High-level expression of branching enzyme II from maize endosperm in Escherichia coli.
The gene that encodes the mature branching enzyme II (BEII) protein from maize (Zea mays L.) endosperm was amplified by means of a polymerase chain reaction technique and inserted into a T7-based expression vector. Although this has been an efficient expression system of maize BEII in Escherichia coli, an example is presented in this report which allows a greater expression of mBEII protein from the bacterial system by changing only one codon. The key to the level of expression appears to be related to the conversion of the third thymine base in the 285 position codon of the mBEII cDNA to cytosine without altering the encoded mBEII protein product. The crude cell extracts of enzyme prepared from E.coli exhibited seven-fold higher expression of branching enzyme activity compared to expression of the native enzyme. The enzymes from wild-type and the silent mutation genes were purified. The proteins were indistinguishable kinetically and immunologically. Thus, we obtained a significantly improved expression of mBEII protein in the bacterial system.
Department of Biochemistry, Michigan State University, East Lansing, Michigan, 48824, USA.
N
N
Libessart
J
J
Preiss
D015061:1,4-alpha-Glucan Branching Enzyme / Q000235:genetics* / Q000302:isolation & purification; D001483:Base Sequence; D003062:Codon / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D018744:DNA, Plant / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D017343:Genes, Plant; D007527:Isoenzymes / Q000235:genetics* / Q000302:isolation & purification; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification; D003313:Zea mays / Q000201:enzymology* / Q000235:genetics*
D003062:Codon; D017931:DNA Primers; D018744:DNA, Plant; D007527:Isoenzymes; D011994:Recombinant Proteins; D015061:1,4-alpha-Glucan Branching Enzyme