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"Fusion Protein Containing a Single-Stranded Dna Binding Protein and Methods for Expression and Purification of the Same" in Patent Application...

September 9, 2014



"Fusion Protein Containing a Single-Stranded Dna Binding Protein and Methods for Expression and Purification of the Same" in Patent Application Approval Process

By a News Reporter-Staff News Editor at Life Science Weekly -- A patent application by the inventor Kong, Daochun (Rockville, MD), filed on December 15, 2013, was made available online on August 28, 2014, according to news reporting originating from Washington, D.C., by NewsRx correspondents (see also Patents).

This patent application has not been assigned to a company or institution.

The following quote was obtained by the news editors from the background information supplied by the inventors: "Due to the availability of entire genomic DNA sequences in numerous organisms and the advance of recombinant DNA technology, it has become simple to clone any genes and subsequently overexpress them in prokaryotic or eukaryotic cells. The biochemical elucidation of protein functions requires obtaining proteins with high purity.

"A number of approaches have been developed for the isolation and purification of proteins, particularly recombinant proteins, from other components of a biological sample. The approaches include ion exchange chromatography based on molecular charges, gel filtration based on molecular size, and affinity chromatography. The affinity chromatography is more specific and much more efficient than the other purification approaches because it makes use of the specific affinity of a protein for a purifying reagent such as an antibody or ligand to which it specifically binds. One member of a binding pair may be used to 'tag' a protein of interest, with the other member used as an affinity ligand. Such a protein 'tag' may be 'fused' recombinantly and expressed to produce a fusion protein with the tag attached. The 'tagged' fusion protein is then affinity purified by interaction with the binding partner of the tag and the tag is then optionally cleaved to release pure protein. However, the known affinity chromatography suffers from the drawbacks such as unsatisfactory purity, time-consuming, high cost and/or unusual elution conditions."

In addition to the background information obtained for this patent application, NewsRx journalists also obtained the inventor's summary information for this patent application: "Therefore, there is a need to provide a method and system for simply, efficiently and economically expressing and purifying recombinant proteins.

"One aspect of the present invention provides an expression vector comprising a promoter and a polynucleotide sequence encoding a fusion protein; wherein the fusion protein-encoding polynucleotide sequence is so operably linked to the promoter that the transcription of the fusion protein-encoding polynucleotide sequence is controlled by the promoter; wherein the fusion protein comprises a single-stranded DNA-binding protein and an interest protein or polypeptide fused directly or indirectly with the COOH-terminus or NH.sub.2-terminus of the single-stranded DNA-binding protein; and wherein the fusion protein is capable of binding to single-stranded DNA.

"Another aspect of the present invention provides a method for purification of an interest protein. In one embodiment, the method comprises contacting a host cell with an expression vector, wherein the expression vector comprises a promoter and a polynucleotide sequence operably linked to the promoter, wherein the polynucleotide sequence encodes a fusion protein comprising a single-stranded DNA binding protein and the interest protein fused directly or indirectly with the COOH-terminus or NH.sub.2-terminus of the single-stranded DNA binding protein, and wherein the fusion protein is capable of binding to single-stranded DNA; culturing the host cell under such conditions that the fusion protein is expressed; lysing the host cell to obtain a cell lysate; contacting, fire cell lysate with a substrate immobilized with single-stranded DNA to allow the fusion protein to bind to the single-stranded DNA of the substrate; washing the substrate to remove impurities; and eluting the bound fusion protein from the substrate; thereby the interest protein is expressed and purified in the form of the fusion protein.

"Another aspect of the present invention provides a fusion protein comprising a single-stranded DNA binding protein and an interest protein or polypeptide fused directly or indirectly with the COOH-terminus or NH.sub.2-terminus of the single-stranded DNA binding protein, wherein said fusion protein is capable of binding to single-stranded DNA; thereby the fusion protein can be purified by a substrate immobilized with single-stranded DNA.

"The present invention, has apparent advantages. First, the fusion protein is soluble and can be purified from cell lysates under physiological condition by affinity chromatography on a column of single-stranded DNA (ssDNA) cellulose. Second, the separation of the said fusion protein from other proteins and impurities existed in cell lysates through one step of ssDNA-cellulose chromatography is highly efficient, apparently more efficient than the other affinity chromatography such as Ni.sup.2--agarose column. Third, the fusion protein can be elated from ssDNA-cellulose by just raising salt (NaCl or KCl) concentration. The recovery of the said fusion proteins from ssDNA-cellulose is very efficient and simple.

"The objectives and advantages of the invention will become apparent from the following detailed description of preferred embodiments thereof in connection with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

"Preferred embodiments according to the present invention will now be described with reference to the Figures, in which like reference numerals denote like elements.

"FIG. 1A shows the structure of the series of expression plasmid vectors of pSSB-B, including the schematic representation of pSSB-B1. This series of plasmids are applicable in bacteria. Also shown are the specific protease cleavage sites among the vectors of pSSB-B1, pSSB-B2, pSSB-B3, pSSB-B4. An additional 6His-tag is located at the N-terminus of the SSB in pSSB-B3 and pSSB-B4 vectors. The multiple enzymatic linkers in pSSB-B1, pSSB-B2, pSSB-B3 and pSSB-B4 are listed as SEQ ID NOs 3, 4, 5, or 6 respectively.

"FIG. 1B shows the structure of the series of expression vectors of pSSB-Y, including the schematic representation of pSSB-Y1. This series of plasmids are applicable in the fission yeast S. pombe. The positions of 6 His tag, SSB, enterokinase or thrombin cleavage site, and multiple cloning sites are also indicated in a portion of pSSB-Y1 or pSSB-Y2 expression vector. The multiple enzymatic linkers in pSSB-Y1 and pSSB-Y2 are listed as SEQ ID NOs 7 or 8 respectively.

"FIG. 1C shows the structure of the series of expression vectors of pSSB-1, including the schematic representation of pSSB-I1. This series of plasmids are applicable in insect cells. Also shown are the specific protease cleavage sites among the vectors of pSSB-I1, pSSB-I2, pSSB-I3, pSSB-I4. An additional 6His-tag is located at the N-terminus of the SSB in pSSB-I3 and pSSB-I4 vectors. The multiple enzymatic linkers in pSSB-I1, pSSB-I2, pSSB-I3 and pSSB-I4 are listed as SEQ ID NOs 9, 10, 11 or 12 respectively.

"FIG. 1D shows the structure of the series of expression vectors of pSSB-H, including the schematic representation of pSSB-H1. This series of plasmids are applicable in human cells. Also shown are the specific protease cleavage sites among the vectors of pSSB-H1, pSSB-H2, pSSB-H3, and pSSB-H4. An additional 6His-tag is located at the N-terminus of the SSB pSSB-H3 and pSSB-H4 vectors. The multiple enzymatic linkers in pSSB-H1, pSSB-H2, pSSB-H3 and pSSB-H4 are listed as SEQ NOs 13, 14, 15 or 16 respectively.

"FIG. 2 shows the expression level of the fusion protein 6His-SSB-Sap1, 6His-Sap1 and 6His-GST-Sap1 that were overexpressed in E. coli BL21(DE3)pLys cells. All these three fusion proteins were soluble in cell lysate as indicated in the lanes of soluble portion. A null control without the expression of the fusion proteins is also shown. The MW in all figures is an abbreviation of molecular weight. The kDa is an abbreviation of kilo-Dalton.

"FIG. 3A shows the purification of the fusion protein 6His-SSB-Sap1 through ssDNA-cellulose column. The total cell extracts were applied to the ssDNA-cellulose column. The unbound proteins and other impurities went through the column. The bound 6His-SSB-Sap1 was eluted from ssDNA-cellulose at .about.0.4 M KCl.

"FIG. 3B shows that 6His-SSB-Sap1 that was already purified by ssDNA-cellulose chromatography bound to Ni.sup.2+-agarose well and was eluted at 250 mM imidazole. This result indicates that 6His-SSB-Sap1 can be further purified with Ni.sup.2+-agarose column if it is needed.

"FIG. 4 shows the purification of the fusion protein SSB-Fen1 that was in cell lysate through ssDNA-cellulose chromatography. The Pen1 is S. pombe flap endonuclease 1. The fusion protein SSB-Fen1 was overexpressed in E. coli cells. The cell extract containing the SSB-Fen1 was applied to ssDNA-cellulose column. The bound SSB-Fen1 was eluted at .about.0.4 M KCl.

"FIG. 5 shows the purification of 6His-GST-Sap1 from cell lysate through glutathione-sepharose 4B. The fusion protein 6His-GST-Sap1 was overexpressed in E. coli cells. The cell extract containing the 6His-GST-Sap1 was applied to Glutathione Sepharose 4B column. After the column was thoroughly washed with a buffer without glutathione, the bound 6His-GST-Sap1 was first eluted with 10 mM glutathione. The 6His-GST-Sap1 that remained binding to the column of Glutathione Sepharose 4B was further eluted by 0.5% SDS.

"FIG. 6 shows the purification of 6His-Sap1 from cell lysate through Ni.sup.2+-NTA agarose column. 6His-Sap1 was overexpressed in E. coli cells. The cell extract containing 6His-Sap1 was applied to Ni.sup.2+-NTA agarose column and the column was washed with several column volumes of buffer containing 20 mM imidazole to remove unbound proteins and other impurities. The bound 6His-Sap1 was eluted at 250 mM imidazole.

"FIG. 7 shows the purification of SSB-Sap1 from human cell lysate. SSB-Sap1 was overexpressed in human cells. The human cell extract containing SSB-Sap1 was applied to ssDNA-cellulose column. By step elution, the majority of the bound SSB-Sap1 was eluted at salt concentration of 0.2 to 1.0 M KCl. The SSB-Sap1 lane in FIG. 7-9 indicates the position of this protein in SDS-PAGE gel.

"FIG. 8 shows the purification of SSB-Sap1 from insect cell lysate. SSB-Sap1 was overexpressed in insect cells. The insect cell extract containing SSB-Sap1 was applied to ssDNA-cellulose column. The majority of the bound SSB-Sap1 was elated at salt concentration of 0.2 to 1.0 M KCl.

"FIG. 9 shows the purification of SSB-Sap1 from yeast cell extract. SSB-Sap1 was overexpressed in the fission yeast S. pombe cells. The yeast cell extract containing SSB-Sap1 was applied to ssDNA-cellulose column. The majority of the bound SSB-Sap1 was elated at salt concentration of 0.2 to 1.0 M KCl.

"FIG. 10 shows the cleavage of 6His-SSB-Sap1 by thrombin and the removal of 6His-SSB by Ni.sup.2+-NTA agarose column."

URL and more information on this patent application, see: Kong, Daochun. Fusion Protein Containing a Single-Stranded Dna Binding Protein and Methods for Expression and Purification of the Same. Filed December 15, 2013 and posted August 28, 2014. Patent URL: http://appft.uspto.gov/netacgi/nph-Parser?Sect1=PTO2&Sect2=HITOFF&u=%2Fnetahtml%2FPTO%2Fsearch-adv.html&r=1872&p=38&f=G&l=50&d=PG01&S1=20140821.PD.&OS=PD/20140821&RS=PD/20140821

Keywords for this news article include: Amino Acids, DNA Research, DNA-Binding Proteins, Glutathione, Oligopeptides, Patents, Peptides.

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Source: Life Science Weekly


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