Claims
- 1. A nucleic acid segment encoding a fusion protein, wherein the segment comprises:
a β-lactamase inhibitor protein; and an affinity carrier forming a fusion protein with said β-lactamase inhibitor protein.
- 2. The nucleic acid segment of claim 1, wherein said nucleic acid segment further comprises a recombinant vector.
- 3. The nucleic acid segment of claim 1, wherein said nucleic acid segment further comprises a recombinant expression vector.
- 4. The nucleic acid segment of claim 1, wherein said nucleic acid segment further comprises an operatively linked promoter.
- 5. The nucleic acid segment of claim 3, wherein said operatively linked promoter comprises the β-lactamase promoter.
- 6. The nucleic acid segment of claim 1, wherein said nucleic acid segment further comprises a signal peptide amino from said β-lactamase inhibitor protein.
- 7. A recombinant host comprising:
a recombinant vector comprising a gene encoding a fusion protein, said fusion protein comprising:
a signal peptide; a β-lactamase inhibitor protein carboxy from said signal peptide; and an affinity carrier, wherein said affinity carrier and said β-lactamase inhibitor protein form a fusion protein.
- 8. The host of claim 7, wherein said recombinant host cell comprises a prokaryotic cell.
- 9. The host of claim 7, wherein said recombinant host cell comprises an E. coli.
- 10. The host of claim 7, wherein said recombinant host cell comprises an M13 phage.
- 11. The host of claim 7, wherein said signal peptide comprises the signal peptide of β-lactamase.
- 12. The host of claim 7, wherein said recombinant vector further comprises a low-level expression promoter upstream from said gene encoding said fusion protein.
- 13. The host of claim 12, wherein said low-level expression promoter comprises the β-lactamase promoter.
- 14. A purified nucleic acid segment encoding a fusion protein, wherein the segment comprises:
a β-lactamase signal sequence; a β-lactamase inhibitor protein carboxy from said β-lactamase signal sequence; and a viral coat protein carboxy from said β-lactamase inhibitor protein, wherein said nucleic acid encodes a viral coat protein that forms part of a phage viral coat.
- 15. The nucleic acid segment of claim 14, wherein said nucleic acid segment further comprises a recombinant vector.
- 16. The nucleic acid segment of claim 14, wherein said nucleic acid segment further comprises a recombinant expression vector.
- 17. The nucleic acid segment of claim 14, wherein said nucleic acid segment further comprises an operatively linked promoter.
- 18. The nucleic acid segment of claim 17, wherein said operatively linked promoter comprises the β-lactamase promoter.
- 19. A polypeptide composition comprising:
a β-lactamase inhibitor protein; and an affinity carrier, wherein said affinity carrier is formed as a fusion protein with said β-lactamase inhibitor protein.
- 20. The polypeptide of claim 19, wherein said affinity carrier comprises g3P.
- 21. The polypeptide of claim 19, wherein said affinity carrier comprises a maltose binding protein.
- 22. The polypeptide of claim 19, wherein said affinity carrier comprises a histidine-tag.
- 23. The polypeptide of claim 19, wherein said polypeptide is expressed in a prokaryotic cell.
- 24. The polypeptide of claim 19, wherein said polypeptide is expressed in E. coli.
- 25. A method of isolating an antimicrobial agent comprising the steps of:
displaying a β-lactamase inhibitor protein on a virus; contacting said virus with a β-lactamase binding protein target; selecting for the virus that has a higher affinity for the target; and testing said β-lactamase inhibitor protein for antimicrobial activity.
- 26. The method of isolating an antimicrobial agent of claim 25, wherein said step of β-lactamase inhibitor protein for antimicrobial activity comprises measuring the growth rate of bacteria to determine if the β-lactamase inhibitor protein is reducing the rate of bacterial cell growth.
- 27. The method of isolating an antimicrobial agent of claim 25, wherein said step of displaying a β-lactamase inhibitor protein is on an M13 phage.
- 28. The method of isolating an antimicrobial agent of claim 25, wherein said step of contacting said virus with a β-lactamase binding protein target comprises immobilizing said target to a solid support.
- 29. The method of isolating an antimicrobial agent of claim 25, wherein said step of contacting said virus with a β-lactamase binding protein target comprises immobilizing said target to oxirane beads.
- 30. The method of isolating an antimicrobial agent of claim 25, wherein said step of selecting for the virus that has a higher affinity for the target further comprises the steps of:
washing said β-lactamase binding protein phage-target complex in a buffer comprising a pH buffered salt solution at about physiologic pH; and eluting said β-lactamase binding protein phage.
- 31. The method of isolating an antimicrobial agent of claim 30, wherein said step of eluting said β-lactamase binding protein phage occurs under conditions that do not significantly affect phage viability.
- 32. The method of isolating an antimicrobial agent of claim 25, wherein said step of testing said β-lactamase inhibitor protein for antimicrobial activity is further defined as comprising the steps of:
contacting the isolated antimicrobial agent with a bacterium; and measuring the viability of the bacterium after a predetermined period of time sufficient to determine said viability.
- 33. A system of identifying, selecting and improving an antimicrobial agent comprising the steps of:
(a) creating a mutant β-lactamase inhibitor protein phage display library; (b) selecting mutant β-lactamase inhibitor protein phage by comparing one or more characteristics of the mutant β-lactamase inhibitor display phage; (c) cloning the selected mutant β-lactamase inhibitor protein phage; (d) conducting mutagenesis on the selected mutant β-lactamase inhibitor protein phage to create a new mutant β-lactamase inhibitor protein phage display:
(d1) evaluating the performance of each β-lactamase inhibitor protein phage by panning for those having a high affinity for a binding target, (d2) eliminating β-lactamase inhibitor protein phage whose performance is less than a specified performance level, and (d3) selecting mutants from the mutant β-lactamase inhibitor protein phage, each mutants of mutant β-lactamase inhibitor protein phage having antimicrobial performance that is equal to or greater than the specified performance level; and (e) repeating steps (b) and (c).
- 34. The system of claim 33, wherein the step of cloning the mutant β-lactamase inhibitor protein phage further includes determining the nucleic acid sequence of the mutant β-lactamase inhibitor protein.
- 35. The system of claim 33, further comprising the step of truncating the mutant β-lactamase inhibitor protein prior to repeating steps (b) and (c).
- 36. The system of claim 33, wherein said binding target used in the step of evaluating the performance of each β-lactamase inhibitor protein phage by panning for those having a high affinity for a binding target comprises one or more β-lactamases.
- 37. The system of claim 33, wherein said binding target used in the step of evaluating the performance of each β-lactamase inhibitor protein phage by panning for those having a high affinity for a binding target comprises one or more penicillin binding proteins.
- 38. The system of claim 33, wherein the step of eliminating β-lactamase inhibitor protein phage whose performance is less than a specified performance level is further defined as comprising the steps of:
isolating the β-lactamase inhibitor protein displayed on the phage to obtain an antimicrobial agent; contacting said isolated antimicrobial agent with a bacterium; and measuring the viability of the bacterium after a predetermined period of time sufficient to determine said viability.
Government Interests
[0001] The government owns certain rights in the present invention pursuant to grant number A132956 from the National Institutes of Health.