Proteins cDNA sequencing

Yeast cells can exist as haploids of opposite mating types (either a or a). When an a and an a cell are allowed to mate, they form a diploid cell (a/a). To study interactions between two proteins, cDNA sequences of a protein of interest (PT1) are expressed as a fusion protein, linked to a DNA-binding domain (DBD) of a yeast gene-transcript activator in a haploid cell (e.g., a). cDNA sequences corresponding to another test protein (PT2) are linked to the Continued on next page)... 

Sherman, D. R, Lloyd, S R., and Chytil, F. (1987) Rat cellular retinol-binding protein. cDNA sequence and rapid retinol-dependent accumulation of mRNA Proc Nat Acad. Sci. USA 84,3209-3213... 

In humans, the structural gene locus is on chromosome 19 (M17), and the gene spans over 40 kilobases (kb) including 18 exons and 17 introns (W2, X2). Neu-roleukin, a protein that acts as both a neurotrophic factor and a lymphokine, has been isolated from mouse salivary glands (G7), and subsequently the primary structure of neuroleukin was found to be identical to that of GPI by comparison of the cDNA sequences (C7, FI). The cDNA sequence encodes 558 amino acid residues. The enzyme consists of two identical subunits with a molecular weight of approximately 63,000 and neuroleukin is active as a monomer. 

The structure and sequence of the catalytic moiety have been determined (06, V6, W6). The enzyme consists of 362 amino acids and 40,638 daltons of the protein predicted by the cDNA sequence. The ADA gene spans 32 kb and consists of 12 exons. The apparent promoter region of the gene lacks the TATA and CAAT sequences often found in eukaryotic promoters and is extremely G/C rich. The location of the ADA gene is on chromosome 20ql2-ql3.11 (Jl). 

Simpson, J. C., Wellenreuther, R., Poustka, A., Pepperkok, R., and Wiemann, S. (2000). Systematic subcellular localization of novel proteins identified by large-scale cDNA sequencing. EMBO Reports 1, 287-292. 

Tabunoki, H., Sugiyama, H., Tanaka, Y. et al. 2002. Isolation, characterization, and cDNA sequence of a carotenoid binding protein from the silk gland of Bombyx mori larvae. J. Biol. Chem., 277(35) 32133-32140. 

Boesten, W.H.J., Raemakers-Franken, PC., Sonke, T. et al. (2003) Protein and cDNA sequences of -//- -amino acid amide racemases cloned from Ochrobactrum anthropi and Arthrobacter nicotianae, W02003106691. 

Based on the above principles, it might be assumed that a therapeutic protein obtained by direct extraction from human sources (e.g. some antibody preparations) or produced via recombinant expression of a human gene/cDNA sequence (e.g. recombinant human hormones or cytokines) would be non-immunogenic in humans whereas foreign therapeutic proteins (e.g. non-engineered monoclonal antibodies) would stimulate a human immune response. This general principle holds in many cases, but not all. So why do therapeutic proteins of human amino acid sequences have the potential to trigger an immune response Potential reasons can include ... 

The translation of the sequence of the cDNA encoding deacetylvindoline 4-O-acetyltransferase compared to other putative plant acetyltransferases revealed a conserved region near the carboxy terminus of the proteins. This sequence was used to design a degenerate antisense oligodeoxynucleotide primer for PCR. The sense primer was based upon an internal peptide sequence of salutaridinol 7-0-acetyltransferase. This approach finally yielded a partial cDNA that encoded salutaridinol 7-O-acetyltransferase. The full-length clone was obtained by RACE-PCR and was functionally expressed in S. frugiperda Sf9 cells.28 The amino acid sequence of salutaridinol 7-O-acetyltransferase is most similar (37% identity) to that of deacetylvindoline acetyltransferase of C. roseus.27... 

A variety of domain or motif families occur only as extensions to other domains. The Bruton s tyrosine kinase motif (BTK), for example, is found only at the C terminus of PH domains. Similarly, a C-terminal extension (the S TK X domain) to some subfamilies of serine/threonine kinases (S TK) is not found in isolation. Cases where only the extension, and not the preceding domain, is found are strong evidence that the proteins are wrongly assembled from genomic sequence or else represent partial cDNA sequences (Fig. 9, see Color insert). Indeed, all five proteins annotated in SMART as containing a S TK X domain with no catalytic domain are noted to be fragments in their corresponding sequence database entries. 

In contrast to P-gp and the MRP proteins, the breast cancer resistance protein (BCRP) contains six transmembrane domains and only one ATP-binding domain. It was first cloned from the breast cancer cell line MCF-7 selected in doxombicin, in the presence of the P-gp inhibitor verapamil. It is found in many human tissues, such as the placenta, small intestine, colon, and liver [133], It is localized to the apical membrane of epithelial cells of the small intestine and colon and to the bile canalicular membrane in the liver and is involved in reducing intestinal uptake, increasing hepatobiliary excretion, etc., leading to diminished oral bioavailability. cDNA sequences identical to BCRP and named MXR and ABCP, respectively, were independently isolated from human colon carcinoma cells and human placenta [134], BCRP requires... 

In vitro transcription from cloned cDNA sequences is required to produce mRNA that can be injected into Xenopus oocytes for expression of the encoded protein and is achieved by standard procedures. While oocyte isolation and injection generally follows the protocols provided below, in vitro transcription into mRNA may be modified in accordance to the instructions provided with... 

Maridor G, Nigg EA (1990) cDna sequences of chicken nucleolin/C23 and No38/B23, two major nucleolar proteins. Nucleic Acids Res 18 1286... 

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