUM> %, at least <NUM> %, at least <NUM> %, at least <NUM> %, at least <NUM> %, at least <NUM> %, at least <NUM> %, at least <NUM> %, at least <NUM> %, at least <NUM> %, at least <NUM> %, at least <NUM> %, at least <NUM> %, at least <NUM> %, at least <NUM> %, at least <NUM> %, at least <NUM> %, at least <NUM> % or <NUM> % identical to SEQ ID NO: <NUM>, as determined using the BestFit software of the Wisconsin sequence analysis package, utilizing the Smith and Waterman algorithm, where gap weight equals <NUM>, length weight equals <NUM>, average match equals <NUM> and average mismatch equals -<NUM>.

According to another embodiment, the protein variant (BSA) comprises an amino acid sequence which is at least <NUM> %, at least <NUM> %, at least <NUM> %, at least <NUM> %, at least <NUM> %, at least <NUM> %, at least <NUM> %, at least <NUM> %, at least <NUM> %, at least <NUM> %, at least <NUM> %, at least <NUM> %, at least <NUM> %, at least <NUM> %, at least <NUM> % or <NUM> % identical to SEQ ID NO: <NUM>, as determined using the BestFit software of the Wisconsin sequence analysis package, utilizing the Smith and Waterman algorithm, where gap weight equals <NUM>, length weight equals <NUM>, average match equals <NUM> and average mismatch equals -<NUM>. Of note, the percentage of homology refers to global homology over the albumin protein sequence.

The protein variant can also be referred to "as human serum albumin (HSA)", which when comprises all the <NUM> mutations (as in Table <NUM>, <NUM>) is referred to herein as "HSA1", "HSA2", "HSA3" or "BSA1", "BSA2", "BSA3". These mutations are only located in the protein core and not on the surface or binding sites of albumin as elaborated hereinbelow and in the Examples section which follows.

The extraordinary ligand binding properties of serum albumin reflect its multidomain organization. The complex mechanism modulating ligand binding to serum albumin represents one of the most important structure-function correlations ever reported for monomeric proteins. Serum albumin is known to carry almost every small molecule, thus it functions as a molecular cargo/or nanovehicle for clinical, biophysical, and industrial purposes. Under physiological conditions, serum albumin binds not only endogenous and exogenous low molecular weight compounds but also peptides and proteins. Thirty-five proteins were found to be associated to serum albumin including both known high and low abundant proteins (e.g., angiotensinogen, apolipoproteins, ceruloplasmin, clusterin, hemoglobin (Hb), plasminogen, prothrombin, and transferrin). Any binding of proteins and peptides to serum albumin impacts proteomics and biomarker discovery studies, since the presence of both unbound and bound states of proteins in serum can affect both the clearance and the detection of the free-state proteins and peptides. The fraction of peptides and proteins bound to serum albumin is defined as "albuminome" (reviewed in <NPL>).

Serum albumin is able to bind up to nine equivalents of long chain FAs, which represent the primary physiological ligands at multiple binding sites (i.e., FA1-FA9). These sites are distributed throughout the protein in an asymmetric way and show different affinity. FA4 and FA5 are high-affinity sites for FAs. FA2, a medium affinity site which lies at the interface between subdomains IA and IIA, is entirely contained within the N-terminal half of the protein, while sites FA4 and FA5 are entirely contained within domain III. These sites provide the most enclosed binding environments on SERUM ALBUMIN that allow the methylene tail of the FA to bind in a nearly linear conformation while the FA carboxyl forms specific salt-bridge interaction(s) with at least one basic amino acid side-chain. FA8 and FA9 are usually considered as supplementary binding sites, as they show ligand occupancy only in the presence of short-chain FAs (i.e., FA8) or in the presence of saturating FA concentration (i.e., FA9). FA binding sites also provide accommodation of several endogenous and exogenous ligands, including a wide variety of drugs, displaying appreciable affinity for one or more binding sites of serum albumin. This issue is of great relevance as binding to albumin improves plasma solubility and half-life of drugs, but at the same time reduces their free active concentration.

Thus, as mentioned, according to a specific embodiment, the mutations are done such that they do not affect albumin binding property. These are at least the <NUM> mutations of HSA1 and possibly any one of HSA2, i.e., H39L, L42M, V120P, K136N, F156Y, D187E, L198H, S202I, V216I, A254M, V310I, V344T, A371S, V381I, V409I, S427A, H440L, A449I, V455I, S470N, K519E, A528F, V547I, A552S, V576I and the equivalents in BSA1 and BSA2, as further described hereinbelow and in the Examples section which follows.

According to a specific embodiment, the albumin protein (e.g., HSA or BSA) exhibits at least about the same binding affinity to fatty acids such as myristate, such as determined by a crystal structure that contains <NUM> myristate molecules. This means that the protein is folded as wild type HSA or BSA (e.g., as compared to commercial HSA in the Examples section which follows) as evidenced by the ability to bind myristate. Myristate typically employs binding domains FA1, FA2, FA3, FA4, FA5.

Binding affinity is typically defined by KD and determined by various methods which are well known in the art.

According to a specific embodiment, binding affinity of albumin to small molecule or proteins ligand is determined by isothermal titration calorimetry (ITC), especially for water soluble molecules.

Specific embodiments of the method are provided in the Examples section which follows.

According to a specific embodiment, the binding affinity of the protein variant is increased by at least <NUM> fold, <NUM> fold, <NUM> fold, <NUM>, fold, <NUM> fold, <NUM> fold, <NUM> fold, <NUM> fold, <NUM> fold, <NUM> fold, <NUM> fold, <NUM> fold, <NUM> fold, <NUM> fold, <NUM> fold, <NUM> fold, <NUM> fold, <NUM> fold, <NUM> fold, <NUM> fold, <NUM> fold, as compared to wild type HSA or BSA and even more when compared to other forms of recombinantly produced albumins such as in yeast or plant systems (see e.g., <NPL>).

According to a specific embodiment, the protein variant binds a small molecule albumin ligand in at least the same affinity as wild type HSA of SEQ ID NO: <NUM> or BSA of SEQ ID NO: <NUM>.

According to a specific embodiment, the small molecule is warfarin which binds Sudlow's site I or Ketoptofen which binds Sudlow's site II. According to a specific embodiment, the protein is HSA1. According to a specific embodiment, the binding affinity to Sudlow's site I or II is unharmed and even improved compared to the wild type protein e.g., wild type HSA.

FA7 (Sudlow's site I) - The hydrophobic cavity of subdomain IIA hosts the seventh FA binding site (i.e., FA7 or Sudlow's site I). This site binds preferentially bulky heterocyclic anions, the prototypical ligand being warfarin. This site is smaller than the analogous binding cavity in subdomain IIIA (i.e., FA3-FA4 or Sudlow's site II). The FA carboxylate is stabilized by polar interaction(s) with the Arg257 residue, thus providing a bridge between FA2 and FA7 sites. Drugs (e.g., warfarin) cluster in the center of Sudlow's site I. Different compounds occupy the apolar compartments of Sudlow's site I to different extents, e.g., warfarin occupies the right-hand and the front sub-chambers.

FA3-FA4 (Sudlow's site II) FA3 and FA4 are composed of six helices and are located in a large cavity in subdomain IIIA that as a whole constitutes Sudlow's site II (<FIG>). This cleft is preferred by aromatic carboxylates with an extended conformation, the non-steroidal antiinflammatory drug ibuprofen representing the prototypical ligand.

According to a specific embodiment, the protein variant supports cell growth in culture, as further described hereinbelow.

According to a specific embodiment, the protein further comprises at least one mutation at albumin binding sites.

For instance, at least one of the mutations in the binding site of HSA:
mutations in the binding sites: V216I (also in HSA2 and HSA3), L250M, A254M (also in HSA2 and HSA3), G328A, V344T (also in HSA2 and HSA3), P384T, K402Y, V415M, V426L, M446L, A449I (also in HSA2 and HSA3), A528F (also in HSA2 and HSA3).

For instance, at least one of the mutations in the binding site of BSA:
mutations in the binding sites (all of these are present BSA2 and BSA3): V240I, H241Y, A253M, A260V, V344L, V414M, V425I, M445L, P485H, A527F, V546I, V551T.

According to a specific embodiment, the at least one mutation (e.g., at least <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>. <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, e.g., <NUM>-<NUM>, <NUM>-<NUM>) is selected from the group of mutations consisting of those in Table <NUM>, where the coordinates are those of SEQ ID NO: <NUM>.

According to a specific embodiment, the at least one mutation (e.g., at least <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>. <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, e.g., <NUM>-<NUM>. <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>) selected from the group of mutations consisting of those in Table <NUM>, where the coordinates are those of SEQ ID NO: <NUM>.

According to a specific embodiment, the protein variant exhibits increased yield when compared to the wild type protein when expressed in bacteria.

As used herein "increased yield" refers to at least <NUM> fold, <NUM> fold, <NUM> fold, <NUM> fold, <NUM> fold, <NUM> fold, <NUM> fold, <NUM> fold, <NUM> fold, <NUM> fold, <NUM> fold, <NUM> fold higher expression of the variant than that of wild type HSA or BSA ( as described below). Commercial WT HSA and BSA are available from Sigma-Aldrich A1653 and A7638, respectively, which were used as control in the Examples section which follows.

As mentioned, the protein variant is characterized by increased thermostability as compared to wild type HSA (SEQ ID NO: <NUM>).

As mentioned the protein variant is characterized by increased thermostability as compared to wild type BSA (SEQ ID NO: <NUM>).

As used herein "increased thermostability" is increased thermal stability by at least <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or more higher than that of wild type HSA or BSA (such as with respect to the controls used in the Examples section which follows).

For instance, HSA1, HSA2 and HSA3 showed <NUM>-<NUM> higher thermal stability relative to the commercial WT HSA (<FIG>). BSA2 and BSA3 designs had TM values of <NUM> and <NUM> respectively, <NUM>-<NUM> higher TM values than the WT BSA (<FIG>).

The solubility, functionality, thermal stability and/or high yield render the albumin variants of some embodiments of the invention, as described herein, highly suitable for recombinant expression.

Thus, according to an aspect there is provided a method of producing albumin, the method comprising expressing in bacteria a nucleic acid sequence encoding the protein variant as described herein, thereby producing albumin.

Accordingly there is provided a polynucleotide comprising a nucleic acid sequence encoding the protein variant as described herein.

To express exogenous nucleic acid sequences in cells, a polynucleotide sequence encoding the protein variant is preferably ligated into a nucleic acid construct suitable for mammalian cell expression. Such a nucleic acid construct includes a promoter sequence for directing transcription of the polynucleotide sequence in the cell in a constitutive or inducible manner.

A variety of prokaryotic can be used as host-expression systems to express the protein variants of some embodiments of the invention. These include, but are not limited to, microorganisms, such as bacteria transformed with a recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vector containing the coding sequence.

According to a specific embodiment, the cell is a bacterial cell such as E. The advantages of using E. coli include, but are not limited, to fast growth kinetics, high cell density cultures easily achieved, rich complex media can be made from readily available and inexpensive components and transformation with exogenous DNA is fast and easy.

Non-limiting examples of bacterial expression vectors include the pET series, pUC series, pQE vectors, ACYC and pBAD series of plasmid of E. coli expression vectors [<NPL>).

The skilled in the art would appreciate that the selection of vector depends on the origin of replication (e.g., pMB1, ColE1, p15A), promoters, selection markers and more.

Promoters - Numerous promoters are known in the art. According to a specific embodiment, the promoter is a constitutive promoter. According to a specific embodiment, the promoter is an inducible promoter. Following are some non-limiting examples: the lac promoter, a key component of the lac operon or its derivative lacUV5. Synthetic hybrids that combine the strength of other promoters and the advantages of the lac promoter are available. For example, the tac promoter consists of the -<NUM> region of the trp (tryptophan) promoter and the -<NUM> region of the lac promoter. This promoter is approximately <NUM> times stronger than lacUV5. Notable examples of commercial plasmids that use the lac or tac promoters to drive protein expression are the pUC series (lacUV5 promoter, Thermo Scientific) and the pMAL series of vectors (tac promoter, NEB). The T7 promoter system present in the pET vectors (pMB1 ori, medium copy number, Novagen) is commonly used for recombinant protein expression. In this system, the gene of interest is cloned behind a promoter recognized by the phage T7 RNA polymerase (T7 RNAP). This highly active polymerase should be provided in another plasmid or, most commonly, it is placed in the bacterial genome in a prophage (λDE3) encoding for the T7 RNAP under the transcriptional control of a lacUV5 promoter. Thus, the system can be induced by lactose or its non-hydrolyzable analog isopropyl β-D-<NUM>-thiogalactopyranoside (IPTG). Basal expression can be controlled by lacIQ but also by T7 lysozyme co-expression. T7 lysozyme binds to T7 RNAP and inhibits transcription initiation from the T7 promoter. In this way, if small amounts of T7 RNAP are produced because of leaky expression of its gene, T7 lysozyme will effectively control unintended expression of heterologous genes placed under the T7 promoter. T7 lysozyme is provided by a compatible plasmid (pLysS or pLysE). After induction, the amount of T7 RNAP produced surpasses the level of polymerase that T7 lysozyme can inhibit. The "free" T7 RNAP can thus engage in transcription of the recombinant gene. Yet another level of control lies in the insertion of a lacO operator downstream of the T7 promoter, making a hybrid T7/lac promoter. All three mechanisms (tight repression of the lac-inducible T7 RNAP gene by lacIQ, T7 RNAP inhibition by T7 lysozyme and presence of a lacO operator after the T7 promoter) make the system ideal for avoiding basal expression.

Promoters that rely on positive control have lower background expression levels. This is the case of the araPBAD promoter present in the pBAD vectors. The AraC protein has the dual role of repressor/activator. In the absence of arabinose inducer, AraC represses translation by binding to two sites in the bacterial DNA. The protein-DNA complex forms a loop, effectively preventing RNA polymerase from binding to the promoter. Upon addition of the inducer. AraC switches into "activation mode" and promotes transcription from the ara promoter. In this way, arabinose is absolutely needed for induction. Another widely used approach is to place a gene under the control of a regulated phage promoter. The strong leftward promoter (pL) of phage lambda directs expression of early lytic genes. The promoter is tightly repressed by the λcI repressor protein, which sits on the operator sequences during lysogenic growth. When the host SOS response is triggered by DNA damage, the expression of the protein RecA is stimulated, which in turn catalyzes the self-cleavage of λcI, allowing transcription of pL-controlled genes. This mechanism is used in expression vectors containing the pL promoter. The SOS response (and recombinant protein expression) can be elicited by adding nalidixic acid, a DNA gyrase inhibitor. Another way of activating the promoter is to control λcI production by placing its gene under the influence of another promoter. This two-stage control system has already been described for T7 promoter/T7 RNAP-based vectors. In the pLEX series of vectors (Life Technologies), the λcI repressor gene was integrated into the bacterial chromosome under the control of the trp promoter. In the absence of tryptophan, this promoter is always "on" and λcI is continuously produced. Upon addition of tryptophan, a tryptophan- TrpR repressor complex is formed that tightly binds to the trp operator, thereby blocking λcI repressor synthesis. Subsequently, the expression of the desired gene under the pL promoter ensues.

Transcription from all promoters described above is initiated by chemical cues. Systems that respond to physical signals (e.g., temperature or pH) are also available. The pL promoter is one example. A mutant λcI repressor protein ( λcI<NUM>) is temperature-sensitive and is unstable at temperatures higher than <NUM>. coli host strains containing the λcI<NUM> protein (either integrated in the chromosome or into a vector) are first grown at <NUM>-<NUM> to the desired density, and then protein expression is induced by a temperature shift to <NUM>-<NUM>. The industrial advantage of this system lies in part in the fact that during fermentation, heat is usually produced and increasing the temperature in high density cultures is easy. This temperature is suitable for expressing thermally stable proteins as in this case. On the other hand, genes under the control of the cold-inducible promoter cspA are induced by a downshift in temperature to <NUM>. The pCold series of plasmids have a pUC118 backbone (a pUC18 derivative) with the cspA promoter.

Selection Markers- To deter the growth of plasmid-free cells, a resistance marker (gene) is added to the plasmid backbone. Thus, antibiotic resistance genes are habitually used for this purpose. Resistance to ampicillin is conferred by the bla gene whose product is a periplasmic enzyme that inactivates the β-lactam ring of β-lactam antibiotics. Other examples of genes that can be used are those which confer resistance to chloramphenicol, kanamycin and tetracycline.

Other elements can be included in the plasmids, and those are selected by the skilled artisan according to various parameters such as the host cell, the scale of production, the intended use of the resultant protein.

For isolating a purified soluble active recombinant protein, it is important to have means to (i) detect it along the expression and purification scheme. (ii) attain maximal solubility, and (iii) easily purify it from the cellular milieu (be it intracellular or secreted). The expression of a heterologous stretch of amino acids (peptide tag, referred to herein as "tag") or a large polypeptide (fusion partner) in tandem with the desired protein, in this case the albumin variant, to form a chimeric protein may allow these three goals to be straightforwardly reached.

Thus, according to an embodiment, the protein is an in-frame fusion with a heterologous tag, not naturally present in albumin.

Peptide tags are less likely to interfere when fused to the protein. Vectors are available that allow positioning of the tag on either the N-terminal or the C-terminal end (the latter option being advantageous when a signal peptide is positioned at the N-terminal end for secretion of the recombinant protein). Common examples of small peptide tags are the poly-Arg-. FLAG-, poly-His-, c-Myc-, S-, and Strep II-tags. Since commercial antibodies are available for all of them, the tagged recombinant protein can be detected by Western blot along expression trials, which is helpful when the levels of the desired proteins are not high enough to be detected by SDS-PAGE. Also, tags allow for one-step affinity purification, as resins that tightly and specifically bind the tags are available. For example, His-tagged proteins can be recovered by immobilized metal ion affinity chromatography using Ni<NUM>+ or Co<NUM>+-loaded nitrilotriacetic acid-agarose resins (see the Examples section which follows), while anti-FLAG affinity gels (Sigma-Aldrich) are used for capturing FLAG fusion proteins.

On the other hand, adding a non-peptide fusion partner has the extra advantage of working as solubility enhancers. The most popular fusion tags are the maltose-binding protein (MBP). N-utilization substance protein A (NusA), thioredoxin (Trx), glutathione S-transferase (GST), ubiquitin and SUMO. calcium binding protein Fh8.

A different group of fusion tags which are also envisaged herein are stimulus-responsive tags, which reversibly precipitate out of solution when subjected to the proper stimulus. The addition of β roll tags to a recombinant protein allows for its selective precipitation in the presence of calcium. The final products present a high purity and the precipitation protocol only takes a couple of minutes. Another protein-based stimulus-responsive purification tags are elastin-like polypeptides (ELPs), which consist of tandem repeats of the sequence VPGXG, where X is Val, Ala, or Gly in a <NUM>:<NUM>:<NUM> ratio. These tags undergo an inverse phase transition at a given temperature of transition (Tt). When the Tt is reached, the ELP-protein fusion selectively and reversibly precipitates, allowing for quick enrichment of the recombinant protein by centrifugation. Precipitation can also be triggered by adjusting the ionic strength of the solution. These techniques represent an alternative to conventional chromatography-based purification methods and can save production costs, especially in large-scale settings.

According to some embodiments, affinity chromatography can be used to recover the protein from the expression system following expression.

Poly-His, MBP or GST can be used to purify the fused protein by affinity chromatography, as poly-His binds to nickel column, MBP binds to amylose-agarose and GST to glutathione-agarose. MBP is present in the pMAL series of vectors from NEB and GST in the pGEX series (GE). A peptide tag is preferably added to the fusion partner-containing protein if an affinity chromatography step is needed in the purification scheme. MBP and GST bind to their substrates non-covalently. On the contrary, the HaloTag7 (Promega) is based on the covalent capture of the tag to the resin, making the system fast and highly specific.

According to another embodiment, the protein variant is expressed tagless, i.e., without a tag. Alternatively, the tag is removed following expression and purification. Regardless, the resultant protein product is tagless (e.g., SEQ ID Nos: <NUM>, <NUM>, <NUM>, <NUM>, <NUM> and <NUM>).

Thus, embodiments envisage isolating the protein from the bacteria or conditioned medium thereof once sufficient levels are achieved.

According to a specific embodiment the tag is removed to avoid interference with protein activity and/or structure, but on the other embodiments they can be left in place even for crystallographic studies. Tags can be eliminated by either enzymatic cleavage or chemical cleavage.

Recovery of the recombinant protein variant is effected following an appropriate time in culture. The phrase "recovering the recombinant polypeptide" refers to collecting the whole fermentation medium containing the polypeptide and need not imply additional steps of separation or purification. Alternatively the protein can be recovered from the intracellular space (e.g., cytosol) following lysis and optionally sonication (see Examples section which follows). Notwithstanding the above, proteins of some embodiments of the invention can be purified using a variety of standard protein purification techniques, such as, but not limited to, affinity chromatography, ion exchange chromatography, filtration, electrophoresis, hydrophobic interaction chromatography, gel filtration chromatography, reverse phase chromatography, concanavalin A chromatography, chromatofocusing and differential solubilization.

In some applications, particularly in applications where there is a desire to use non-animal origin components, it is advantageous to combine recombinantly produced albumin with some or all of the following compounds when they are produced recombinantly or naturally by microbial systems: insulin, transferrin, IGF1, EGF or other proteins, growth factors and metabolites.

Thus the protein variants can be used in cell culture media component in diagnostic kits, stabilizer of protein solutions applications within the pharmaceutical area such as blood expanders and excipients, digestive support, removal of toxins, imaging-radiologic or ultrasonic imaging, drug delivery, coating of surfaces e.g. medical devices, invitro fertilization-both as storage medium of egg alone, sperma alone, but also for culturing of egg+sperma.

The serum albumin according to the invention can replace albumin derived from any animal species, most particular from human or bovine sources, or recombinant animal albumins, at an equivalent or better function for all uses of albumin. The reasons for this includes: <NUM>) The invention, as it is herein described, naturally creates beneficial species of small molecules bound to the albumin molecules, including, but not limited to, molecules such as fatty acids, vitamins, amino acids, phospholipids and cations; and <NUM>) It does not contain the high amounts of caprylic acid and N-acetyl DL tryptophan that many manufacturers of native and recombinant albumin use as stabilizers. It is well known that fatty acid and cation binding to albumin produce conformational changes which further affect both cooperative and competitive interactions of fatty acids and drugs. Excessive amounts of caprylic acid and/or N-acetyl DL tryptophan often have unwanted or adverse effects in many albumin applications. The use of recombinant human albumin in critically ill patients has thus been shown to increase mortality. (<NPL>; <NPL>; <NPL>).

According to an aspect there is provided a composition comprising the protein variant.

According to some embodiments, the protein (with or without a tag, or as a chimera where it is fused to another active protein) can be purified such that it constitutes at least <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> % of the proteins in the composition w/w or w/v.

When formulated with another protein (but not covalently) it can be present at any level even such as lower than <NUM> % w/w or w/v.

Thus, according to an aspect, there is provided a composition comprising the protein variant and an active ingredient, which can also be referred to as "payload".

According to an embodiment, the active ingredient is a protein.

According to a specific embodiment, the proteinaceous active ingredient is a peptide, a growth factor, an antibody, a hormone, a cytokine or an interleukin.

According to a specific embodiment, the antibody is an intact antibody or a fragment thereof such as a Fac fragment or a single chain (ScFv) antibody.

For instance, the protein variant can be used to target to a tumor site or to increase the half-life of the active ingredient in the serum.

Antibody fragments such as ScFvs have a molecular mass lower than <NUM> KDa and are cleared rapidly from the circulation. <NUM> ScFv-HAS fusion was shown to accumulate in tumors (reviewed in <NPL>)).

According to another embodiment, the active ingredient is a non-proteinaceous agent such as a small molecule, a nucleic acid agent, a fatty acid or a lipid, an ion [e.g., HSA displays a wide variety of binding sites for several metal ions, including Mg(II), Al(III), Ca(II), Mn(II), Co(II/III), Ni(II), Cu(I/II), Zn(II), Cd(II), Pt(II), Au(I/II), Hg(II), and Tb(III)].

According to another embodiment, the active ingredient is a drug e.g., propranolol, salicylate, diazepam, valproic acid, and sulfafurazole, warfarin. Thus, the high affinity variants described herein can be used to modulate the pharmacokinetics of drugs.

According to some embodiments, the active ingredient is attached to the protein in a covalent or non-covalent manner.

According to a specific embodiment, the composition is shaped as a tube. Fanali <NUM> supra describes organic nanotubes which have been modified to include albumin in the inner or outer surface of the tube and can be used as a carrier for payload binding.

When used in the clinic, the composition can be used as a pharmaceutical composition where the albumin is the active agent (e.g., in the case of reduced blood volume) or the drug-carrier.

On the basis of clinical evidence, the use of albumin can be indicated in acute conditions, in which it is necessary to expand the volume and maintain the circulation, and in some chronic states of low serum albumin; there are some widely shared and fully agreed indications for the appropriate use of human albumin and indications that are occasionally appropriate, that is, when other criteria are fulfilled. Albumin is also used in all cases in which there is a contraindication to the use of non-protein colloids.

The albumin protein variant of some embodiments of the invention can be administered to an organism per se, or in a pharmaceutical composition where it is mixed with suitable carriers or excipients.

As used herein a "pharmaceutical composition" refers to a preparation of one or more of the active ingredients described herein with other chemical components such as physiologically suitable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.

Herein the term "active ingredient" refers to the agent accountable for the biological and/or pharmaceutical effect.

Hereinafter, the phrases "physiologically acceptable carrier" and "pharmaceutically acceptable carrier" which may be interchangeably used refer to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound. An adjuvant is included under these phrases.

Herein the term "excipient" refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient. Examples, without limitation, of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.

Techniques for formulation and administration of drugs may be found in "<NPL>on.

Suitable routes of administration may, for example, include oral, rectal, transmucosal, especially transnasal, intestinal or parenteral delivery, including intramuscular, subcutaneous and intramedullary injections as well as intrathecal, direct intraventricular, intracardiac, e.g., into the right or left ventricular cavity, into the common coronary artery, intravenous, intraperitoneal, intranasal, or intraocular injections.

Compositions of some embodiments may, if desired, be presented in a pack or dispenser device, such as an FDA approved kit, which may contain one or more unit dosage forms containing the active ingredient. The pack may, for example, comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. The pack or dispenser may also be accommodated by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions or human or veterinary administration. Such notice, for example, may be of labeling approved by the U. Food and Drug Administration for prescription drugs or of an approved product insert. Compositions comprising a preparation formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition, as is further detailed above.

Due to expression in bacteria the protein preparations are devoid of xeno animal/eukaryotic contaminations rendering them beneficial for clinical applications as well as the food industry, biotechnological industry and research.

Further in vitro and in vivo applications of the protein variants according to the present invention include but are not limited to:.

As such also provided herein are research compositions which comprise the albumin. Thus, according to an aspect there is provided a composition comprising a research reagent and the albumin protein as described herein.

According to some embodiments, the research reagent is selected from the group consisting of a buffer, an enzyme (e.g., restriction enzyme, polymerase) and a cell culture medium.

Examples of buffers includes, PBS, DPBS, HEPES, Tris, RIPA, MOPS, ME, MOPSO, ACES, TAPS, Bicine or Tricine.

In some applications, particularly applications where albumin is used as a cell culture ingredient which can be combined into a base culture medium, it is advantageous to load the albumin molecule with one or more ligands, including but not limited to fatty acids, vitamins, hormones and ions--e.g. copper, zinc etc..

Examples of base media for tissue culturing include, but are not limited to, MEM DMEM. RPMI-<NUM>. IMDM, F12, Ham's F12, EMEM.

Typically albumin is added at an amount of <NUM>-<NUM> gr/L medium (e.g., <NUM> gr/L).

According to a particular embodiment, there is provided a method of cell culturing, the method comprising culturing cells in the presence of an albumin protein variant as described herein.

Any type of cell culturing method is envisaged herein, e.g., D2, 3D, large scale, small scale, suspension (without any matrix adherence) or adherent cultures.

According to a specific embodiment, said culturing is in serum-free medium or in the presence of serum up to <NUM> %.

According to a specific embodiment, said cells are eukaryotic cells and optionally mammalian cells.

According to a specific embodiment, said cells are hybridoma cells.

According to a specific embodiment, said cells are bovine cells, chicken cells, duck cells, fish cells or pig cells.

According to a specific embodiment, said cells are stem cells or progenitor cells.

According to a specific embodiment, said cells are differentiated cells.

According to a specific embodiment, the albumin protein is formulated with any of a fatty acid, a vitamin, a hormone or an ion.

When reference is made to particular sequence listings, such reference is to be understood to also encompass sequences that substantially correspond to its complementary sequence as including minor sequence variations, resulting from, e.g., sequencing errors, cloning errors, or other alterations resulting in base substitution, base deletion or base addition, provided that the frequency of such variations is less than <NUM> in <NUM> nucleotides, alternatively, less than <NUM> in <NUM> nucleotides, alternatively, less than <NUM> in <NUM> nucleotides, alternatively, less than <NUM> in <NUM> nucleotides, alternatively, less than <NUM> in <NUM> nucleotides, alternatively, less than <NUM> in <NUM>,<NUM> nucleotides, alternatively, less than <NUM> in <NUM>,<NUM> nucleotides.

Computational design. HSA and BSA proteins (SEQ ID Nos: <NUM> and <NUM>, respectively) were stabilized using a proprietary algorithm. For HSA, design was performed on two structures: pdb ID 2bx8 - with <NUM> azopropazone ligands, and pdb ID 2bxi - with <NUM> myristate ligands and <NUM> azopropazone ligands. Positions interacting with ligands were restricted to design. Three designs with reduced sets of mutations were constructed, based on a proprietary algorithm design7 (SEQ ID NO: <NUM>).

For BSA, design was performed on three structures (having different ligands and thus different conformations): pdb ID 6qs9 (with ketoprofen), 4jk4 (with <NUM>-hydroxy <NUM>,<NUM>-diiodobenzoic acid), and 4or0 (with naproxen). As in the case of HSA, three designs were tested experimentally, all based on design6 coming from 4or0 run (SEQ ID NO: <NUM>).

Protein expression and purification. Several constructs of HSA and BSA were tested. WT HSA and BSA (SEQ ID Nos: <NUM> and <NUM>, respectively), and the designed genes (SEQ ID Nos: <NUM> and <NUM>, respectively) were ordered from Twist Bioscience and cloned into a pET29b vector with C-terminal 6xHis tag (His-tag version). HSA1 and BSA2 variants (SEQ ID Nos: <NUM> and <NUM>, respectively) were also cloned into a pET28-SUMO vector with cleavable N-terminal bdSUMO tag (SUMO version), and HSA3 variant (SEQ ID NO: <NUM>) was also cloned into a pET29b vector with deleted <NUM>-His tag (no-His version). The His tag was added at C-term, SUMO tag was added at N-term.

All the variants were transformed into SHuffle T7 cells (NEB) and plated on LB plates with kanamycin (kana) and spectinomycin (spec). <NUM> of 2YT medium supplemented with <NUM> ug/ml kana and <NUM> ul/ml spec were inoculated with a single colony and grown overnight at <NUM>. Then, 2YT medium supplemented with kana and spec (<NUM>-<NUM> of 2YT medium) was inoculated <NUM>:<NUM> with the overnight culture and grown at <NUM> until OD of ~<NUM>. Overexpression was induced with <NUM> IPTG, with which the cultures were grown for <NUM>-<NUM>/<NUM>, harvested (<NUM> rpm/<NUM>/<NUM>) and the pellet was frozen at -<NUM>. The cells were dispersed in lysis buffer (phosphate buffer saline (PBS) + <NUM>:<NUM> benzonase (Sigma-Aldrich #E1014)) and lysed by sonication.

His-tagged variants purification: The supernatant obtained after centrifugation (<NUM>,<NUM> rpm/<NUM>/<NUM>) was supplemented with <NUM> imidazole and mixed with Ni-Nta resin (<NUM>-<NUM>/<NUM>), washed with <NUM> imidazole in PBS and the protein was eluted with <NUM> imidazole in PBS.

SUMO-tagged variants purification: The supernatant was purified on Ni-Nta resin as described before, but the elution was done by mixing overnight at <NUM> in PBS + <NUM> DTT with 5µg/ml bdSENP1 protease (<NUM>). The unbound fraction contained the tagless BSA/HSA variant.

No-His variants purification: The supernatant of HSA3 was incubated for <NUM> at <NUM> and cleared by centrifugation (<NUM>,<NUM> rpm/<NUM>/<NUM>).

At the final stage, all the variants were purified by gel filtration chromatography, using HiLoad Superdex <NUM> column for preps coming from large scale expression (><NUM> culture) and HiLoad Superdex <NUM> column for preps coming from medium scale expression (<NUM>-<NUM> culture).

Thermal stability measurements. Thermal stability of BSA and HSA variants (His-tagged form) and of commercial WT HSA and BSA (Sigma-Aldrich A1653 and A7638, respectively) was measured by nanoscale differential scanning fluorimetry (nanoDSF), in Prometheus NT. <NUM> instrument (NanoTemper). The temperature was increased from <NUM> to <NUM> at <NUM>/min ramp. Thermal stability of HSA variants was also measured by differential scanning calorimetry (DCS) in VP-DSC instrument (Malvern), with heating from <NUM> to <NUM> at 1C°/min and cooling at the same rate.

Ligand binding measurements. Ligand binding to commercial WT HSA (non-defatted, Sigma-Aldrich A1653) and to HSA1 variant (His-tagged) was measured at <NUM> by isothermal titration calorimetry (ITC, MicroCal iTC instrument, Malvern), in PBS supplemented with <NUM> % DMSO. HSA concentration was maintained at <NUM> in the cell, and <NUM> of warfarin (Sigma-Aldrich A2250) or ketoprofen (Sigma-Aldrich K1751) were loaded into the injector. The proteins were titrated with <NUM> ul ligands with a <NUM>-min equilibration time. Titration of ligand to buffer (PBS with <NUM> % DMSO) was used as a blank control, and one-site model was used to calculate the binding constants.

Crystallization and structure determination of HSA1 and Warfarin. Tagless HSA1 protein obtained from SUMO-HSA production was concentrated to <NUM>/ml in Tris buffer pH <NUM> and supplemented with <NUM> warfarin from <NUM> stock in DMSO. Crystals of HSA1 and warfarin were obtained using the hanging-drop vapor diffusion method with a Mosquito robot (TTP LabTech). All datasets were collected under cryogenic conditions at the European Synchrotron Radiation Facility (ESRF), Grenoble, France at beamline ID30B. The crystals were grown from <NUM> NaCl, <NUM> % PEG <NUM> and <NUM> Tris pH=<NUM>. The crystals formed in the space group P1, with two monomers per asymmetric unit and diffracted to <NUM>. 0Å resolution. The integrated reflections were scaled using the AIMLESS program (<NUM>) from the CCP4i2 program suite (<NUM>). HSA1 structure determined by molecular replacement with PHASER (<NUM>) using the structure of human serum albumin in complex with aristolochic acid (PDB code 6HSC). All steps of atomic refinement were carried out with the CCP4i2/REFMAC5 program (<NUM>) and by PHENIX. refine (<NUM>). The models were built into 2mFobs - DFcalc, and mFobs - DFcalc maps by using the COOT program (<NUM>). The model was optimized using PDB_REDO (<NUM>, <NUM>) and was evaluated with MOLPROBITY (<NUM>). Electron density revealed unambiguous density for the bound warfarin. Details of the refinement statistics of HSA1 and Warfarin structure are described in Table <NUM>.

The crystal structure was deposited in the PDB-ID code 8A9Q.

Cell culture experiments. Tagless albumin variants were used for all the cell culture experiments. Hybridoma cells producing anti-GST monoclonal antibody (IgG1, Igk) were cultured for <NUM>-<NUM> days in the presence of <NUM>% horse serum with or without <NUM>/ml of the following proteins: commercial HSA, HSA1, HSA3, commercial BSA, and BSA2. The number of live cells and the levels of produced antibody were analyzed via flow cytometry and ELISA respectively. For flow cytometry, cells were collected every <NUM> hours stained with NucBlue™ Live Cell Stain (ThermoFisher LTD), according to manufacturer protocol and analyzed using LSRII cell analyzer plate reader. For ELISA, sups were collected every <NUM> hours and binding to GST was assessed using standard anti GST produced in our facility.

HEK293T adherent cells were grown in DMEM supplemented with GlutaMAX, NEAA and <NUM>% FBS (all from Gibco) at <NUM>, <NUM>%CO<NUM>. To test the viability of the cultured cells in the presence of PROSS BSA2, HSA1 and HSA3 in comparison to the commercial BSA or HSA, respectively, cells (~3x <NUM><NUM>) were seeded in <NUM>-well plate and followed each day for <NUM> days. All the albumin variants were added to the cells at <NUM>/ml in duplicates, and their effect was analyzed. As a control, no protein was added to the growth medium. In each time point, cells were collected and the number of total cells, live cells and % of viable cells were determined automatically using Brightfield Cell Counter DeNovix.

DNA Restriction. pET29b vector with 900bp insert was restricted with NcoI and XhoI enzymes (NEB) overnight at <NUM>, using the following buffers: rCutsmart buffer (NEB, contains <NUM>/ml rBSA), buffer <NUM> (NEB, identical composition to rCutsmart buffer, but no BSA), and buffer <NUM> supplemented with HSA1, BSA2, and HSA3 variants (tagless, after gel filtration and ion exchange purification) at <NUM>/ml.

Samples of pET28b(+) vector (<NUM> µg) were restricted by BsiEI (10U, NEB) in <NUM> µl reaction volumes containing the Cutsmart buffer (NEB) or a reconstituted equivalent (<NUM> Potassium acetate, <NUM> Tris-acetate, Mg-acetate, pH <NUM>) supplemented with <NUM>/ml of either commercial BSA (Sigma), HSA1, BSA2, or HSA3 variants (tagless, after gel filtration and ion exchange purification) at temperatures of <NUM>-<NUM> for <NUM> using a gradient PCR (SensoQuest). Samples were then supplemented with <NUM> µl DNA sample buffer (<NUM> TrisHCl pH <NUM>, <NUM>% SDS, <NUM>% Glycerol, <NUM>% Bromophenol Blue), heated (<NUM>, <NUM>), and resolved on a <NUM>% agarose-TAE gel containing <NUM>µg/mL Ethidium Bromide for <NUM> at 120V.

In the most conservative design (HSA1 and BSA <NUM>=design <NUM>), the present inventors allowed mutations only in the protein core and away from any of its binding sites. In the next design (HSA2 and BSA2=design <NUM>), the present inventors also enabled design in surfaces outside the binding pockets. Binding pockets are amino acids in contact with small molecule ligands, which were observed in the crystal structures. Specifically, it is Sudlow sites I and II, and binding sites of myristate molecules. , and in HSA3 and BSA3 (also termed design <NUM>), mutations were allowed throughout the protein (<FIG>, Tables <NUM>, <NUM> and <NUM>). It is envisioned that design <NUM> may find uses in settings in which all solvent-accessible surfaces must remain intact, such as in raising albumin-targeting antibodies. Design <NUM> can be used when only the ligand binding sites need to be conserved, and design <NUM> in those cases in which albumin is used for its osmotic or surface-binding properties. Furthermore, crystallographic analyses reveal two major conformations for albumin (compact and myristate-bound), and the present inventors used both in the design process, selecting mutations that are observed in both structures.

As expected, WT HSA did not express in a soluble manner, but the designed HSA variants were soluble, and the expression levels increased with the number of stabilizing mutations (not shown), from ~ <NUM>/L culture for HSA <NUM> to ~ <NUM>/L culture for HSA3. A similar behavior was obtained for BSA variants, but WT BSA had some soluble expression, and BSA <NUM> showed only a modest increase in expression level. Size exclusion chromatography demonstrated that the His-tagged proteins are monomeric (<FIG>).

The thermal stability of HSA and BSA variants was tested by nanoDSF, and compared to that of commercial (WT) HSA and BSA samples (Table <NUM>). As expected, thermal stability increased with the number of stabilizing mutations. HSA variants had TM values of <NUM>-<NUM>, an increase of <NUM>-<NUM> relative to the commercial WT HSA (<FIG>). BSA1 variant didn't melt, and BSA2 and BSA3 designs had TM values of <NUM> and <NUM> respectively, <NUM>-<NUM> higher TM values than the WT BSA (<FIG>).

Encouraged by the soluble expression and the increased thermostability of HSA and BSA variants, the present inventors proceeded to further functional characterization with the most conserved improved variants, HSA <NUM> and BSA2. To obtain tagless proteins, the present inventors have expressed the HSA1 and BSA2 in fusion with the SUMO tag (<NPL>), which was cleaved without any damage to the protein during purification. Gel filtration profiles demonstrated that HSA <NUM> protein purified from SUMO-fused constructs was completely monomeric, and BSA2 protein in some preps had similar proportions of monomeric and dimeric fractions (<FIG>). Dimerization of albumin is a known process in mammalian albumins (<NUM>), and since no aggregates were observed, it was concluded that the present recombinant HSA <NUM> and BSA2 could be purified in a tagless form.

Binding of various ligands is the primary biological activity of HSA. Ligand binding capacity of HSA1, the variant with no mutations in the active sites, was compared to that of the commercial WT HSA. Binding of two site-specific drug ligands, warfarin, which binds to Sudlow site I, and ketoprofen, which binds to Sudlow site II (<NPL>), was measured by ITC. Binding affinities of HSA1 for these ligands were similar and even better than those obtained for the commercial WT HSA (Table <NUM>, <FIG>). This indicates that the stabilized HSA1 variant retained the ligand binding capacity in the two primary binding sites.

In order to validate the structure of HSA1 variant, it was crystallized in complex with warfarin. The crystals had two monomers per asymmetric unit and diffracted to <NUM>. 0Å resolution. The warfarin is indeed occupying its binding site (<FIG>), as in the plasma-derived WT HSA structure (pdb ID 2bxd). In addition, four molecules of myristate fatty acid are present in the structure, in the same locations as in the structure of WT HSA (pdb ID 2bxi). Since no myristate was added during protein production, the source of myristate the 2YT medium, and/or the E. coli bacteria. The overall structure of HSA <NUM> overlaps well with the open conformation structure of plasma-derived WT HSA with myristate (pdb ID 2bxd, rmsd <NUM>. All the <NUM> disulfide bonds were identified in the structure, as well as the free Cys34, and no significant changes in the side chain conformations were observed. These results demonstrate that stabilized HSA expressed in E. coli is folded to the same structure as the HSA from blood serum, with the capacity of myristate binding, as well as other ligands of HSA.

Stabilized HSA and BSA variants in cell culture and in vitro applications To test whether the albumin designs are not toxic to human cells and can be used in cell culture medium, they were added to the growth medium of HEK293T cells and hybridoma cells. In the case of HEK293T cells, there was no adverse effect of HSA <NUM>, HSA3, or BSA2 variants, as well as of the commercial plasma derived HSA and BSA, on both the number of cells and their viability (<FIG>). Similarly, all the HSA and BSA design did not have any adverse effect on the number of live hybridoma cells, as well as on the produced antibody titers (<FIG>). Based on these results, it can be concluded that the stabilized recombinant albumin variants are not toxic to HEK and hybridoma cells and could be considered for use in cell culture media.

To test whether the designed albumins can be used in molecular biology applications, restriction reactions were performed in which a buffer containing commercial BSA was compared to a buffer supplemented with stabilized albumins, HSA1, HSA3, or BSA2. In one case, pET28b(+) plasmid was digested using the enzyme BsiEl (<FIG>and in another case, pET29b(+) plasmid was digested with NcoI and XhoI enzymes (not shown). In both cases, the designs and commercial BSA performed equally well, producing the same restriction pattern with the same efficiency. The albumin designs can be used in reactions performed at extreme temperatures, such as PCR reactions, since their apparent melting temperatures are <NUM>-<NUM>.

Claim 1:
An albumin protein variant comprising:
(i) an amino acid sequence at least <NUM> % identical to SEQ ID NO: <NUM>;
(ii) mutations set forth in H39L, L42M, V120P, F156Y, D187E, L198H, S202I, V310I, A371S, V381I, V409I, S427A, V455I, K519E, A552S and V576I, where the positions correspond to said SEQ ID NO: <NUM>; and
wherein the albumin protein variant is soluble when expressed in bacteria and characterized by increased thermostability as compared to wild type human serum albumin (HSA) of SEQ ID NO: <NUM>.