Chromosomal sequencing using a PCR-based biotin-capture method allowed isolation of the complete gene for the outer membrane protein A of Klebsiella pneumoniae

Gene
T N NguyenS Ståhl

Abstract

By employing a novel biotin- and PCR-assisted capture method, which allows determination of unknown sequences on chromosomal DNA, the gene for the outer membrane protein A (OmpA) of Klebsiella pneumoniae has been isolated and sequenced to completion. The method involves linear amplification of DNA from a biotinylated primer annealing to a region with known sequence. After capture of the amplified single-stranded DNA on to paramagnetic beads, unspecifically annealing primers, i.e. arbitrary primers, were used to generate sequences with only partly determined nt sequences. The homology of the sequenced gene to ompA of related bacteria is discussed, and the gene fragment was assembled for intracellular expression in Escherichia coli, and two different fusion proteins were produced and recovered with good yields. The importance of the novel chromosomal sequencing method for gene isolation in general and the potential use of the OmpA fusion proteins are discussed.

References

Jul 1, 1976·European Journal of Immunology·G Köhler, C Milstein
Jan 1, 1992·Trends in Biotechnology·M UhlénT Moks
Aug 1, 1991·Journal of General Microbiology·J G LawrenceD L Hartl
Jun 11, 1991·Nucleic Acids Research·J D ParkerG C Burmer
Apr 1, 1988·Journal of Molecular Recognition : JMR·P A NygrenE Palmcrantz
May 1, 1994·Trends in Biotechnology·P A NygrenM Uhlén
May 1, 1997·Trends in Biotechnology·S Ståhl, M Uhlén
Nov 5, 1997·Protein Expression and Purification·J NilssonP A Nygren

❮ Previous
Next ❯

Citations

Apr 12, 2000·Journal of Immunological Methods·C AnderssonS Ståhl
Jul 27, 2001·Journal of Immunological Methods·C AnderssonS Ståhl
Jun 1, 2002·Journal of Biotechnology·Patrik SamuelsonStefan Ståhl
May 19, 2000·Infection and Immunity·E Ofori-DarkoJ L Merchant
Feb 10, 2000·The Journal of Peptide Research : Official Journal of the American Peptide Society·A BeckA Van Dorsselaer
Feb 20, 2002·Biologicals : Journal of the International Association of Biological Standardization·A BeckN Corvaïa
Jun 3, 2005·The Journal of Biological Chemistry·Emilisa FrirdichChris Whitfield
Mar 20, 2001·The Journal of Immunology : Official Journal of the American Association of Immunologists·I MiconnetT Renno

❮ Previous
Next ❯

Related Concepts

Trending Feeds

COVID-19

Coronaviruses encompass a large family of viruses that cause the common cold as well as more serious diseases, such as the ongoing outbreak of coronavirus disease 2019 (COVID-19; formally known as 2019-nCoV). Coronaviruses can spread from animals to humans; symptoms include fever, cough, shortness of breath, and breathing difficulties; in more severe cases, infection can lead to death. This feed covers recent research on COVID-19.

Blastomycosis

Blastomycosis fungal infections spread through inhaling Blastomyces dermatitidis spores. Discover the latest research on blastomycosis fungal infections here.

Nuclear Pore Complex in ALS/FTD

Alterations in nucleocytoplasmic transport, controlled by the nuclear pore complex, may be involved in the pathomechanism underlying multiple neurodegenerative diseases including Amyotrophic Lateral Sclerosis and Frontotemporal Dementia. Here is the latest research on the nuclear pore complex in ALS and FTD.

Applications of Molecular Barcoding

The concept of molecular barcoding is that each original DNA or RNA molecule is attached to a unique sequence barcode. Sequence reads having different barcodes represent different original molecules, while sequence reads having the same barcode are results of PCR duplication from one original molecule. Discover the latest research on molecular barcoding here.

Chronic Fatigue Syndrome

Chronic fatigue syndrome is a disease characterized by unexplained disabling fatigue; the pathology of which is incompletely understood. Discover the latest research on chronic fatigue syndrome here.

Evolution of Pluripotency

Pluripotency refers to the ability of a cell to develop into three primary germ cell layers of the embryo. This feed focuses on the mechanisms that underlie the evolution of pluripotency. Here is the latest research.

Position Effect Variegation

Position Effect Variagation occurs when a gene is inactivated due to its positioning near heterochromatic regions within a chromosome. Discover the latest research on Position Effect Variagation here.

STING Receptor Agonists

Stimulator of IFN genes (STING) are a group of transmembrane proteins that are involved in the induction of type I interferon that is important in the innate immune response. The stimulation of STING has been an active area of research in the treatment of cancer and infectious diseases. Here is the latest research on STING receptor agonists.

Microbicide

Microbicides are products that can be applied to vaginal or rectal mucosal surfaces with the goal of preventing, or at least significantly reducing, the transmission of sexually transmitted infections. Here is the latest research on microbicides.