Ian G. Sumner

Nucleotide sequence and expression in Escherichia coli of cDNAs encoding papaya proteinase omega from Carica papaya.

We have cloned and sequenced two similar, but distinct, cDNAs from both fruit and leaf tissues of Carica papaya. The C-terminal portion of the predicted amino acid (aa) sequence of one of the clones has complete identity with the mature enzyme sequence of the cysteine proteinase papaya proteinase omega (Pp omega). The second clone contains ten individual bp changes compared with the first and encodes a protein with three single-aa substitutions, only one of which is located in the mature sequence, but most noticeably carries an additional 19-aa C-terminal extension. The clones encode pre-pro precursor isoforms of Pp omega. The former of these clones has been expressed in Escherichia coli using a T7 polymerase expression system to produce insoluble pro-enzyme which has been solubilized and refolded to yield auto-activable pro-Pp omega.

cDNA cloning and expression of Carica papaya prochymopapain isoforms in Escherichia coli

Chymopapain is one of the four known cysteine proteinases found in the latex of Carica papaya. DNA sequencing of clones derived from a leaf cDNA library identified five cDNA types coding for precursor chymopapains. All of these isoforms have a free cysteine residue at position 117, characteristic of chymopapain. Two of the isoforms possess a further free cysteine residue, which is not likely to be involved in disulphide bonds or the active site apparatus. Another amino acid substitution found in two of the isoforms at position 133 is predicted to lie in the S2 subsite of the substrate binding cleft. One of the prochymopapain isoforms was expressed in Escherichia coli. Protein was expressed as insoluble inclusion body material. This protein was solubilised, refolded and autocatalytically cleaved to yield mature chymopapain that had comparable kinetic constants to authentic native enzyme.

Modification of the stability of phospholipase A2 by charge engineering.

Electrostatic interactions play an important role in stabilizing the folded conformation of globular proteins. Here we predict the change in stability of charge engineered mutants, construct these mutants and compare the predicted change in stability with that observed. The change in stability was correctly predicted for two of the three mutants and the factors responsible for the discrepancy between observation and prediction for the third mutant are discussed.

A novel method for the purification of porcine phospholipase A2 expressed in E. coli.

Porcine phospholipaseA2 expressed in E. coli as a fusion protein was isolated, renatured and specifically cleaved by trypsin as described in (1). Active phospholipaseA2, was purified to homogeneity on a column of PBE-94 over a pH region 7.4-4.5. Using this method, several phospholipase A2 mutant enzymes have now been purified in a single step and all behaved identically during chromatofocusing. The method will therefore be extremely useful not only for those interested in understanding the structure-function relationships of phospholipaseA2 but also for preparing the enzyme in large quantities for industrial and pharmaceutical purposes.

Simplified methods for the synthesis of 2-hexadecanoylthio-l-ethylphosphorylcholine and for the determination of phospholipase A2 activity

A simple and straight forward method was developed for the synthesis of 2-hexadecanoylthio-1-ethyl phosphorylcholine (HEPC). The new procedure, which used p-toulenesulfonate instead of 2-bromoethyl phosphorylcholine, not only reduced the reaction time but also allowed the reaction to proceed under mild conditions. Using HEPC as a substrate, we have also developed a microplate assay for measuring phospholipase A2 activity which is rapid and will be useful for analyzing a large number of samples in a very short time. The applicability of this assay method for assessing phospholipases A2 from two different sources and determining their kinetic constants is also demonstrated. This method can also be extended for measuring lipases and lysophospholipases using a suitable thioester. Thus, both synthesis and assay methods will be useful in basic and applied research on phospholipases and related enzymes.

Physiological consequence of expression of soluble and active hen egg white lysozyme in Escherichia coli

Hen egg white lysozyme was expressed as a protein fusion with the OmpA signal sequence and an octapeptide linker in Escherichia coli. The expression yielded soluble and enzymatically active lysozyme. Lysozyme activity was detected in the periplasmic space, in the cytosol and in the insoluble cytosolic fraction of E. coli. The results indicate that the environmental conditions in both the cytosol and the periplasmic space of E. coli were sufficient for correct protein folding and disulphide bond formation of eukaryotic recombinant lysozyme. However, the expression of active enzyme in E. coli consequently led to bacterial cell lysis due to hydrolysis of the peptidoglucan.

Kinetic analysis of papaya proteinase Ω

Abstract Papaya proteinase Ω (ppΩ) has been purified from dried latex both by immunoaffinity and traditional methods. Kinetic analysis revealed that (1), the ppΩ-catalysed hydrolysis of N- benzoyl- l -arginine p- nitroanilide (BApNA) has a lower specificity (kcat/Km) than the same reaction catalysed by papain; (2), the ppΩ-catalysed hydrolysis of a tripeptide substrate having phenylalanine at the second position (S2-site) showed a more similar specificity to that catalysed by papain; (3), the significant difference between the two enzymes is that steady state kinetics with both l -BApNA and a tripeptide enables the identification in ppΩ of other ionizations affecting binding. The active sites of papain and ppΩ can therefore be distinguished by pH-dependence of kcat/Km.

Analysis of catalytic properties of hen egg white lysozyme during renaturation from denatured and reduced material

Catalytic properties of hen egg white lysozyme were analyzed during the renaturation of the enzyme from completely reduced and denatured material. The formation of intermediate folding products and the generation of native lysozyme was monitored by acetic acid/urea electrophoresis. The results showed that during the beginning of renaturation almost all reduced and denatured lysozyme is converted to forms possessing lower compactness than native lysozyme, probably as a result of formation of only one or two disulfide bonds. Kinetic analysis of lysozyme during renaturation showed that the generation of lysozyme with four disulfide bonds was not necessarily equivalent to the formation lysozyme with native-like catalytic properties. It appeared that the formation rate of the structures of the structures of the substrate binding site and of the catalytic site were limited by the generation of four disulfide bonds containing lysozyme. The catalytic properties of intermediate folding products made it evident that the final structures of the substrate binding site and of the catalytic site were formed after the generation of all disulfide bonds.

Making a small enzyme smaller; removing the conserved loop structure of hen lysozyme

Engineering a smaller lysozyme is a challenge for both random and site‐directed mutagenesis. This paper illustrates the power of knowledge‐based protein engineering in the design of a smaller lysozyme that folds correctly and has activity against bacterial cell walls. In this smaller lysozyme the conserved disulphide bridged loop is replaced by a short loop. The long loop was selected because it buries a predominantly hydrophilic surface. The short loop was discovered by searching for appropriate fragments in the protein databank. This approach is important in the design of small enzymes useful to the food industry.