Co-expression of the Thermotoga neapolitana aglB gene with an upstream 3'-coding fragment of the malG gene improves enzymatic characteristics of recombinant AglB cyclomaltodextrinase.
A cluster of Thermotoga neapolitana genes participating in starch degradation includes the malG gene of sugar transport protein and the aglB gene of cyclomaltodextrinase. The start and stop codons of these genes share a common overlapping sequence, aTGAtg. Here, we compared properties of expression products of three different constructs with aglB from T. neapolitana. The first expression vector contained the aglB gene linked to an upstream 90-bp 3'-terminal region of the malG gene with the stop codon overlapping with the start codon of aglB. The second construct included the isolated coding sequence of aglB with two tandem potential start codons. The expression product of this construct in Escherichia coli had two tandem Met residues at its N terminus and was characterized by low thermostability and high tendency to aggregate. In contrast, co-expression of aglB and the 3'-terminal region of malG (the first construct) resulted in AglB with only one N-terminal Met residue and a much higher specific activity of cyclomaltodextrinase. Moreover, the enzyme expressed by such a construct was more thermostable and less prone to aggregation. The third construct was the same as the second one except that it contained only one ATG start codon. The product of its expression had kinetic and other properties similar to those of the enzyme with only one N-terminal Met residue.
Lunina
Natalia A
NA
Institute of Molecular Genetics, Russian Academy of Sciences, 2 Kurchatov Sq., Moscow 123182, Russia. lumina@img.ras.ru
Agafonova
Elena V
EV
Chekanovskaya
Lyudmila A
LA
Dvortsov
Igor A
IA
Berezina
Oksana V
OV
Shedova
Ekaterina N
EN
Kostrov
Sergey V
SV
Velikodvorskaya
Galina A
GA
D018528:ATP-Binding Cassette Transporters / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D006026:Glycoside Hydrolases / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D020539:Sequence Analysis, Protein; D046949:Thermotoga neapolitana / Q000201:enzymology* / Q000235:genetics
D018528:ATP-Binding Cassette Transporters; D011993:Recombinant Fusion Proteins; D006026:Glycoside Hydrolases; C048745:cyclomaltodextrinase