Construction Of Vectors For The Overexpression Of Recombinant Human Growth Hormone In Bacillus Megaterium
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Date
2009-01
Authors
Abd. Rashid, Norhashima
Journal Title
Journal ISSN
Volume Title
Publisher
Universiti Sains Malaysia
Abstract
In order to express the hGH gene in Bacillus megaterium, 3 constructs were
designed. The constructs were named as Construct 1 (M7hGH), Construct 2
(R2L4sphGH) and Construct 3 (spBsubhGH). Construct 1 was designed with
regulatory features [stop codon, ribosome binding site (RBS), TAACA sequence and
start codon] meant for intracellular expression. Construct 2, was created to express
human growth hormone (hGH) extracellularly by using the signal peptide from
Bacillus brevis. For Construct 3, the signal peptide from B. subtilis CU1065 was used
to express hGH extracellularly but the regulatory gene [stop codon, ribosome binding
site (RBS) and start codon between xylA protein and hGH] was excluded to create a
fusion protein combining xylA and the hGH gene. However, no expressions were
obtained from Construct 1 and 2 probably due to the presence of unfavourable
codons. The synthetic hGH gene contains 10 arginine codons (AGA) (4.8%) in which
the frequency is much higher than the 1.7% AGA and 0.9% AGG estimated for E.
coli. Construct 3 yielded an unidentified ~20 kDa protein and further analysis is
required to identify the protein. The average percentage of cells carrying plasmid
was calculated among these 3 constructs in order to determine the plasmid stability
For Construct 1 (M7hGH), the average rate of reduction of cells that carry plasmid is
10% for each generation, Construct 2 (R2L4sphGH) is 5% for each generation and
Construct 3 (spBsubhGH) is 4.8% for each generation.
Description
Keywords
Vectors For The Overexpression , Recombinant Human Growth Hormone In Bacillus Megaterium