A human cDNA library for high-throughput protein expression screening.
We have constructed a human fetal brain cDNA library in an Escherichia coli expression vector for high-throughput screening of recombinant human proteins. Using robot technology, the library was arrayed in microtiter plates and gridded onto high-density filter membranes. Putative expression clones were detected on the filters using an antibody against the N-terminal sequence RGS-His(6) of fusion proteins. Positive clones were rearrayed into a new sublibrary, and 96 randomly chosen clones were analyzed. Expression products were analyzed by SDS-PAGE, affinity purification, matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry, and the determined protein masses were compared to masses predicted from DNA sequencing data. It was found that 66% of these clones contained inserts in a correct reading frame. Sixty-four percent of the correct reading frame clones comprised the complete coding sequence of a human protein. High-throughput microtiter plate methods were developed for protein expression, extraction, purification, and mass spectrometric analyses. An enzyme assay for glyceraldehyde-3-phosphate dehydrogenase activity in native extracts was adapted to the microtiter plate format. Our data indicate that high-throughput screening of an arrayed protein expression library is an economical way of generating large numbers of clones producing recombinant human proteins for structural and functional analyses.
Büssow
K
K
Max Planck Institute of Molecular Genetics, Ihnestrasse 73, Berlin, 14195, Germany. buessow@molgen.mpg.de
Nordhoff
E
E
Lübbert
C
C
Lehrach
H
H
Walter
G
G
D000465:Algorithms; D000911:Antibodies, Monoclonal / Q000276:immunology; D001921:Brain / Q000378:metabolism; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics*; D016208:Databases, Factual; D004591:Electrophoresis, Polyacrylamide Gel; D005333:Fetus; D015870:Gene Expression; D018507:Gene Expression Regulation, Developmental; D015723:Gene Library; D006801:Humans; D011506:Proteins / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D017422:Sequence Analysis, DNA; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D000911:Antibodies, Monoclonal; D018076:DNA, Complementary; D011506:Proteins; D011993:Recombinant Fusion Proteins