Yeast expression vectors for production of ITF

The invention features ITF expression vectors and methods of producing ITF.

BACKGROUND OF THE INVENTION

Intestinal trefoil factor (“ITF”) expression vectors and methods of overexpressing ITF have been described (see, for example, Thim et al., Biochemistry, 34:4757-4764, 1995, and Kanai et al., Proc. Natl. Acad. Sci. USA, 95:178-182, 1998). One useful expression system involves the use of the yeastPichia pastoris, which allows overexpression and secretion of heterologous genes at high levels (see, for example, Tschopp et al., Bio/Technology, 5:1305-1308, 1987, and Cregg et al., Bio/Technology, 11:905-910, 1993).

A need exists in the art for new methods of producing ITF in quantity, especially without extraneous amino acid sequences (FIGS. 1-2). The present invention provides such vectors and methods.

SUMMARY OF THE INVENTION

In one aspect, the invention features an expression vector including a nucleic acid encoding a biologically active intestinal trefoil factor (ITF) polypeptide. This expression vector encodes a polypeptide that includes an N-terminal fusion sequence, a protease cleavage site, and the ITF polypeptide, such that the cleavage site is contiguous with the ITF polypeptide and is recognized by a protease that cleaves immediately C-terminal to the cleavage site. In particular embodiments, the N-terminal fusion sequence includes an MFα signal sequence or an MFα presequence.

In particular embodiments, the cleavage site is recognized by the protease KEX2. Desirably, the cleavage site includes a polypeptide having the sequence of SEQ ID NO: 401.

In particular embodiments, the cleavage site is recognized by yeast aspartic protease (Yap3), Type IV dipeptidyl aminopeptidase (DPAP), yeast glycosyl-phosphatidylinositol-linked aspartyl protease (Mkc 7), pepsin, trypsin, chymotrypsin, or subtilisin.

In particular embodiments, the ITF polypeptide used in the vector of the invention is hITF15-73. Desirably, the vector includes a nucleic acid having the sequence of SEQ ID NO: 111. Desirably, the vector includes a nucleic acid having the sequence of SEQ ID NO: 3.

In particular embodiments, the ITF polypeptide used in the vector of the invention is hITF1-73. Desirably, the vector includes a nucleic acid having the sequence of SEQ ID NO: 116. Desirably, the vector includes a nucleic acid having the sequence of SEQ ID NO: 4.

In particular embodiments, the ITF polypeptide used in the vector of the invention is hITF21-62, hITF21-70, hITF21-72, hITF21-73, hITF22-62, hITF22-70, hITF22-72, hITF22-73, hITF25-62, hITF25-70, hITF25-72, or hITF25-73.

In particular embodiments, the ITF polypeptide used in the vector of the invention is pITF22-80or pITF1-80. Desirably, the vector includes a nucleic acid having the sequence of SEQ ID NO: 112 or SEQ ID NO: 117.

In particular embodiments, the ITF polypeptide used in the vector of the invention is dITF22-80or dITF1-80. Desirably, the vector includes a nucleic acid having the sequence of SEQ ID NO: 113 or SEQ ID NO: 118.

In particular embodiments, the ITF polypeptide used in the vector of the invention is rITF23-81or rITF1-81. Desirably, the vector includes a nucleic acid having the sequence of SEQ ID NO: 114 or SEQ ID NO: 119.

In particular embodiments, the ITF polypeptide used in the vector of the invention is mITF23-81or mITF1-81. Desirably, the vector includes a nucleic acid having the sequence of SEQ ID NO: 115 or SEQ ID NO: 120.

In a second aspect, the invention features an expression vector that includes a nucleic acid having at least 90% sequence identity to nucleotides 1 to 1,191 of SEQ ID NO: 2, a nucleic acid encoding a biologically active ITF polypeptide, and a nucleic acid having at least 90% sequence identity to nucleotides 1,218 to 8,001 of SEQ ID NO: 2.

In a third aspect, the invention features an expression vector that includes a nucleic acid having at least 90% sequence identity to nucleotides 1 to 1,008 of SEQ ID NO: 5, a nucleic acid encoding a biologically active ITF polypeptide, and a nucleic acid having at least 90% sequence identity to nucleotides 1,035 to 7,818 of SEQ ID NO: 5.

In a fourth aspect, the invention features a cell transformed with the vector of any of the previous aspects.

In a fifth aspect, the invention features a composition that includes a cell transformed with the vector of any of the previous aspects and a cell culture medium. In particular embodiments of the above aspect, the cell isPichia pastoris. Desirably, the cell is a (Mut+) GS115 strain or a (his4-) GS115 strain.

In a sixth aspect, the invention features a method of culturing a cell of the fourth aspect so as to express the encoded ITF polypeptide and recover this polypeptide from the culture medium. In particular embodiments of the above aspect, the ITF polypeptide is secreted from the cell. Desirably, the expressed polypeptide is proteolytically processed in vivo prior to secretion from said cell, resulting in secretion of the ITF polypeptide substantially free of extraneous residues. Alternatively, the secreted polypeptide is contacted with a purified proteolytic enzyme in a reaction chamber, thereby producing the ITF polypeptide substantially free of extraneous residues.

In a seventh aspect, the invention features a method of culturing a cell of the fourth aspect so as to express the encoded ITF polypeptide and recover this polypeptide from the culture medium. In particular embodiments of the above aspect, the ITF polypeptide is secreted from the cell. Desirably, the expressed polypeptide is proteolytically processed in vivo prior to secretion from said cell. Alternatively, the secreted polypeptide is contacted with a purified proteolytic enzyme in a reaction chamber.

In an eighth aspect, the invention features a method for producing a biologically active ITF polypeptide. This method includes the step of culturing yeast transformants containing recombinant plasmids encoding a polypeptide that includes a ITF polypeptide, such that the yeast produce and secrete the ITF polypeptide unaccompanied by an extraneous EA amino acid sequence. The method also includes steps of isolating and purifying the ITF polypeptide. In particular embodiments, the ITF polypeptide is hITF15-73, hITF1-73, hITF21-62, hITF21-70, hITF21-72, hITF21-73, hITF22-62, hITF22-70, hITF22-72, hITF22-73, hITF25-62, hITF25-70, hITF25-72, hITF25-73, pITF1-80, pITF22-80, dITF1-80, dITF22-80, rITF1-81, rITF23-81, mITF1-81, or mITF22-81.

In a ninth aspect, the invention features a polypeptide that includes an N-terminal fusion sequence, a protease cleavage site, and the ITF polypeptide, such that the cleavage site is contiguous with the ITF polypeptide and is recognized by a protease that cleaves immediately C-terminal to the cleavage site. In particular embodiments, the sequence of the polypeptide comprises SEQ ID NO: 306, SEQ ID NO: 307, SEQ ID NO: 308, SEQ ID NO: 309, SEQ ID NO: 310, SEQ ID NO: 311, SEQ ID NO: 312, SEQ ID NO: 313, SEQ ID NO: 314, SEQ ID NO: 315, SEQ ID NO: 316, SEQ ID NO: 317, SEQ ID NO: 318, SEQ ID NO: 319, or SEQ ID NO: 320.

In a tenth aspect, the invention features an expression vector that includes a nucleic acid having the sequence of SEQ ID NO: 2.

In an eleventh aspect, the invention features an expression vector that includes a nucleic acid having the sequence of SEQ ID NO: 5.

By “intestinal trefoil factor” (“ITF”) is meant any protein that is substantially homologous to human ITF (FIG. 14A) and that is expressed in the large intestine, small intestine, or colon to a greater extent than it is expressed in tissues other than the small intestine, large intestine, or colon. Also included are: allelic variations; natural mutants; induced mutants; proteins encoded by DNA that hybridizes under high or low stringency conditions to ITF encoding nucleic acids retrieved from naturally occurring material; and polypeptides or proteins retrieved by antisera to ITF, especially by antisera to the active site or binding domain of ITF. The term also includes other chimeric polypeptides that include an ITF.

In addition to substantially full-length polypeptides, the term ITF, as used herein, includes biologically active fragments of the polypeptides. As used herein, the term “fragment,” which applies to a polypeptide unless otherwise indicated, will ordinarily be at least 10 contiguous amino acids, typically at least 20 contiguous amino acids, more typically at least 30 contiguous amino acids, usually at least 40 contiguous amino acids, preferably at least 50 contiguous amino acids, and most preferably 59 or more contiguous amino acids in length. Fragments of ITF can be generated by methods known to those skilled in the art and described herein. The ability of a candidate fragment to exhibit a biological activity of ITF can be assessed by methods known to those skilled in the art and are described herein. Also included in the term “fragment” are biologically active ITF polypeptides containing amino acids that are normally removed during protein processing, including additional amino acids that are not required for the biological activity of the polypeptide, or including additional amino acids that result from alternative mRNA splicing or alternative protein processing events.

An ITF polypeptide, fragment, or analog is biologically active if it exhibits a biological activity of a naturally occurring ITF, e.g., the ability to alter gastrointestinal motility in a mammal, the ability to restitute gastrointestinal, respiratory, or uterine epithelium, or the ability to enhance dermal or corneal epithelial wound healing.

Particularly useful ITF polypeptides that retain biological activity include the polypeptides corresponding to amino acid residues 1-73 of SEQ ID NO: 301 (full-length human ITF, also designated hITF1-73), amino acid residues 15-73 of SEQ ID NO: 301 (hITF15-73), amino acid residues 21-62 of SEQ ID NO: 301 (hITF21-62), amino acid residues 21-70 of SEQ ID NO: 301 (hITF21-70), amino acid residues 21-72 of SEQ ID NO: 301 (hITF21-72), amino acid residues 21-73 of SEQ ID NO: 301 (hITF21-73), amino acid residues 22-62 of SEQ ID NO: 301 (hITF22-62), amino acid residues 22-70 of SEQ ID NO: 301 (hITF22-70), amino acid residues 22-72 of SEQ ID NO: 301 (hITF22-72), amino acid residues 22-73 of SEQ ID NO: 301 (hITF22-73), amino acid residues 25-62 of SEQ ID NO: 301 (hITF25-62), amino acid residues 25-70 of SEQ ID NO: 301 (hITF25-70), amino acid residues 25-72 of SEQ ID NO: 301 (hITF25-72), amino acid residues 25-73 of SEQ ID NO: 301 (hITF25-73), amino acid residues 1-80 of SEQ ID NO: 302 (full-length pig ITF, also designated pITF1-80), amino acid residues 22-80 of SEQ ID NO: 302 (pITF22-80), amino acid residues 1-80 of SEQ ID NO: 303 (full-length dog ITF, also designated dITF1-80), amino acid residues 22-80 of SEQ ID NO: 303 (dITF22-80), amino acid residues 1-81 of SEQ ID NO: 304 (full-length rat ITF, also designated rITF1-81), amino acid residues 23-81 of SEQ ID NO: 304 (rITF23-81), amino acid residues 1-81 of SEQ ID NO: 305 (full-length mouse ITF, also designated mITF1-81), and amino acid residues 23-81 of SEQ ID NO: 305 (mITF23-81).

By “MFα prepropeptide sequence” or “MFα signal sequence” is meant the DNA sequence spanned by nucleotides 949 to 1,191 of SEQ ID NO: 2 or the protein sequence encoded therein.

By “MFα presequence” is meant the DNA sequence spanned by nucleotides 949 to 1,008 of SEQ ID NO: 5 or the protein sequence encoded therein.

By “modify,” when applied to an expression vector, is meant to alter the sequence of such a vector by addition, deletion, or mutation of nucleotides. A modified vector will generally exhibit at least 70%, more preferably 80%, more preferably 90%, and most preferably 95% or even 99% sequence identity with the unmodified vector.

By “N-terminal fusion sequence” is meant an amino acid sequence fused to the N terminus of a protein of interest, or a nucleotide sequence encoding such an amino acid sequence. This could include the MFα signal sequence, the MFα presequence, or other signal or leader sequences. The term also encompasses fusion partners that enhance size, solubility, or other desirable characteristics of the expressed fusion protein.

By “polypeptide” or “peptide” or “protein” is meant any chain of at least two naturally-occurring amino acids, or unnatural amino acids (e.g., those amino acids that do not occur in nature) regardless of post-translational modification (e.g., glycosylation or phosphorylation), constituting all or part of a naturally-occurring or unnatural polypeptide or peptide, as is described herein.

Polypeptides or derivatives thereof may be fused or attached to another protein or peptide, for example, as an α-Factor signal sequence fusion polypeptide.

Sequence identity is typically measured using sequence analysis software with the default parameters specified therein (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705). This software program matches similar sequences by assigning degrees of homology to various substitutions, deletions, and other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine, valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.

By “protease” or “proteolytic enzyme” is meant an enzyme that catalyzes the cleavage, or proteolysis, of proteins into smaller peptide fractions and amino acids. Some proteases recognize and bind to particular amino acid sequences and cleave specific peptide bonds within or outside the recognition sequence, while other proteases cleave nonspecifically.

By “protease cleavage site” or “protease recognition site” is meant an amino acid sequence to which a protease is capable of binding, thereby leading to proteolysis. Desirably, a protease will bind specifically to a corresponding recognition site.

By “vector” or “plasmid” is meant a DNA molecule into which fragments of DNA may be inserted or cloned. A vector will contain one or more unique restriction sites, and may be capable of autonomous replication in a defined host or vehicle organism such that the cloned sequence is reproducible.

By “expression vector” or “construct” is meant any autonomous element capable of replicating in a host independently of the host's chromosome, after additional sequences of DNA have been incorporated into the autonomous element's genome.

All nucleotide sequences presented herein should be understood to read from the 5′ end to the 3′ end unless otherwise indicated. Likewise, all amino acid sequences should be understood to read from the N-terminal end to the C-terminal end unless otherwise indicated.

DETAILED DESCRIPTION OF THE INVENTION

A vector designed for expression inP. pastorisis shown inFIG. 3, and the corresponding protein product is shown inFIG. 2. This construct includes an N-terminal mating factor α (MFα) signal sequence fused to residues 15-73 of human ITF. The MFα signal sequence is cleaved in vivo, and the cleaved protein is secreted from the cell into the expression medium. Signal peptidase cleaves at the junction between the MFα pre sequence and the MFα pro sequence, while KEX2 cleaves near the N-terminal end of the hITF1573 polypeptide, between residues Arg and Glu (FIG. 2). The protease STE13 cleaves the remaining Glu-Ala sequence from the ITF15-73polypeptide, but it does so only with approximately 70% efficiency, resulting in approximately 30% of the expression product containing an extraneous dipeptide fusion at the N-terminal end (FIG. 1). These extraneous residues result in greater heterogeneity and potentially greater antigenicity of the expression product. In addition, because hITF has a cysteine residue near the C-terminal end, causing the peptide to form dimers by disulfide-bonding, the complexity in the resulting preparation is increased by the presence of two homodimers (hITF15-73:hITF15-73and EA-hITF15-73:EA-hITF15-73) and a heterodimer (EA-hITF15-73:hITF15-73). This reduces the yield of the native homodimeric hITF15-73.

In order to facilitate the preparation of expression vectors capable of producing ITF free of extraneous residues, the vector pPICGIco is created (FIGS.5and11A-11B). pPICGIco, which encodes the MFα signal sequence and has the sequence of SEQ ID NO: 2, is generated by linearizing the plasmid pPIC9 (Invitrogen) (FIG. 4) with the restriction endonucleases Xho I and SnaB I in accordance with the vendor's instructions (New England Biolabs, Inc.). This reaction eliminates the DNA segment between the Xho I sites and the SnaB I sites that code for the KEX2 recognition sequence and the Glu-Ala (EA) spacer. The 5′-overhang is filled in with DNA polymerase in the presence of dNTPs to produce blunt ends. The blunt-ended vector is circularized by blunt-end ligation in the presence of DNA ligase (New England Biolabs, Inc.). The DNA preparation is then transformed into a bacterial host (E. coliHB101) and transformants selected from LB agar plates containing ampicillin. The resulting vector, pPICGIco, is isolated from these transformants and identified by restriction endonuclease analysis, in which the loss of the SnaB I site and retention of the Xho I and EcoR I sites in the multiple cloning site are tested.

It is also desirable to create fusion proteins in which the MFα presequence is fused to the protein of interest, rather than the full MFα prepropeptide sequence. Thus, a second expression vector encoding the MFα presequence, pPICpre, is created based on pPICGIco (FIGS.8and12A-12C). pPICpre, which has the sequence of SEQ ID NO: 5, is generated by linearizing pPICGIco with the restriction endonucleases BamH I and Xho I in accordance with the vendor's instructions (New England Biolabs, Inc.). This reaction eliminates the DNA segment between the BamH I and Xho I sites that code for the MFα signal sequence. An insert containing the MFα presequence is prepared by PCR reaction using primers having the sequences of SEQ ID NO: 241 and SEQ ID NO: 242, with pPICGIco used as a template (FIG. 12B). The resulting fragment has the sequence of SEQ ID NO: 131. This PCR fragment is cleaved with the restriction enzymes BamH I and Xho I to generate ends compatible with subcloning into pPICGIco previously cleaved with BamH I and Xho I. The linearized pPICGIco vector and PCR insert are ligated in the presence of DNA ligase. The DNA preparation is transformed into a bacterial host (E. coliHB101) and transformants selected from LB agar plates containing ampicillin. The resulting vector, pPICpre, is isolated from these transformants.

Below, examples are described in which the vectors pPICGIco or pPICpre are utilized to prepare numerous improved ITF expression vectors of the invention. The examples are provided for the purpose of illustrating the invention and are not meant to limit the invention in any way.

Yeast Expression Vectors for Production of Mammalian ITF

In order to generate an expression vector encoding MFα-hITF15-73, the following protocol is followed. Total RNA isolated from human intestine, which includes RNA molecules having the sequence of SEQ ID NO: 101, is used as a template in an RT-PCR reaction including Taq polymerase (New England Biolabs, Inc.) and primers having the sequence of SEQ ID NO: 201 and SEQ ID NO: 202. This reaction results in a PCR product having the sequence of SEQ ID NO: 106.

The resulting PCR product is then subcloned into the bacterial plasmid vector pCR2.1 (Invitrogen) using standard ligation reaction conditions in the presence of T4 DNA ligase (New England Biolabs, Inc.). The resulting clone, pCR2.1-hITF, may be sequenced using standard M13 primers adjacent to the cloning site.

Next, using pCR2.1-hITF as the template, a nucleotide sequence encoding hITF15-73, with a KEX2 recognition sequence operably linked to the N terminus of hITF15-73, may be obtained by performing a PCR reaction using Taq polymerase and primers having the sequence of SEQ ID NO: 211 and SEQ ID NO: 212. This reaction results in a PCR product having the sequence of SEQ ID NO: 111.

The resulting PCR product includes a Xho I site and KEX2 recognition sequence at its 5′ end, and an EcoR I site at its 3′ end. This PCR product is then digested with the restriction endonucleases Xho I and EcoR I. In a separate reaction, the vector pPICGIco is similarly digested with Xho I and EcoR I. The digested PCR product is subcloned into the linearized pPICGIco vector using standard ligation reaction conditions in the presence of T4 DNA ligase. The resulting clone, pPICGIco-hITF15-73(FIG. 6), is identified by restriction endonuclease mapping and DNA sequencing.

Other embodiments of the invention may be generated by following a similar protocol (see, e.g.,FIGS. 7,9, and10). For example, vectors expressing fusion proteins containing alternative fragments of human ITF may be created. In addition, ITF from different species may be used. Desirable embodiments include, but are not limited to, vectors expression fusion proteins containing the following: hITF1-73, hITF21-62, hITF21-70, hITF21-72, hITF21-73, hITF22-62, hITF22-70, hITF22-72, hITF22-73, hITF25-62, hITF25-70, hITF25-72, hITF25-73, pITF1-80, pITF22-80, dITF1-80, dITF22-80, rITF1-81, rITF23-81, mITF1-81, and mITF22-81.

Table 1 lists DNA sequences used in generating several desirable embodiments of the invention (seeFIGS. 13A-21E). For each embodiment represented therein, the sequences shown may be substituted into the protocol described above for generating a vector expressing MFα-hITF15-73. Embodiments 1-10 make use of the pPICGIco vector in the final subcloning step, while embodiments 11-15 instead make use of the pPICpre vector in the final subcloning step.

Variants of the above-described embodiments of the invention are possible. For example, alternative protease cleavage sites may be used in place of the KEX2 site. Cleavage sites recognized by any of the following enzymes would be useful: yeast aspartic protease (Yap3), Type IV dipeptidyl aminopeptidase (DPAP), yeast glycosyl-phosphatidylinositol-linked aspartyl protease (Mkc7), pepsin, trypsin, chymotrypsin, and subtilisin. Yap3 cleaves immediately C-terminal to Arg residues (Bourbonnais et al., Biochimie, 76:226-233, 1994) and cleaves following Arg-Arg and Lys-Arg sites, though it cleaves poorly after three or more consecutive basic residues (Ledgerwood et al., FEBS Lett., 383:67-71, 1996); DPAP cleaves immediately C-terminal to Ala or Pro residues, including Leu-Pro and Val-Pro sites (Brenner et al., Proc Natl Acad Sci U.S.A., 89:922-926, 1992); Mkc7 cleaves immediately C-terminal to Lys-Arg (Komano et al., Proc Natl Acad Sci USA, 92:10752-10756, 1995); pepsin cleaves immediately C-terminal to Tyr, Phe, or Trp residues; trypsin cleaves immediately C-terminal to Arg or Lys residues; and chymotrypsin cleaves immediately C-terminal to Tyr, Phe, or Trp residues.

Vectors of the invention are designed so that, as with the KEX2 site in the vectors described above, no extraneous residues are present between an alternative protease cleavage site and the sequence encoding the ITF polypeptide to be expressed. Cleavage of the resulting MFα-ITF fusion protein may occur in vivo prior to secretion; for example, this could occur with cleavage sites recognized by proteases that occur naturally in the host, such as Yap3, DPAP, or Mkc7. Alternatively, uncleaved fusion protein may be secreted by the host cell if no endogenous enzyme recognizes the cleavage site of the fusion construct; in this case, cleavage may be achieved in vitro in a reaction chamber by contacting purified secreted fusion protein with a purified endoprotease such as pepsin, trypsin, chymotrypsin, subtilisin, or other enzymes. Mature ITF is known to be resistant to the action of such proteases in solution (Kinoshita et al., Mol Cell Biol., 20:4680-4690, 2000) and so will remain intact following the reaction. Subsequently, the resulting ITF polypeptide may be purified away from protease and reaction products.

Additionally, alternative embodiments are possible in which an N-terminal fusion sequence other than an MFα signal sequence or an MFα presequence is used. Any N-terminal fusion sequence that results in secretion of the expressed ITF polypeptide is useful in the methods of the invention. N-terminal fusion sequences that do not result in secretion are also possible; in such cases, the cells in which expression occurs would be lysed prior to protein extraction and purification. Signal and leader sequences for yeast expression include the yeast K28 virus preprotoxin secretion signal sequence (Eiden-Plach et al., Appl Environ Microbiol., 70:961-966, 2004), Sacchromyces cerevisiae acid phosphatase signal sequence (Akeboshi et al., Biosci Biotechnol Biochem., 67:1149-1153, 2003),Aspergillus nigerisopullulanase signal sequence (Akeboshi et al., Biosci Biotechnol Biochem., 67:1149-1153, 2003), chimeric yeast alpha factor andstreptomycesmobaraensis transglataminase propeptide (Yurimoto et al., Biosci Biotechnol Biochem., 68:2058-2069, 2004), modified signal peptide forrhizopus oryzaeglucoamylase (Liu et al, Biochem Biophys Res Commun., 326:817-824, 2005),Kluyveromyces lactiskiller toxin signal sequence (Tokunaga et al., Yeast, 9:379-397, 1993), and Map2 secretion sequence (Giga-Hama et al., Biotech Appl Biochem., 30:235-244, 1999).

In each instance described above, as well as in other embodiments of the invention, standard methods of protein purification may be used. See, for example, the purification methods and activity assays described in Thim et al., Biochemistry, 34:4757-4764, 1995, and U.S. Ser. No. 10/698,572, each of which are hereby incorporated by reference.

Organisms produced according to the invention may be employed in industrial scale production of human ITF, yielding product in quantities and for applications hitherto unattainable.

Expression Of pPICpre-hITF15-73

The pPICpre-hITF15-73construct was transformed intoPichia pastorisstrain GS115(Mut+), and transformants were selected. Expression of hITF15-73(mol weight: ˜6.5 KDa) secreted into the media after 96-120 hours of growth in shake flasks was assessed using SDS-PAGE (FIG. 22). A construct expressing EA-hITF15-73was used as a control. Four GS115 clones were tested, and clone pre-ITF #1 showed expression of a band comparable in size to that of hITF15-73. These data demonstrate that the pPICpre-hITF15-73construct directs expression of hITF15-73.

Expression of pPICGIco-hITF15-73

The pPICGIco-hITF15-73construct was transformed intoPichia pastorisstrains GS115(Mut+), and transformants were selected. Expression of hITF15-73(mol weight: ˜6.5 KDa) secreted into the media after 120 hours of growth in shake flasks was assessed using SDS-PAGE (FIGS. 23A and 24A) and Western blot (FIGS. 23B and 24B). A construct expressing EA-hITF15-73was used as a control. Nine GS115 clones were tested; clones 3-19, 3-24, 3-25, and 3-26 showed expression of a band comparable in size to that of hITF15-73, and Western blotting confirmed that this band contained hITF. The data demonstrate that the pPICGIco-hITF15-73construct directs expression of hITF15-73.

The invention provides ITF expression vectors and methods of their use for treating epithelial cell lesions. Lesions amenable to treatment using the expression products and methods of this invention include epithelial lesions of the dermis and epidermis (skin), alimentary canal including the epithelia of the oral cavity, esophagus, stomach, small and large intestines (anal sphincter, rectum, and colon, particularly the sigmoid colon and the descending colon), genitourinary tract (particularly the vaginal canal, cervix, and uterus), trachea, lungs, nasal cavity, and the eye.

Other Embodiments