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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: 2003-10-07
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 pnuemoniae pbp-nv.sup.S Gene in a Heterolocous Host

Plasmid JAH142 (See Figure) is an approximately 7300 base pair expression vector suitable for expressing a modified pbp-nv.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 tranformation procedure, and further comprises the T7 promoter in operable linkage to the coding region of said PBP gene. Parent plasmid pET11A (obtained from Novogen, Madison, Wis.) was linearized by digestion with endonucleases NdeI and HindIII. Linearized pET11A was ligated to a DNA fragment bearing NdeI and HindIII sticky ends, comprising a modified pbp-nv.sup.s gene. The pbp-nv.sup.s gene ligated into pJAH142 was modified at the 5' end in order to simplify purification of the encoded protein product. For this purpose, an oligonucleotide encoding 8 histidi...

example 2

Construction of a DNA Vector for Expression of pbp-nv in a heteroloaous host

The plasmid construction method outlined in Example 1 is followed to construct a vector for expressing PBP-Nv in a heterologous host such as E. coli. A "his-tag" oligonucleotide comprising the same structure and sequence disclosed in Example 1 is inserted at the 3' end of the ATG initiation codon at nucleotide position 3 of SEQ ID NO.1.

example 3

Expression of Streptococcus pneumoniae pbp-nvS Gene in Echerichia coli

Expression plasmid JAH142 was 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 were tested for the presence of pJAH142 by agarose gel electrophoresis using quick plasmid preparations. Id. Transformants were grown overnight at 37.degree. C. in LB medium supplemented with 100 .mu.g / ml ampicillin. The overnight culture was 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-nvS gene was induced by adding 0.4 mM IPTG for a period of 3 hours. The induced-culture was 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

BACKGROUND OF THE INVENTIONThis invention relates to recombinant DNA technology. In particular the invention pertains to the cloning of a gene, pbp-nv, encoding a novel high molecular weight penicillin binding protein (PBP), PBP-Nv, from Streptococcus pneumoniae and the use of said gene and its encoded protein in a screen for new inhibitors of bacterial cell wall biosynthesis.The 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 multi-drug...

Claims

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Application Information

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Patent Type & Authority Patents(United States)
IPC IPC(8): C07K14/195C07K14/315A61K38/00G01N33/50C12N1/15C12N1/19C12N1/21C12N5/10C12N15/09C12N15/31C12P21/02C12R1/46G01N33/15G01N33/566
CPCA61K38/00C07K14/3156
Inventor HOSKINS, JO ANNJASKUNAS, JR., S. RICHARDZHAO, GENSHIROCKEY, PAMELA K.ROSTECK, JR., PAUL R.NORRIS, FRANKLIN H.
Owner ELI LILLY & CO
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