Trypsin, purification

Reddy, S., N. J. Bibby, P. A. Smith, and R. B. Elliott. 1985. Rat trypsin Purification radioimmunoassay and age related serum levels in normal and spontaneously diabetic BB Wistar rats. Australian Journal of Experimental Biology and Medical Science 63 667-681. 

From animal tissue, especially bovine lung and liver (e. g. autolysis of comminuted tissue parts, heating with ammonium sulfate in alkaline solution, filtration and acidification yield heparin as complex with protein, removal of fat with alcohol and treatment with trypsine for the purpose of decomposition of proteins, precipitation with alcohol and various purification methods). 

Titani, K., Sasagawa, T., Resing, K., and Walsh, K. A., A simple and rapid purification of commercial trypsin and chymostrypsin by reverse-phase high-performance liquid chromatography, Anal. Biochem., 123, 408, 1982. 

Figure 2.7. Identification ofphosphoproteins by site-specific chemical modification. A. Method of Zhou et al. (2001) involves trypsin digest of complex protein mixture followed by addition of sulfhydryl groups specifically to phosphopeptides. The sulfhydryl group allows capture of the peptide on a bead. Elution of the peptides restores the phosphate and the resulting phosphopeptide is analyzed by tandem mass spectrometry. B. Method of creates a biotin tag in place of the phosphate group. The biotin tag is used for subsequent affinity purification. The purified proteins are proteolyzed and identified by mass spectrometry.

Proteins have, to date, only rarely been purified by SMB. The first attempt was made by Huang et al. in 1986 [42]. They isolated trypsin from porcine pancreas extracts using an SMB made of only six columns. In addition, this example also demonstrates that SMB systems with a very limited number of columns can be efficient. Another example for a successful protein-separation by SMB is the purification of human serum albumin (HSA) using two SMB-systems connected in series [43]. The first SMB was used for removing the less strongly retained components and the second one for removing the more strongly retained components of the sample matrix. 

For further purification, primary cultures of PBEC are passaged at the third day of culture by gentle trypsinization at room temperature. This enzymatic treatment selectively releases endothelial cells, leaving behind contaminating cells, such as pericytes and smooth muscle cells. Usually, contamination by nonendothelial cells should be below 1-3%. Endothelial cells are then seeded at a density of 30,000-50,000 cells/cm2 on rat-tail collagen-coated cell culture inserts (Figure 17.5). 

Native RNase is quite resistant to digestion with trypsin, even at a w/w ratio of 1 20, but small or unfolded fragments would be expected to be digested. When the synthetic enzyme was treated with trypsin, a 70% recovery of protein with a specific activity of 61% was obtained. Treatment of this material with saturated ammonium sulfate (diluted 16 26), pH 4.6, gave 33% of amorphous precipitate and 66% of soluble RNase A. The overall yield from the first Val residue was only 3%, but the specific activity was quite high at 78%. This is as far as the purification was carried out at that time. 

Trypsin Porcine pancreas Tetraoxyethylene monodecylether/ Extraction and purification [64]... 

Materials and Methods. Fully deuterated phycocyanin and protio phycocyanin from Ph. luridum were used. The method of purifying phycocyanin was identical to that used previously (15, 16). The purity of the phycocyanin preparations, the complete substitution of deuterium for hydrogen in the fully deuterated phycocyanin, and the reversibility of the aggregation phenomenon were ascertained as previously (4, 16). Purified bovine trypsin, soybean trypsin inhibitor, and bovine liver catalase were obtained from the Worthington Biochemical Corp., Freehold, N. J., and used without further purification. Bovine a -casein B was kindly supplied by Chien Ho of the University of Pittsburgh. 

Figure B3.1.1 A 15% SDS-polyacrylamide gel stained with Coomassie brilliant blue. Protein samples were assayed for the purification of a proteinase, cathepsin L, from fish muscle according to the method of Seymour et al. (1994). Lane 1, purified cathepsin L after butyl-Sepharose chromatography. Lane 2, cathepsin L complex with a cystatin-like proteinase inhibitor after butyl-Sepharose chromatography. Lane 3, sarcoplasmic fish muscle extract after heat treatment and ammonium sulfate precipitation. Lane 4, sarcoplasmic fish muscle extract. Lanes M, low-molecular-weight standards aprotinin (Mr 6,500), a-lactalbumin (Mr 14,200), trypsin inhibitor (Mr 20,000), trypsinogen (Mr 24,000), carbonic anhydrase (Mr 29,000), gylceraldehyde-3-phosphate dehydrogenase (Mr 36,000), ovalbumin (Mr 45,000), and albumin (Mr 66,000) in order shown from bottom of gel. Lane 1 contains 4 pg protein lanes 2 to 4 each contain 7 pg protein.

In addition to the proposed regulatory role of ATP and pyrophosphate, some possibility exists that 3, 5 -cyclic phosphate diesterase is under physiological control. Such ideas arose through observations of Cheung (43, 62) that the partially purified enzyme from beef brain was markedly activated by snake venom. The stimulatory factor was labile at extreme pH it was not dialyzable and appeared to be a protein. A similar activating factor is also present in brain tissue (63) and is removed during purification of the diesterase. It seems to interact stoichiometrically with the enzyme. The activator is destroyed by trypsin and is not proteolytic itself. The precise role of this protein in regulating the phosphodiesterase in vivo is not yet established, however. 

Preparation of Human Insulin. Porcine insulin can be converted to the human insulin sequence by an enzyme-catalyzed transpeptidation reaction (10,11). Under appropriate conditions trypsin acts preferentially at LysB29 rather than ArgB22 to yield a covalent des[B30]insulin/trypsin complex (acyl—enzyme intermediate). In the presence of high concentrations of organic co-solvents and the /-butyl ester of threonine, transpeptidation predominates over hydrolysis to yield the /-butyl ester of human insulin. Following appropriate purification steps and acidolytic removal of the ester, human insulin suitable for treating patients is obtained. 

Brattsand M, Egelrud T. Purification, molecular cloning, and expression of a human stratum corneum trypsin-like serine protease with possible function in desquamation. J Biol Chem 1999 274 30033-30040. 

Jayakumar A, Kang Y, Mitsudo K, et al. Expression of LEKTI domains 6-9 in the baculovirus expression system Recombinant LEKTI domains 6-9 inhibit trypsin and subtilisin A. Protein Expr Purif 2004 35 93-101. 

The purification and partial characterization of a novel trypsin-like cysteine protease, Pr4 from M. anisopliae was reported by Cole et al. (1993). The enzyme, with an isoelectric point of 4.6 and a molecular mass of 26.7 kDa, exhibited trypsin-like specificity, but according to... 

Cole, S. C. J., Chamley, A. K., and Cooper, R. M. (1993). Purification and partial characterization of a novel trypsin-like cysteine protease from Metarhizium anisopliae. FEMSMicrobiology Letters 113, 189-196. 

---