Construction of a new family of high efficiency bacterial expression vectors: identification of cDNA clones coding for human liver proteins.

Abstract

Construction of a family of bacterial expression vectors, pEX1‐3, is described. These vectors are derived from a cro‐lacZ gene fusion plasmid which expresses large quantities of fusion protein under the control of the PR promoter of bacteriophage lambda. A polylinker has been engineered into the 3′ end of the lacZ gene in all three translational reading frames, and stop signals for transcription and translation inserted, so that any open reading frame DNA may be expressed as a hybrid beta‐galactosidase protein. cDNA fragments cloned in these vectors can be detected with an efficiency of greater than 1 in 3, thus enabling the detection of rare cDNA molecules. In addition, the low solubility of hybrid proteins leads to a rapid isolation procedure allowing antibodies of pre‐determined specificity to be made against expressed regions of cloned DNA. We describe the cloning of albumin and complement C9 genes from a human cDNA library using polyclonal and monoclonal antibodies.

A recombinant autoantigen derived from the human (u1) small nuclear rnp‐specific 68‐kd protein

“…A human liver complementary DNA (cDNA) library constructed with expression vector pEXl (19) was screened with serum from a patient known to have high-titer autoantibodies to U1 RNP and Sm antigens, as well as to several other as-yet-unidentified nuclear components. All positive clones (n = 384) were picked onto master plates and screened with 7 sera from patients with SLE or MCTD and with 4 sera from apparently healthy individuals.…”

Yeast counterparts of subunits S5a and p58 (S3) of the human 26S proteasome are encoded by two multicopy suppressors of nin1-1.

“…Escherichia coli strain DH1 (recAl endAl gyr96 thi-1 hsdR17 supE44 relAl) and HB101 (recA13 hsdS20 supE44 proA2 rpsL20; Takara Shuzo, Kyoto, Japan) were used for construction and propagation of plasmids. POP2136 (clts857; Stanley and Luzio, 1984) was used as a host for production of a large amount of lacZ-fusion protein whose expression is under the control of the promoter of phage A according to the procedure supplied by the manufacturer (P & S Biochemicals Inc., Gaithersburg, MD). To construct the lacZ-SUN2 fusion gene, we amplified the ORF of the SUN2 gene, flanked by the EcoRI site just upstream of the initiation codon and the Sall site just down stream of the termination codon, by polymerase chain reaction (PCR) according to the method described by Saiki et al (1988).…”

“…To construct the lacZ-SUN2 fusion gene, we amplified the ORF of the SUN2 gene, flanked by the EcoRI site just upstream of the initiation codon and the Sall site just down stream of the termination codon, by polymerase chain reaction (PCR) according to the method described by Saiki et al (1988). Then, the ORF was inserted between the EcoRI-SalI sites of the expression vector pEX2 (Stanley and Luzio, 1984) to construct the lacZ-SUN2 fusion gene. The ORF of the SUNI gene that was flanked by the EcoRI site and the BglII site at the 5' and 3' side, respectively, was amplified by PCR and the resulting fragment was digested with EcoRI and BglII and inserted in-frame between the EcoRI and BglII sites pGEX-5X-3 (Pharmacia Biotech, Piscataway, NJ).…”

The chimeric nature of the genome of pea enation mosaic virus: the independent replication of RNA 2

“…The three 3'-terminal ORFs of RNA 2 (corresponding to the 25K, 27K and 15K protein-encoding reading frames; Fig. 3) were expressed as flgalactosidase fusion proteins in vitro using the pEX vector system (Stanley & Luzio, 1984). Vectors expressing the overlapping 25K and 27K protein-encoding reading frames were constructed from an AsnI (nucleotide 2795)-BamHI (nucleotide 3632) fragment of clone p80 in which the 5' AsnI overhang had been repaired with the Klenow fragment of DNA polymerase.…”