Post-translational processing of barley beta-glucan endohydrolases in the baculovirus-insect cell expression system.
Two cDNAs encoding barley (1-->3,1-->4)-beta-glucanase (EC 3.2.1.73) isoenzymes EI and EII have been expressed in Spodoptera frugiperda (Sf9) cell cultures using the baculovirus AcNPV vector. Modifications to both the 5' and 3' ends of the cDNAs were required before satisfactory levels of expression were obtained. The modified cDNAs directed high levels of (1-->3,1-->4)-beta-glucanase expression in the Sf9 insect cell cultures, with yields of approximately 10 mg/liter of isoenzyme EI (expEI) and 15 mg/liter of isoenzyme EII (expEII). Amino acid sequence analyses showed that the expressed enzymes were processed correctly at their amino termini. However, affinity chromatography of the isoenzyme expEII on concanavalin-A (conA)-Sepharose indicated that, although the enzyme is glycosylated, the structures of the carbohydrate chains differ from those of the native enzyme. When a cDNA encoding the homologous barley (1-->3)-beta-glucanase (EC 3.2.1.39) isoenzyme GII was expressed in insect cells, aberrant amino-terminal processing of the nascent polypeptide was sometimes observed. The forms with incompletely removed signal peptides retained their substrate specificity, but exhibited slightly reduced catalytic efficiency, altered chromatographic behavior, and reduced stability at elevated temperatures. The results show that high levels of expression of recombinant plant proteins can be obtained in insect cells, but they emphasize the need to characterize thoroughly the products that are expressed in the heterologous insect cell system before comparisons are made with the native enzyme or with engineered enzyme mutants.
Doan
D N
DN
Department of Biochemistry, La Trobe University, Bundoora, Victoria, Australia.
Høj
P B
PB
Collins
A
A
Din
N
N
Hoogenraad
N J
NJ
Fincher
G B
GB
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae; D002460:Cell Line; D003001:Cloning, Molecular; D004247:DNA / Q000032:analysis; D015971:Gene Expression Regulation, Enzymologic; D005822:Genetic Vectors; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001467:Hordeum / Q000201:enzymology / Q000235:genetics*; D006358:Hot Temperature; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D009036:Moths; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017421:Sequence Analysis; D013379:Substrate Specificity; D014158:Transcription, Genetic
D007527:Isoenzymes; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D004247:DNA; D006026:Glycoside Hydrolases; C028020:licheninase