Abstract
In order to carry out studies on structure and function relationships of porcine pepsinogen using site-directed mutagenesis approaches, the cDNA of this zymogen was cloned, sequenced, expressed in Escherichia coli, and the protein refolded, and purified to homogeneity. Porcine pepsinogen cDNA, obtained from a lambda gt10 cDNA library of porcine stomach contains 1364 base pairs. It contains leader, pro, and pepsin regions of 14, 44, and 326 residues, respectively. In addition, it also contains 5'- and 3'-untranslated regions. Four differences are present between the sequence deduced from the cDNA and the pepsinogen sequence determined previously by protein chemistry methods. Residues P19 (in the pro region) and 263 are asparagines in the cDNA sequence instead of aspartic acids. Isoleucine 230 is not present in the cDNA sequence and residue 242 is a tyrosine in the cDNA instead of an aspartic acid. Porcine pepsinogen cDNA was placed under the control of a tac promoter in a plasmid and expressed in E. coli. The synthesis of pepsinogen was optimized to about 50 mg/liter of culture. The recombinant (r-) pepsinogen, which was insoluble, was recovered by centrifugation, washed, dissolved in 6 M urea in Tris-HCl, pH 8, and refolded by rapid dilution. r-pepsinogen was purified to homogeneity after chromatography on Sephacryl S-300 and fast protein liquid chromatography on a monoQ column. r-pepsinogen contains an additional methionine residue at the NH2 terminus as compared to native (n-) pepsinogen. However, r- and n-pepsinogens are indistinguishable in their intramolecular activation constants. After activation, r- and n-pepsins have the same NH2-terminal sequences as well as Km values. Based on these data, r-pepsinogen was judged suitable for mutagenesis studies. A mutant pepsinogen (D32A) with the active site aspartic acid changed to an alanine was produced and purified. D32A-pepsinogen did not convert to pepsin in acid solution but it bound to pepstatin with an apparent KD of about 5 x 10(-10) M. D32A-pepsinogen possesses no detectable proteolytic activity. These results indicate that (i) intramolecular pepsinogen activation is accomplished by the pepsin active site, and (ii) unlike subtilisin (Carter, P., and Wells, J. A. (1988) Nature 332, 564-568), the active site mutant of pepsin is not enzymically active.
Highlights
From the$Laboratory of Protein Studies, Oklahoma Medical Research Foundation and the TDepartment of Biochemistry and Molecular Biology, University of Oklahoma Health Science Center, Oklahoma City, Oklahoma 73104
We have changed, by site-directed detectable proteolytic activity. These results indicate mutagenesis, pepsin active site Asp3’ to an Ala3’ in order to that (i)intramolecular pepsinogen activation is accom- determine its effect on pepsinogen activation
From the nucleotide sequence and predicted amino acid sequence shown in Fig. 1, it is clear that thecDNA clone Xpg6 of porcine pepsinogen corresponds to anear full length mRNA
Summary
Isolation and Sequencing of Porcine Pepsinogen cDNA-Procedures for the mRNA purification, porcine stomach cDNA library constitution, isolation, and sequence determination of porcine pepsinogen cDNA are described in the Miniprint Supplement. Kinetic Measurements-The intramolecular activation constant of pepsinogen, k l , was measured according to previously described procedures [8].K,,, measurements for n- and r-pepsin were carried out as described previously by Marciniszyn et al [15]. In both cases, 14Cmethylated bovine hemoglobin wasused as substrate. The cellswere pelleted and Pepstatin-A competitive assay was developed to determine the by centrifugation at 3,500 X g for 30 min and resuspended to 100 ml dissociation constant, KO,,, between themutant pepsinogen and with T N buffer (0.05M Tris-HC1,pH 7.2, and 0.15 M NaCl). The value of Ks,,,,is unknown, it is reasonable to assume that mutant pepsinogen binds substrate with equal or less affinity than n-pepsin i.e. Ks.,,, > K,,, as shown inTable 111, different values of Ks,,,,were used to calculate [E,,,]and KO,,,,
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
