A general bacterial expression system for functional analysis of cDNA-encoded proteins.
A general system for functional analysis of cDNA-encoded proteins is described. The basic concept involves the expression in Escherichia coli of selected portions of cDNAs in an approach toward the understanding of the function of the corresponding proteins. A selected cDNA is expressed as part of a fusion protein used for immunization to elicit antibodies, and a corresponding fusion protein, having the cDNA-encoded portion in common, for purification of target protein-specific antibodies. This antiserum could be used for functional analysis of the cDNA-encoded protein, e.g., by immunohistology. Two general expression vector systems for E. coli have been constructed, both (i) designed with multiple cloning sites in three different reading frames, (ii) having their protein production controlled by the tightly regulated T7 promoter, and (iii) enabling affinity purification of the expressed target proteins by fusions to IgG-binding domains derived from staphylococcal protein A or a serum albumin-binding protein derived from streptococcal protein G, respectively. This novel system has been evaluated by expressing five cDNAs, isolated from prepubertal mouse testis by a differential cDNA library screening strategy. All five clones could be expressed intracellularly in E. coli as fusion proteins with high production levels, ranging from 4 to 500 mg/liter, and affinity purification yielded essentially full-length products. Characterization of affinity-purified antibodies revealed that there exists no cross-reactivity between the two fusion systems and that such antibodies indeed could be used for immunohistology. The implications for the described system for large-scale functional analysis of cDNA libraries are discussed.
Larsson
M
M
Department of Biochemistry and Biotechnology, Royal Institute of Technology (KTH), Stockholm, Sweden.
Brundell
E
E
Nordfors
L
L
Höög
C
C
Uhlén
M
M
Ståhl
S
S
D000818:Animals; D002847:Chromatography, Agarose; D017931:DNA Primers / Q000737:chemistry; D004274:DNA, Recombinant / Q000737:chemistry / Q000235:genetics; D012321:DNA-Directed RNA Polymerases / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D005822:Genetic Vectors; D015698:Genomic Library; D005854:Germ Cells / Q000737:chemistry; D007106:Immune Sera / Q000276:immunology / Q000302:isolation & purification; D015151:Immunoblotting; D007074:Immunoglobulin G / Q000737:chemistry / Q000276:immunology; D007150:Immunohistochemistry; D008297:Male; D051379:Mice; D016133:Polymerase Chain Reaction; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D017422:Sequence Analysis, DNA; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry; D013737:Testis / Q000737:chemistry
D017931:DNA Primers; D004274:DNA, Recombinant; D007106:Immune Sera; D007074:Immunoglobulin G; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D012967:Sodium Dodecyl Sulfate; D012321:DNA-Directed RNA Polymerases