High-level temperature-induced synthesis of an antibody VH-domain in Escherichia coli using the PelB secretion signal.
We have constructed a temperature-inducible Escherichia coli expression vector (pPOW) for enhanced secretion of antibody (Ab) domains and other foreign proteins. The vector contains the lambda pRpL promoters in tandem, and the cI857 gene encoding the temperature-sensitive repressor which provide tight control over protein production. The PelB secretion signal directs the synthesized foreign protein through the cytoplasmic membrane. A mouse Ab fragment (the variable heavy (VH) domain of NC41) was synthesized efficiently by this vector and accumulated with the cell membranes (not as inclusion bodies) at levels of 30 mg/l. This represents the highest yields reported to date for Ab fragments with a native N terminus. An octapeptide (FLAG) tail was fused to the C terminus of the VH domain to aid in purification, and remained intact throughout the protein purification process. The optimum conditions for protein production were controlled by the type of culture medium used, the age of the bacterial population at the time of induction, and the period of synthesis of the protein product. The purified Ab VH fragment showed binding affinity (Ka less than 10(4)/M) to its target antigen (neuraminidase).
Power
B E
BE
CSIRO Division of Biomolecular Engineering, Parkville, Victoria, Australia.
Ivancic
N
N
Harley
V R
VR
Webster
R G
RG
Kortt
A A
AA
Irving
R A
RA
Hudson
P J
PJ
D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods; D004926:Escherichia coli / Q000235:genetics*; D005822:Genetic Vectors; D007074:Immunoglobulin G / Q000235:genetics*; D007143:Immunoglobulin Heavy Chains / Q000096:biosynthesis / Q000235:genetics*; D007135:Immunoglobulin Variable Region / Q000096:biosynthesis / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D011133:Polysaccharide-Lyases; D011401:Promoter Regions, Genetic; D021382:Protein Sorting Signals / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis; D015183:Restriction Mapping; D013696:Temperature
D001426:Bacterial Proteins; D007074:Immunoglobulin G; D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D011133:Polysaccharide-Lyases; C074124:pectin lyase B