A new series of trpE vectors that enable high expression of nonfusion proteins in bacteria.
Expression of recombinant proteins in bacteria has facilitated the characterization of many gene products. However, the biochemical characterization of recombinant proteins is limited since the bacterially expressed proteins are often synthesized as fusion polypeptides. The presence of bacterial sequences in fusion proteins further limits the use of these proteins for generating antibodies since the bacterial sequences are also antigenic. We describe two new bacterial expression vectors based on the pATH series of plasmids. These vectors were made by precisely deleting all of the trpE coding sequences found in pATH. The new vectors have enabled us to express eukaryotic genes as nonfusion polypeptides. These altered plasmids can be used to insert any DNA sequence of interest through a multiple cloning site located just 3' of an ATG start codon. Protein expression is still under the control of the trp operon and is carried out at great efficiency when the bacteria are tryptophan deprived. Studies presented here test the expression system with neurofilament subunits, NF-L and NF-H. Large amounts of recombinant nonfusion proteins were produced. Also, a time course of induction shows that the production of the nonfusion proteins was under the control of the trp operon which is readily inducible after tryptophan starvation and addition of indoleacrylic acid. These vectors may be useful for the overexpression of many proteins in a form closely approximating their native state.
Mercy
M R
MR
Medical Biotechnology Center, Maryland Biotechnology Institute, Baltimore.
Troncoso
J C
JC
Monteiro
M J
MJ
D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli; D015870:Gene Expression; D005822:Genetic Vectors; D051379:Mice / Q000235:genetics; D008969:Molecular Sequence Data; D016900:Neurofilament Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D009876:Operon; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D014367:Tryptophan Synthase / Q000235:genetics*
D001426:Bacterial Proteins; D016900:Neurofilament Proteins; D011994:Recombinant Proteins; C044882:neurofilament protein L; D014367:Tryptophan Synthase