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Penicillin binding protein from Streptococcus pneumoniae

a technology of penicillin and pneumoniae, which is applied in the field of recombinant dna technology, can solve the problems of mdr organisms that are a real threat to humans, children and the elderly, and emergence and rapid spread of beta-lactam resistance in streptococcus pneumoniae, and achieves the effects of reducing the number of mdr organisms, and reducing the number of mdl

Inactive Publication Date: 2002-05-07
ELI LILLY & CO
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The emergence and rapid spread of beta-lactam resistance in Streptococcus pneumoniae has been particularly problematic.
These multi-drug resistant (MDR) organisms are a real threat to humans, particularly children and the elderly.
Without the crosslinking step the peptidoglycan structure is severely weakened and susceptible to degradation.

Method used

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  • Penicillin binding protein from Streptococcus pneumoniae

Examples

Experimental program
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Effect test

example 1

Construction of a DNA Vector for Expressing Streptococcus pneumoniae pbp-nv2.sup.S Gene in a Heterologous Host

Plasmid pJAH242 (See FIG. 1) is an approximately 7600 base pair expression vector suitable for expressing a modified pbp-nv2.sup.S in procaryotic host E. coli. This plasmid contains an origin of replication (Ori), an ampicillin resistance gene (Amp), useful for selecting cells that have incorporated the vector following a transformation procedure, and further comprises the T7 promoter in operable linkage to the coding region of said PBP gene. Parent plasmid pET16b (obtained from Novogen, Madison, Wis.) is linearized by digestion with endonucleases NdeI and EcoRI. Linearized pET16b is ligated to a DNA fragment bearing NdeI and EcoRI cohesive ends, comprising a modified pbp-nv2.sup.S gene. The pbp-nv2.sup.S gene is ligated into pET16b which contains a region just downstream of the ATG initiation codon that encodes 10 histidine residues followed by a factor Xa cleavage site, Il...

example 2

Construction of a DNA Vector for Expression of pbp-nv2 in a Heterologous Host

The plasmid construction method outlined in Example 1 is followed to construct a vector for expressing PBP-Nv2 in a heterologous host such as E. coli.

example 3

Expression of Streptococcus pneumoniae pbp-nv2.sup.S Gene in Escherichia coli

Expression plasmid pJAH242 can be transformed into E. coli BL21 (DE3) (F.sup.- ompT[lon]hsdS r.sub.B.sup.- m.sub.B.sup.-) using standard methods (See e.g. Sambrook et al. Supra). Transformants, chosen at random are tested for the presence of pJAH242 by agarose gel electrophoresis using quick plasmid preparations. Id. Transformants are grown overnight at 37.degree. C. in LB medium supplemented with 100 .mu.g / ml ampicillin. The overnight culture is diluted into fresh LB medium and allowed to grow to an O.D..sub.600 of 0.4 to 0.6. At that point, expression of the vector-bound pbp-nv2.sup.S gene can be induced by adding 0.4 mM IPTG for a period of 3 hours. The induced-culture is then pelleted by centrifugation in preparation for protein purification (See Example 4).

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Abstract

The invention provides isolated nucleic acid compounds encoding a novel high molecular weight PBP of Streptococcus pneumoniae. Also provided are vectors and transformed heterologous host cells for expressing the PBP and a method for identifying compounds that bind and / or inhibit the enzymatic activity of the PBP.

Description

FIELD OF THE INVENTIONThis invention relates to recombinant DNA technology. In particular the invention pertains to the cloning of a gene, pbp-nv2, encoding a novel high molecular weight penicillin binding protein (PBP), PBP-Nv2, from Streptococcus pneumoniae and the use of the gene and its encoded protein in a screen for new inhibitors of bacterial cell wall biosynthesis.BACKGROUNDThe emergence of antibiotic resistance in common pathogenic bacterial species has justifiably alarmed the medical and research communities. The emergence and rapid spread of beta-lactam resistance in Streptococcus pneumoniae has been particularly problematic. This organism is responsible for many respiratory tract infections, and resistance to beta-lactam drugs has been attributed to a modification of one or more of the penicillin-binding proteins (PBPs). Furthermore, penicillin-resistant Streptococcus pneumoniae are frequently resistant to other commonly used antibiotics, such as erythromycin. These mult...

Claims

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Application Information

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Patent Type & Authority Patents(United States)
IPC IPC(8): C07K14/195C07K14/315
CPCC07K14/3156
Inventor HOSKINS, JOANNJASKUNAS, JR., STANLEY RICHARDZHAO, GENSHIROCKEY, PAMELA KAY
Owner ELI LILLY & CO
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