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  • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
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  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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  • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
  • C07K14/4732—Casein
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  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/64—General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host
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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/67—General methods for enhancing the expression
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  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/70—Vectors or expression systems specially adapted for E. coli
• • C—CHEMISTRY; METALLURGY
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  • C07K2319/00—Fusion polypeptide
  • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
  • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence

Definitions

• human ⁇ —casein The primary structure of human ⁇ —casein was determined by Greenberg et al . (Journal of Biological Chemistry 259:5132-5138, 1984) . It was shown to be a phosphorylated protein with phosphorylation sites at specific seryl and threonyl residues located near the amino terminus. Comparison of human and bovine ⁇ - caseins showed 47% identity. The sequence of human
• Hansson et al demonstrated that recombinant human ⁇ - casein was expressed in the yeast, S . cerevisiae, using the pYES 2.0 vector (Invitrogen Corp., San Diego, CA) . Production levels were estimated to be approximately 10% of the production found in E. coli .
• recombinant ⁇ -casein obtained from S . cerevisiae a eukaryotic cell that has endogenous enzymes capable of phosphorylating proteins, was phosphorylated, but the protein produced by E. coli , a prokaryotic cell that lacks the ability in its native state to phosphorylate, was non-phosphorylated.
• recombinant human casein kinase II (rhCKII) produced in and purified from E. coli can phosphorylate protein substrates in vi tro (Shi et al. , Proceeding of the National Academy of Sciences, USA 91:2767-2771, 1994) .
• One specific embodiment of the present invention uses a nucleotide sequence encoding a recombinant human casein kinase II in a single construct with nucleotide sequence encoding ⁇ -casein to transform E. coli and produce phosphorylated ⁇ -casein.
• exogenous proteins capable of being modified through the process of the present invention include but are not limited to human caseins, including ⁇ -casein, cell receptor proteins, fatty acylated proteins including palmitoylated proteins, mammalian muscle proteins, the gag polyproteins of retroviruses, and mammalian proteins targeted by retroviral src kinases.
• Transmembrane glycoproteins that acquire covalent palmitate after synthesis include the insulin, ⁇ 2 -adrenergic and transferrin receptors.
• Figure 1 shows physical maps of expression vectors pS637 and pRJB-6 constructed for inducible intracellular expression in E. coli . 191 base pairs were removed from pS637 to produce PRJB-6.
• lane 2 native human ⁇ -casein
• lane 3 induced HMS174 (DE3 )pLysS (pRJB-9)
• lane 4 induced HMS174 (DE3 )pLysS (pS637)
• lane 5 induced HMS174 (DE3 )pLysS (pS637)
• Nucleotide sequences encoding ⁇ —casein in several different expression formats, were evaluated for expression of recombinant human ⁇ -casein in an E. coli strain. After a series of experiments, it was determined that recombinant human ⁇ -casein was efficiently phosphorylated when sequences encoding human ⁇ —casein were placed in a single construct with sequences encoding human casein kinase CKII ⁇ . Efficiency of phosphorylation was not compromised when both genes were placed in tandem in one plasmid when compared with experimental systems in which sequences encoding the kinase and the ⁇ -casein were placed in two separate vectors.
• Electrophoresis and Detection of Recombinant ⁇ -Casein Cells were pelleted by centrifugation and the pellet from 1 ml of culture was dissolved in 100 ⁇ l of sample buffer, which contains Tris, glycerol, SDS, dithiothreotol (DTT) , and bromophenol blue.
• sample buffer which contains Tris, glycerol, SDS, dithiothreotol (DTT) , and bromophenol blue.
• the proteins were separated by SDS-PAGE as described in Laemmli (Nature 227:680-685, 1970) .
• Gradient gels were cast and run in the discontinuous buffer system in a Protean (Bio-Rad, Richmond, CA) electrophoresis unit. Gels were stained as described in Laemmli. Immunoblotting was performed according to the specifications of the manufacturer (Bio-Rad) .
• the Western blot shown in Figure 8 shows reduced production of recombinant CKII ⁇ -casein by induced cells containing pRJB-7 when compared with cells containing pRJB-9. This is seen by comparing lane 4 (induced pRJB-7) with lane 8 (induced pRJB-9) . Although both pRJB-7 and pRJB-9 are derived from pS637, only pRJB-9 produced amounts of CKII ⁇ -casein equivalent to the parent construct. The presence of an additional T7 promoter before the CKII genes in the hybrid construct had the effect of both reducing cell growth and consequently reducing recombinant protein production.
• Figure 9 shows a Western blot analysis in which the lysates were developed with phosphoserine antibody to detect phosphorylated protein. Induced E. coli
• HMS174 (DE3)LysS cells containing pET-lld- CKII ⁇ , pRJB-9 (hybrid construct with one T7 promoter) , pRJB-7 (hybrid construct with two T7 promoters) , or pS637 (contains ⁇ -casein encoding sequence but not CKII ⁇ encoding sequence) were compared for production of phosphorylated recombinant ⁇ -casein. Phosphorylated ⁇ - casein was produced only in cells containing pRJB-9 (lane 6) . No phosphorylated protein was detected in lane 7, which contains the lysate of cells containing pRJB-7.
• Example 4 Production of human ⁇ -casein in E. coli K-12: Construct pRJB-9 containing both ⁇ -casein encoding sequence and CKII ⁇ encoding sequences
• the present invention uses a single construct expressing both the information for transferring functional groups to specific sites and the protein to be modified.
• the transferred functional group is phosphate.
• the transfer is accomplished by a kinase that is demonstrated to mediate phosphorylation of specific sites on recombinant human ⁇ -casein in vivo .
• This invention demonstrates that not only can human ⁇ —casein be specifically phosphorylated in vivo by E. coli , but that a single-construct with a promoter located before the sequence encoding ⁇ -casein and having the advantages of a single-construct system can successfully mediate this function.
• H. influenzae has been identified as a causative factor for otitis media (Murray et al . , 1994) . Since it has been demonstrated in the experiments described above that recombinant human ⁇ -casein phosphorylated in at least three sites under the direction of the plasmid of the invention inhibits adhesion of H. influenzae to human cells, it is concluded that phosphorylated recombinant human ⁇ -casein, as described above, may be used in the prevention and treatment of otitis media in humans, particularly in human infants.
• This invention will allow commercial-scale production of phosphorylated, recombinant mammalian proteins in microorganisms.
• the method of the invention can be used to produce recombinant exogenous proteins, including but not limited to, recombinant human ⁇ — casein, in large quantities.
• Phosphorylation of ⁇ - casein in a bioreactor makes possible large-scale synthesis in a fermentor of recombinant ⁇ -casein that is equivalent to native human ⁇ -casein. This will facilitate the production of infant formula containing human ⁇ -casein in its native phosphorylated state.
• the method of the invention can also be used for phosphorylation of cell proteins, including receptors which are regulated by phosphorylation and dephosphorylation and thereby act as signals in cell metabolism.
• the invention provides a cost-effective method of phosphorylating peptide receptors and will be useful in the manufacture of pharmaceutical drugs.
• the single plasmid system is preferable to a two-plasmid system for industrial production of fermented proteins such as recombinant, phosphorylated human ⁇ —casein.
• MOLECULE TYPE genomic DNA

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• 1996
  • 1996-02-27 EP EP96906664A patent/EP0812362A1/de not_active Withdrawn
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