Proteins cloning

The cDNAs for the muscarinic receptors encode apparent glycoproteins of 55-70 kDa, which contain seven predicted transmembrane-spanning regions, similar to what is seen for the [3-adrenergic receptor and other receptors that couple to G proteins (Fig. 11-11). There is only 38% amino acid identity between the proteins cloned from porcine brain and heart. The cDNA encoding the receptor initially cloned from the brain has been termed m whereas that cloned from the heart has been termed m2, in keeping with the names (Mj and M2) applied to the purified recombinant receptors. 

Somatostatin is a polypeptide hormone involved in regulating growth it is important in the treatment of various human growth disorders. The hormone is small, only 14 amino acids in length, making it a good candidate for cloning. In fact, it was the hrst human protein cloned in E. coli. 

The leader sequence of the Serratia marcescens extracellular nuclease has been removed and the gene for the resulting nontransportable protein cloned behind the leftward promoter (PL) of lambda (Ahrenholtz, Lorenz Wackernagel, 1994). In the presence of a temperature-sensitive repressor (cl857), the cells can be induced to produce this altered enzyme by an increase in temperature. This produces the nuclease within the cell and, because it cannot be exported, its intracellular concentrations rise, rapidly degrading the cellular genome. Limitations to this system are that cell survival is reduced to only 2 x 10 5. 

Gao, Y., Wells, L., Comer, F. I., Parker, G. J., and Hart, G. W. (2001) Dynamic O-glycosylation of nuclear and cytosolic proteins cloning and characterization of a neutral, cytosolic beta-N-acetylglucosaminidase from human brain. J. Biol. Chem. 276, 9838-9845. 

The second alternative function of PDI again comes from sequence analysis. This time a clone isolated as a lodothyronine 5 -monodeiodinase (5 -MD, thyroxine deiodinase) from rat codes for a protein that is identical to PDI in all but two residues (Boado et ai, 1988). The clone was isolated by screening a library with polyclonal antibodies raised against rat liver microsomal proteins. Clones coding for 5 -MD were selected by... 

Carr, D. W., Hausken, Z. E., Fraser, I. D. C., et al. (1992). Association of the type II cAMP-dependent protein kinase with a human thyroid Rll-anchoring protein. Cloning and characterization of the Rll-binding domain. ]. Biol. Chem. 267, 13376-13382. 

Reflect and Apply One of the first human proteins cloned was interferon. Why would it be important to be able to produce interferon in a lab ... 

Inagaki N, Yamamoto Y, Mori H et al. Carboxyl-terminal processing protease for the D1 precursor protein Cloning and sequencing of the spinach cDNA. Plant Mol Biol 1996 30 39-50. 

The most important task of the Edman degradation is still delivering sequence information for the synthesis of oligonucleotides. This preparatory work for protein cloning used to be done as follows. The protein purifier prepared a clean product and handed the preparation to a group specializing in Edman degradation. 

Zimmer, S., Stocker, A., Sarbolouki, M.N., Spycher, S.R, Sassoon, J., and Azzi, A., A novel human tocopherol-associated protein cloning, in vitro expression, and characterization, J. Biol. Chem. 275 (33), 25672—25680, 2000. 

Synthetic gene coding for a SMT3-chemokine fusion protein cloned into the B zl and Hindlll/Pstl of either pQE30 or pET28a. 

Stoner, C M and Gudas, L J. (1989) Mouse cellular retinoic acid binding protein cloning, complimentary DNA sequence, and messenger RNA expression during retinoic acid-induced differentiation of F9 wild type and RA-3-10 mutant teratocarcinoma cells Cancer Res 49,1497-1504... 

L.K. Kreppel, M.A. Blomberg and G.W. Hart, J. Biol Chem., 1997, 272, 9308-9315, Dynamic glycosylation of nuclear and cytosolic proteins—Cloning and characterization of a unique D-GlcNAc transferase with multiple tetratricopeptide repeats. 

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