A DNA fragment encoding the green fluorescent protein (GFP) was isolated via PCR from a jellyfish Aequorea victoria cDNA, cloned and sequenced. Subsequently, a recombinant baculovirus bearing the coding region of the GFP under the transcriptional control of the Autographa californica nuclear polyhedrosis virus (AcMNPV) polyhedrin gene promoter was constructed and isolated. High-level expression of GFP could be easily monitored in Spodoptera frugiperda (Sf9) insect cells after infection with recombinant baculovirus, due to the intrinsic fluorescence (lambda(max) = 508 nm) of the recombinant protein after excitation with blue light (lambda(max) = 400 nm). The functional recombinant GFP displayed an apparent molecular mass of approximately 43 kDa and the fluorescence emission spectrum of the recombinant protein was virtually identical to that of the native green fluorescent protein.

A new set of versatile advanced baculovirus (BV) vectors for the production of fused proteins in insect cells, under the control of the strong polyhedrin promoter of the Autographa california nuclear polyhedrosis virus (AcMNPV), has been constructed. The vectors contain peptide tags which allow immunological detection, as well as purification of recombinant protein produced via the BV expression system.

The cDNA for the human D2S-dopamine receptor has been functionally expressed in the unicellular yeast Saccharomyces cerevisiae. Two expression plasmids pRS421D2 (original D2S-gene coding region) and pRS421D2S (the first 24 aa of the yeast STE2-gene are fused to the N-terminus of the D2S-gene) were constructed and transformed into the protease deficient S. cerevisiae strain cI3-ABYS-86. Northern blot analysis of total RNA from transformed yeast clones revealed that for both constructs the D2S-gene was constitutively transcribed from the plasmids PMA1 promoter. Membranes prepared from recombinant S. cerevisiae exhibited saturable binding with the antagonist [3H]methylspiperone. Competition studies revealed pharmacological properties for these sites which were comparable to those reported for the D2-receptor heterologously expressed in mammalian cells. The expression of the receptor was monitored by Western blot analysis using an antiserum raised against a peptide from the third intracellular domain of the receptor protein and by ligand binding assay.

Complementary DNA encoding the Na/Ca,K-exchanger was isolated from bovine retina cDNA libraries. The complete full-length cDNA is approximately 4 kb long and contains an open reading frame of 3597 bp. The deduced amino acid sequence corresponds to a protein of 1199 amino acids with a calculated molecular weight of approximately 130 kDa. Hydrophobicity analysis revealed the presence of two alternating sets of hydrophobic and hydrophilic domains. There also exists a hydrophobic region at the N-terminus which may be part of a cleavable signal peptide. The protein shares limited sequence homology with the Na/Ca-exchanger from cardiac sarcolemma. Northern blot analysis indicates that the approximately 6 kb transcript is highly specific for retinal tissue. Insect cells infected with recombinant baculovirus bearing the full-length cDNA express a functional Na/Ca,K-exchanger with an apparent relative molecular weight of approximately 210 kDa, as determined by Western blotting.

A recombinant baculovirus bearing the cDNA coding for the rat muscarinic acetylcholine receptor subtype M3 placed under the control of the Autographa californica nuclear polyhedrosis virus polyhedrin gene promoter, was constructed. Polymerase chain reaction screening was used to identify the recombinant baculovirus. Northern blot analysis of total RNA from insect cells infected with the recombinant baculovirus indicated that the transcripts were abundant. Binding assays carried out with the muscarinic antagonist [3H]quinuclidinyl benzilate indicated that more than 1 × 106 receptors were produced per cell. Immunofluorescence microscopy showed that the receptor is located on the cell surface.

A human cDNA fragment bearing the complete coding region for the beta 2-adrenergic receptor was introduced into the genome of Autographa california nuclear polyhedrosis virus under the control of the polyhedrin promoter. Binding studies using [125I]iodocyanopindolol showed that Sf9 insect cells infected with the recombinant virus expressed approximately 1 x 10(6) beta 2-adrenergic receptors on their cell surface. Photoaffinity labeling of whole cells and membranes revealed a molecular weight of approximately 46,000 for the expressed receptor. The receptor produced in insect cells is glycosylated but the extent and pattern differ from that of the receptor from human tissue. The heterologously expressed receptor was purified by alprenolol affinity chromatography, and was able to activate isolated Gs-protein.