biotechnology symposium 2005 abstracts - Universität Leipzig

4.2 A different approach for cloning and purifi cation
of the 5S subunit of transcarboxylase
multienzyme complex
Rakesh Kumar Bhat, Stefan Berger
Transcarboxylase (EC2.1.3.1) (TC) from Propionibacterium shermanii is a complex
biotin-containing enzyme composed of 30 polypeptides of three different types. It is
composed of six dimeric outer subunits associated with a central cylindrical hexameric
subunit through 12 biotinyl subunits with three outer subunits on each face of the central
hexamer. Each outer dimer is termed a 5S subunit, which is associated with two biotinyl
subunits. The enzyme catalyzes a two-step reaction in which methylmalonyl-CoA and
pyruvate form propionyl-CoA and oxalacetate, the 5S subunit specifi cally catalyzing one
of these reactions. We report here our attempts for cloning, sequencing and expression of
the monomer of the 5S subunit. The gene was identifi ed by matching nucleotide sequences
from the database available, isolated from authentic 5S peptides with the deduced sequence
of an open reading frame present on a cloned P. shermanii genomic fragment. The cloned
5S gene encodes a protein of 519 amino acids, M, 57,793.
Initially we successfully cloned the gene into the pET 28a(+) vector. But there were
problems while purifi cation of the protein. After cleaving the His–Tag, the protein gets
precipated and the cleavage was also not 100 %, which may give problems during NMR
studies. Currently we tried to clone and purify the gene by a different strategy based on
IMPACT-I system.
Rakesh Kumar Bhat
Universität Leipzig
Faculty of Chemistry and Mineralogy
Institute of Analytical Chemistry
E-Mail: bhat@chemie.uni-leipzig.de
www.uni-leipzig.de/~nmr/ANALYTIK/
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