Difference between revisions of "Part:BBa K3038001"

Revision as of 12:44, 18 October 2019

Alcohol dehydrogenase - Geobacillus stearothermophilus_Cmyc and 6His tags_Cterminal

Description

Alcohol dehydrogenase, ADH or ADR N-term, is a BioBrick C-Myc and 6-His tagged in N-term.

ADR is a thermophilic NAD+ dependent alcohol dehydrogenase. This enzyme bears mainly an ethanol-dehydrogenase activity.

GenBank

ADR : GenBank: P42327
https://www.ncbi.nlm.nih.gov/protein/P42327

Protein Sequence

MEQKLISEEDLNSAVDHHHHHHVKAAVVNEFKKALEIKEVERPKLEEGEVLVKIEACGVCHTDLHAAHGD WPIKPKLPLIPGHEGVGIVVEVAKGVKSIKVGDRVGIPWLYSACGECEYCLTGQETLCPHQLNGGYSVDG GYAEYCKAPADYVAKIPDNLDPVEVAPILCAGVTTYKALKVSGARPGEWVAIYGIGGLGHIALQYAKAMG LNVVAVDISDEKSKLAKDLGADIAINGLKEDPVKAIHDQVGGVHAAISVAVNKKAFEQAYQSVKRGGTLV VVGLPNADLPIPIFDTVLNGVSVKGSIVGTRKDMQEALDFAARGKVRPIVETAELEEINEVFERMEKGKI NGRIVLKLKED

Reaction

Primary alcohol + NAD+ = Aldehyde + NADH + H+ Secondary alcohol + NAD+ = ketone + H+ + NADH EC:1.1.1.1

Alcohol dehydrogenase; Short= ADH or ADR

Thermophilic NAD+-dependent alcohol dehydrogenase

Bears mainly an ethanol-dehydrogenase activity.

Reaction

a secondary alcohol + NAD+ = a ketone + H+ + NADH EC:1.1.1.1

Protein Sequence

MVKAAVVNEFKKALEIKEVERPKLEEGEVLVKIEACGVCHTDLHAAHGDWPIKPKLPLIPGHEGVGIVVEVAKGVK SIKVGDRVGIPWLYSACGECEYCLTGQETLCPHQLNGGYSVDGGYAEYCKAPADYVAKIPDNLDPVEVAPILCAGV TTYKALKVSGARPGEWVAIYGIGGLGHIALQYAKAMGLNVVAVDISDEKSKLAKDLGADIAINGLKEDPVKAIHDQ VGGVHAAISVAVNKKAFEQAYQSVKRGGTLVVVGLPNADLPIPIFDTVLNGVSVKGSIVGTRKDMQEALDFAARGK VRPIVETAELEEINEVFERMEKGKINGRIVLKLKEDEQKLISEEDLNSAVDHHHHHH

Usage and Biology

PCR amplification of the PCR products

Electrophoresis photography following deposits on agarose gel 0.8% of enzymatic digestion products. The migration was performed at 100 volts for 30 minutes in TAE 1X. The marker used during the migration is the NEB 1 kb Plus Ladder. Lane 1 corresponds to the marker, lane 2 to the digested N-ter,ADR lane 3 to the digested C-ter ADR and lane 4 to the digested plasmid pSB1A3.

Plasmid construction

Design of ADR N-ter/pSB1A3 and ADR C-ter/pSB1A3 with Geneious software. This map shows the pBAD promoter and its terminator flanking the coding sequence of the ADR protein. Also present in N-ter or C-ter are 6-His and c-myc tag. Finally, in the plasmid is present and ampicillin resistance cassette.

Expression of the CMYC-6HIS-ADR and ADR-CMYC-6HIS recombinant proteins

NI : Not induced I: Induced

After sequencing, induction was performed on the thermocompetent bacteria JM109. The objective was to verify if the cloned gene leads to the production of a protein. The expected size of the ADR protein is 40 kDa. A very strong expression of the ADR protein was observed at this size when the pBAD promoter is induced with arabinose. The gene has therefore been correctly cloned into the strain and the protein is produced.

Activity

Sequence and Features