Polyelectrolyte precipitation of beta-galactosidase fusions containing poly-aspartic acid tails.
Protein recovery from industrial microbial processes can be very expensive, often exceeding the cost of protein production. We have genetically engineered 3 beta-galactosidase (beta-gal) fusion proteins containing poly-aspartic acid tails to test the effect of the tails on recovery by the relatively inexpensive method of polyelectrolyte precipitation. The fusion proteins, designated T1, T2, and T3, were constructed with C-terminal tails of 5, 11, and 16 aspartic acid residues, respectively. The fusion proteins were expressed in Escherichia coli, and purified by affinity chromatography. T1 and T2 had specific activities similar to that of wildtype beta-gal, whereas the specific activity of T3 was about half that of T1 and T2. The increased net charge of the fusion proteins compared to wildtype beta-gal was indicated both by ion-exchange chromatography and their migration pattern in non-denaturing polyacrylamide gel electrophoresis. All three tails enhanced polyethyleneimine (PEI) precipitation of the fusion proteins compared to wildtype beta-gal. At a low PEI/protein ratio (0.01, g g-1), recovery by precipitation of T2 and T3 was more than 2 X that of the beta-gal control, whereas that of T1 was only slightly greater than that of the control. At a higher PEI/protein ratio (0.03, g g-1) the amount of precipitation of all three fusion proteins was nearly the same, about 1.5 X that of the control.
Department of Genetics, Iowa State University, Ames 50011.
J Y
JY
Zhao
C F
CF
Ford
C E
CE
Glatz
M A
MA
Rougvie
S M
SM
Gendel
D001224:Aspartic Acid / Q000737:chemistry; D001483:Base Sequence; D011232:Chemical Precipitation; D004573:Electrolytes; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D011094:Polyethyleneimine; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification; D013379:Substrate Specificity; D001616:beta-Galactosidase / Q000737:chemistry / Q000302:isolation & purification*
D004573:Electrolytes; D011993:Recombinant Fusion Proteins; D001224:Aspartic Acid; D011094:Polyethyleneimine; D001616:beta-Galactosidase