Characterization of midgut trypsin-like enzymes and three trypsinogen cDNAs from the lesser grain borer, Rhyzopertha dominica (Coleoptera: Bostrichidae)

Protein digestion in the lesser grain borer, Rhyzopertha dominica (F.) (Coleoptera: Bostrichidae), results from the action of a complex of serine proteinases present in the midgut. In this study we partially characterized trypsin-like enzyme activity against N-alpha-benzoyl-L-arginine p-nitroanilide (BApNA) in midgut preparations and cloned and sequenced three cDNAs for trypsinogen-like proteins. BApNAase activity in R. dominica midgut was significantly reduced by serine proteinase inhibitors and specific inhibitors of trypsin, whereas BApNAase activity was not sensitive to specific inhibitors of chymotrypsin or aspartic proteinases. However, trans-epoxysuccinyl-L-leucylamido-(4-guanidino) butane (E-64) inhibited BApNAase activity by about 30%. BApNAase was most active in a broad pH range from about pH 7 to 9.5. The gut of R. dominica is a tubular tract approximately 2.5 mm in length. BApNAase activity was primarily located in the midgut region with about 1.5-fold more BApNAase activity in the anterior region compared to that in the posterior region. Proteinases with apparent molecular masses of 23-24 kDa that were visualized on casein zymograms following electrophoresis were inhibited by TLCK. Three cDNAs for trypsinogen-like proteins were cloned and sequenced from mRNA of R. dominica midgut. The full cDNA sequences consisted of open reading frames encoding 249, 293, and 255 amino acid residues for RdoT1, RdoT2, and RdoT3, respectively. cDNAs RdoT1, RdoT2, and RdoT3 shared 77-81% sequence identity. The three encoded trypsinogens shared 54-62% identity in their amino acid sequences and had 16-18 residues of signal peptides and 12-15 residues of activation peptides. The three predicted mature trypsin-like enzymes had molecular masses of 23.1, 28, and 23.8 kDa for RdoT1, RdoT2, and RdoT3, respectively. Typical features of these trypsin-like enzymes included the conserved N-terminal residues IVGG62-65, the catalytic amino acid triad of serine proteinase active sites (His109, Asp156, Ser257), three pairs of conserved cysteine residues for disulfide bridges, and the three residues (Asp251, Gly274, Gly284) that determine specificity in trypsin-like enzymes. In addition, RdoT2 has both a PEST-like sequence at the C-terminus and a free Cys158 near the active site, suggesting instability of this enzyme and/or sensitivity to thiol reagents. The sequences have been deposited in GenBank database (accession numbers AF130840 for RdoT1, AF130841 for RdoT2, and AF130842 for RdoT3).     

Molecular cloning and partial characterization of a trypsin-like protein in salivary glands of Lygus hesperus (Hemiptera: Miridae)

Trypsin-like enzymes from the salivary gland complex (SGC) of Lygus hesperus Knight were partially purified by preparative isoelectric focusing (IEF). Enzyme active against Nalpha-benzoyl-L-arginine-p-nitroanilide (BApNA) focused at approximately pH 10 during IEF. This alkaline fraction gave a single activity band when analyzed with casein zymograms. The serine proteinase inhibitors, phenylmethylsulfonyl fluoride (PMSF) and lima bean trypsin inhibitor, completely inhibited or suppressed the caseinolytic activity in the crude salivary gland extract as well as the IEF-purified sample. Chicken egg white trypsin inhibitor also inhibited the IEF-purified sample but was not effective against a major caseinolytic band in the crude salivary gland extract. These data indicated the presence of serine proteinases in the SGC of L. hesperus. Cloning and sequencing of a trypsin-like precursor cDNA provided additional direct evidence for serine proteinases in L. hesperus. The encoded trypsin-like protein included amino acid sequence motifs, which are conserved with five homologous serine proteinases from other insects. Typical features of the putative trypsin-like protein from L. hesperus included residues in the serine proteinase active site (His(89), Asp(139), Ser(229)), conserved cysteine residues for disulfide bridges, residues (Asp(223), Gly(252), Gly(262)) that determine trypsin specificity, and both zymogen signal and activation peptides.     

Partial characterization of trypsin-like protease and molecular cloning of a trypsin-like precursor cDNA in salivary glands of Lygus lineolaris

Based on substrate specificity, an alkaline pH optimum, sensitivity to selected proteinase inhibitors, and molecular analysis, we provide evidence for the presence of a trypsin-like serine proteinase in the salivary gland complex (SGC) of the tarnished plant bug, Lygus lineolaris (Palisot de Beauvois) (Heteroptera: Miridae). The predominant activity in extracts of the SGC against N(2)-benzoyl-L-arginine-p-nitroanilide (L-BApNA) was at pH 10, but a minor peak of activity also occurred at pH 5. The major BApNAase activity focused at 10.4 during preparative isoelectric focusing and was eluted with an apparent molecular weight of 23,000 from a calibrated gel filtration column. The BApNAase fraction gave a single major band when analyzed on a casein zymogram. The activity was completely suppressed by the serine protease inhibitors, phenylmethylsulfonyl fluoride (PMSF) and lima bean trypsin inhibitor. A cDNA coding for a trypsin-like protein in the salivary glands of L. lineolaris was cloned and sequenced. The 971bp cDNA contained an 873-nucleotide open reading frame encoding a 291-amino acid trypsin precursor. The encoded protein included amino acid sequence motifs that are conserved with four homologous serine proteases from other insects. Typical features of the putative trypsin-like protein from L. lineolaris included the serine protease active site (His(89), Asp(139), Ser(229)), conserved cysteine residues for disulfide bridges, the residues (Asp(223), Gly(252), Gly(262)) that determine trypsin specificity, and both zymogen signal and activation peptides. Cloning and sequencing of a trypsin-like precursor cDNA provided additional direct evidence for trypsin like enzymes in the salivary glands of L. lineolaris.     

Host hemolymph proteins and protein digestion in larval Habrobracon hebetor (Hymenoptera: braconidae)

Host plasma proteins and protein digestion in larval parasitoids were studied during trophic interactions of the ectoparasitoid Habrobracon hebetor Say (Hymenoptera: Braconidae), with a host, larvae of the Indianmeal moth, Plodia interpunctella Hübner (Lepidoptera: Pyralidae). We could detect no apparent differences in host hemolymph protein patterns up to 72 h after paralysation and/or parasitization by H. hebetor. A 190 kDa putative apolipophorin I present in host hemolymph could not be detected in the midguts of feeding H. hebetor larvae indicating that it is rapidly digested. The major 60 kDa storage proteins (putative hexamerins) in host hemolymph were detected in the parasitoid midgut and were completely digested 24 h after cessation of feeding and the beginning of cocoon formation. Host hemolymph had a pH of about 6.4. The pH optima of the midgut proteinases in the larval parasitoid were in the alkaline region, but midgut fluid in feeding parasitoid larvae was about pH 6. 8. Based on enzyme activity against selected artificial proteinase substrates including azocasein, N-alpha-benzoyl-L-Arg p-nitroanilide (BApNA), succinyl-Ala-Ala-Pro-Phe p-nitroanilide (SAAPFpNA), succinyl-Ala-Ala-Pro-Leu p-nitroanilide (SAAPLpNA), and inhibition by selected proteinase inhibitors, serine proteinases appear to be the predominant class of enzymes involved in protein digestion in the midguts of H. hebetor. There is also an active aminopeptidase (LpNA) associated with the microsomal fraction of midgut preparations. There was no evidence for preoral digestion or ingestion of proteinases from host hemolymph by the parasitoid larva. There was a very active BApNAase in the soluble fraction of midgut extracts. This activity increased on a per midgut basis up to 24 h after the beginning of cocoon formation but decreased rapidly by 48 h. Two major (P1 and P3) and several minor proteinases were detected in midgut extracts of H. hebetor analysed with gelatin zymograms. The apparent molecular mass of P1 varied from 95 to 49 kDa depending on protein loading. P3 had an apparent molecular mass of 39 kDa that was independent of protein loading. In summary, electrophoretic evidence indicates that host hemolymph protein patterns do not change significantly for at least 72 h after paralysation by H. hebetor. The role, if any, of envenomization in preventing breakdown of hemolymph proteins during this time remains to be determined. Because the predominant host hemolymph proteins, a putative apolipophorin I and the putative hexamerins, are readily digested by the serine proteinases present in the midguts of this parasitoid larva, these or similar proteins would provide an easily digested source of dietary amino acids that could be used for development of artificial diets for this beneficial insect.     

Analysis of midgut proteinases from Bacillus thuringiensis-susceptible and -resistant Heliothis virescens (Lepidoptera: Noctuidae)

Insects with altered proteinases can avoid intoxication by Bacillus thuringiensis (Bt) toxins. Therefore, proteinase activities from gut extracts of Bt-susceptible (YDK) and -resistant (YHD2-B, CXC and KCBhyb) Heliothis virescens strains were compared. The overall pH of gut extracts from YDK and CXC were statistically similar (9.56 and 9.62, respectively), while the pH of extracts from KCBhyb and YHD2-B were significantly more alkaline (9.81 and 10.0, respectively). Gut extracts from YHD2-B and CXC larvae processed Cry1Ac and Cry2Aa protoxin slower than extracts from YDK larvae, suggesting that differences in proteolysis contribute to resistance in these strains. Casein zymogram analysis of gut extracts revealed both qualitative and quantitative differences in caseinolytic activities among all strains, but the overall caseinolytic activity of YHD2-B gut extract was lower. Kinetic microplate assays with a trypsin substrate (l-BApNA) demonstrated that proteinases in YDK gut extract had increased alkaline pH optima compared to resistant strains YHD2-B, CXC and KCBhyb. Gut extracts from YHD2-B had reduced trypsin-like activity, and activity blots indicated that YHD2-B had lost a trypsin-like proteinase activity. In assays with a chymotrypsin substrate (SAAPFpNA), enzymes from all Bt-resistant strains had increased pH optima, especially those from KCBhyb. Activity blots indicated that CXC had lost a chymotrypsin-like proteinase activity. Because serine proteinases are a critical component of Bt toxin mode of action, these differences may contribute to decreased toxicity in the Bt-resistant strains.     

Protein digestion in larvae of the red oak borer Enaphalodes rufulus

Abstract In the Ozark Mountains of the U.S.A., the red oak borer Enaphalodes rufulus contributes to the destruction of red oaks. To understand nutrient digestion in E. rufulus larvae, digestive proteinases are compared in both larvae fed heartwood phloem and those transferred to artificial diet. The pH of gut extracts is approximately 6.3 in the midgut and foregut and decreases to 5.5 in the hindgut region. The hydrolysis of casein by midgut extracts from E. rufulus larvae fed either artificial diet or phloem from tree sections increases in buffers greater than pH 6.19, with maximum hydrolysis being observed at pH 10.1. Casein zymogram analysis reveals two major proteinase activities in larval midgut extracts of diet‐fed larvae, with molecular masses of approximately 25 and 40–60 kDa, whereas phloem‐fed larvae have proteinase activities corresponding to 40, 45, 60, 80 and >100 kDa. Substrate analysis indicates at least one major trypsin‐like activity in both gut extracts with a molecular mass of >100 kDa, but two chymotrypsin‐like activities of approximately 25 and >200 kDa are found only in diet‐fed larvae. Inhibitors of serine proteinases are most effective in reducing the general proteolytic activity of midgut extracts from larvae fed either food source. The data indicate that serine proteinase inhibitors have the potential to reduce E. rufulus larval damage to oaks. In particular, transgenic technologies incoporating trypsin inhibitors may be effective in reducing protein digestion in phloem‐feeding larvae.     

Cloning and molecular characterization of a cubilin-related serine proteinase from the hard tick Haemaphysalis longicornis

Serine proteinases are one of the largest proteolytic families of enzymes, and have diverse cellular activities in mammalian tissues. We report here the cloning and molecular characterization of a cDNA encoding the serine proteinase of the hard tick Haemaphysalis longicornis (HlSP). The HlSP cDNA is 1570 bp long and the deduced precursor protein consists of 464 amino acids with a predicted molecular mass of 50.4 kDa and a pI of 8.2. The preprotein, consisting of 443 amino acids, was predicted to include a complement C1r/C1s, Uegf, and bone morphogenic protein-1 domain, a low-density lipoprotein receptor class A domain, and a catalytic domain. HlSP sequence analysis showed high similarity to serine proteinases reported from arthropods and vertebrate animal species. Two-dimensional immunoblot analysis revealed endogenous HlSP in adult tick extracts at 50 kDa. Endogenous HlSP was also expressed in all lifecycle stages of H. longicornis. Immunohistochemical studies detected the endogenous enzyme in the midgut epithelial cells of an adult tick. The Escherichia coli-expressed recombinant HlSP was demonstrated to degrade bovine serum albumin and hydrolyze the substrate Bz-L-Arg-pNA at the rate of 30.2 micromol/min/mg protein. Further, HlSP expression was up-regulated during a blood-feeding process, indicating its involvement in the digestion of host blood components.     

Nucleotide and protein sequences of a proteinase inhibitor from the vitelline envelope of dace (Tribolodon hakonensis) eggs

Our experimental purpose is to probe the structure(s) of the chorionic proteinase inhibitor and its cDNA sequence(s) and to develop the application of safe medicines for protection of human and other animal bodies from pathogenic microbe attacks. In this study, chorionic proteinase inhibitor protein was isolated, sequenced and used to base the design of PCR primers, which were then used to amplify DNA using RT-PCR. A cDNA clone of the protein which inhibited the activities of serine proteinases and thermolysin was obtained on the basis of mRNA extracted from ovarian tissue of dace, Tribolodon hakonensis, and the deduced amino acid sequence was determined. Chorionic proteinase inhibitor (TribSPI) peptides of about 9.0 kDa (TribSPI) and 14 kDa (TribSPI-S) were purified from vitelline envelope extracts by thermolysin-immobilized affinity-chromatography. The cloned TribSPI cDNA was 1806 bp in length, and the open reading flame (ORF) was 915 bp encoding a protein of 305 amino acid residues. The inhibitor protein had a molecular mass of 33,550 daltons and was composed of five similar domains. Each domain contained eight cysteine residues, and it's deduced amino acid sequence was only 33 approximately 34% identical to those of human and porcine antileukoproteinases (hALP and pALP, respectively). A possible binding-site for serine proteinases, Arg-Ile, was contained in three domains.     

Identification of six chymotrypsin cDNAs from larval midguts of Helicoverpa zea and Agrotis ipsilon feeding on the soybean (Kunitz) trypsin inhibitor

Lepidopteran insects like Helicoverpa zea and Agrotis ipsilon produce STI-insensitive trypsins in the midgut following ingestion of dietary plant proteinase inhibitors like STI [Broadway, R. M., J. Insect Physiol. 43(9) (1997) 855-874]. In this paper, the effects of dietary STI on a related family of midgut serine proteinases, the chymotrypsins, were investigated. STI-insensitive midgut chymotrypsins were detected in larvae of H. zea and A. ipsilon feeding on diets containing 1% STI while STI-sensitive chymotrypsins were present in larvae feeding on diets containing 0% STI. These chymotrypsins were unaffected by TPCK, a diagnostic inhibitor of mammalian chymotrypsins but were fully inhibited by chymostatin. Four midgut cDNA libraries were constructed from larvae of each species fed either 0% STI or 1% STI diets. Six full-length cDNAs(1) encoding diverse preprochymotrypsins were isolated (three from H. zea and three from A. ipsilon) with certain sequence motifs that set them apart from their mammalian counterparts. Northern blots showed that some chymotrypsin mRNA were detected at higher levels while others were down-regulated when comparing insects reared on 0% STI and 1% STI diets. Southern hybridizations suggested that (like mammals) both species contained several chymotrypsin genes. A full-length chymotrypsin gene(1) from H. zea was sequenced for the first time and the presence of four introns was deduced. A first time comparison of 5' upstream regions(1) from three chymotrypsin genes and two trypsin genes of A. ipsilon indicated the presence of putative TATA boxes and regulatory elements. However a lack of consensus motifs in these upstream regions suggested the likelihood of multiple trans factors for regulation of genes encoding digestive proteinases and a complex response mechanism linked to ingestion of proteinase inhibitors.     

Pen c 1, a novel enzymic allergen protein from Penicillium citrinum. Purification, characterization, cloning and expression.

A 33-kDa alkaline serine protease secreted by Penicillium citrinum strain 52-5 is shown to be an allergenic agent in this fungus. The protein, designated Pen c 1, was purified by sequential DEAE-Sepharose and carboxymethyl (CM)-Sepharose chromatographies. Pen c 1 has a molecular mass of 33 kDa and a pI of 7.1. The caseinolytic enzyme activity of this protein was studied. The protein binds to serum IgE from patients allergic to Penicillium citrinum. The cDNA encoding Pen c 1 is 1420 bp in length and contains an open reading frame for a 397-amino-acid polypeptide. Pen c 1 codes for a larger precursor containing a signal peptide, a propeptide and the 33-kDa mature protein. Sequence comparison revealed that Pen c 1 possesses several features in common with the alkaline serine proteases of the subtilisin family. The essential Asp, His, and Ser residues that make up the catalytic triad of serine proteases are well conserved. Northern blots demonstrated that mRNAs transcribed from this gene are present at early stages of culture. The allergen encoded by Pen c 1 gene was expressed in Escherichia coli as a fusion protein bearing an N-terminal histidine-affinity tag. The protein, purified by affinity chromatography with a yield of 130 mg of pure protein per liter of culture, was able to bind to both a monoclonal anti-Pen c 1 antibody and IgE from the serum of patients allergic to Penicillium. Recombinant Pen c 1 can therefore be expressed in E. coli in large quantities and should prove useful as a standardized specific allergen for immuno-diagnosis of atopic disorders. In addition, full caseinolytic enzyme activity could be generated in the purified recombinant protein by sulfonation and renaturation, followed by removal of the affinity tag, indicating that the refolded protein can assume the same conformation as the native protein.     

Peptidoglycan inducible expression of a serine proteinase homologue from kuruma shrimp (Marsupenaeus japonicus)

A cDNA encoding a serine proteinase homologue of kuruma shrimp (Marsupenaeus japonicus) was cloned. The 1257 bp cDNA encodes a 339 amino acid putative peptide, with a signal sequence of 16 amino acid residues. The deduced amino acid sequence is 42-67% similar to the immune-related serine proteinases and serine proteinase homologues of arthropods. It contains catalytic triad residues in the putative catalytic domain except for one substitution of Ser by a Gly residue. The six cysteine residues that form three disulphide bridges in most serine proteinases were conserved. The M. japonicus serine proteinase homologue was mainly expressed in haemocytes, in which expression dramatically increased after 3 days feeding with peptidoglycan at 0.2 mg kg(-1) shrimp body weight per day.     

Trypsinogen-like cDNAs and quantitative analysis of mRNA levels from the Indianmeal moth, Plodia interpunctella

Two cDNA fragments encoding full-length trypsinogen-like proteins were cloned from larvae of two strains (RC688s and HD198r) of the Indianmeal moth, Plodia interpunctella (Hübner), which differed in their sensitivity to Bacillus thuringiensis protoxins. One cDNA fragment contained 874 nucleotides, including a 780-nucleotide open reading frame that encoded a trypsinogen-like protein (PiT2b). Another cDNA fragment amplified from both P. interpunctella strains contained 864 nucleotides including a 780 bp open reading frame encoding a second trypsinogen-like protein (PiT2c). The cDNA sequence of PiT2b shared 89% sequence identity with PiT2a, a trypsinogen-like protein cloned previously from this species. The cDNA sequences of PiT2a and PiT2c shared 83% identity. The cDNA sequence identity between PiT2b and PiT2c was 80%. The cDNA for PiT2b from strain RC688s was different at six nucleotide positions from that of PiT2b from strain HD198r. Five nucleotide replacements occurred in the open reading frame leading to amino acid changes at all five positions. There were five nucleotide differences in the cDNAs for PiT2c trypsinogen-like proteins from the two strains. Two nucleotide substitutions in the open reading frame resulted in replacements of two amino acid residues in the deduced protein sequences. Amino acid sequences for PiT2a and PiT2b shared 84% identity, but only 50% identity was observed between PiT2c and the other two trypsinogen-like proteins. The deduced amino acid sequences for PiT2b and PiT2c included both signal and zymogen activation peptides and amino acid sequence motifs which are conserved in seven homologous trypsinogen-like proteins from other insects. Typical features of the putative trypsinogen-like proteins from P. interpunctella included the serine proteinase active site triad (His(81), Asp(133), and Ser(233)), three pairs of cysteine residues for disulfide bridges, and three residues, Asp(227), Gly(250), and Gly(260), that help to confer trypsin-like specificity to the enzymes. Quantitative RT-PCR analyses showed that, in fourth instar larvae, RC688s had 1.6-fold higher PiT2a trypsinogen-like mRNA than did HD198r. Expression of PiT2b mRNA was 3.4-fold higher in HD198r than in RC688s. Expression of PiT2c mRNA was 2.8-fold higher in RC688s than in HD198r. Mean accumulation levels of mRNAs for all three trypsinogen-like proteins were slightly higher in RC688s than in HD198r based on total RNA, and 1.3-fold higher in RC688s than in HD198r based on wet weight of larval body tissues.     

Characterization of midgut trypsin-like enzymes and three trypsinogen cDNAs from the lesser grain borer, Rhyzopertha dominica (Coleoptera: Bostrichidae)

Protein digestion in the lesser grain borer, Rhyzopertha dominica (F.) (Coleoptera: Bostrichidae), results from the action of a complex of serine proteinases present in the midgut. In this study we partially characterized trypsin-like enzyme activity against N-α-benzoyl- -arginine p-nitroanilide (BApNA) in midgut preparations and cloned and sequenced three cDNAs for trypsinogen-like proteins. BApNAase activity in R. dominica midgut was significantly reduced by serine proteinase inhibitors and specific inhibitors of trypsin, whereas BApNAase activity was not sensitive to specific inhibitors of chymotrypsin or aspartic proteinases. However, trans-epoxysuccinyl- -leucylamido-(4-guanidino) butane (E-64) inhibited BApNAase activity by about 30%. BApNAase was most active in a broad pH range from about pH 7 to 9.5. The gut of R. dominica is a tubular tract approximately 2.5 mm in length. BApNAase activity was primarily located in the midgut region with about 1.5-fold more BApNAase activity in the anterior region compared to that in the posterior region. Proteinases with apparent molecular masses of 23–24 kDa that were visualized on casein zymograms following electrophoresis were inhibited by TLCK.Three cDNAs for trypsinogen-like proteins were cloned and sequenced from mRNA of R. dominica midgut. The full cDNA sequences consisted of open reading frames encoding 249, 293, and 255 amino acid residues for RdoT1, RdoT2, and RdoT3, respectively. cDNAs RdoT1, RdoT2, and RdoT3 shared 77–81% sequence identity. The three encoded trypsinogens shared 54–62% identity in their amino acid sequences and had 16–18 residues of signal peptides and 12–15 residues of activation peptides. The three predicted mature trypsin-like enzymes had molecular masses of 23.1, 28, and 23.8 kDa for RdoT1, RdoT2, and RdoT3, respectively. Typical features of these trypsin-like enzymes included the conserved N-terminal residues IVGG^62–65, the catalytic amino acid triad of serine proteinase active sites (His¹⁰⁹, Asp¹⁵⁶, Ser²⁵⁷), three pairs of conserved cysteine residues for disulfide bridges, and the three residues (Asp²⁵¹, Gly²⁷⁴, Gly²⁸⁴) that determine specificity in trypsin-like enzymes. In addition, RdoT2 has both a PEST-like sequence at the C-terminus and a free Cys¹⁵⁸ near the active site, suggesting instability of this enzyme and/or sensitivity to thiol reagents. The sequences have been deposited in GenBank database (accession numbers AF130840 for RdoT1, AF130841 for RdoT2, and AF130842 for RdoT3).     

Production of chymotrypsin-resistant Bacillus thuringiensis Cry2Aa1 delta-endotoxin by protein engineering.

Cleavage of the Cry2Aa1 protoxin (molecular mass, 63 kDa) from Bacillus thuringiensis by midgut juice of gypsy moth (Lymantria dispar) larvae resulted in two major protein fragments: a 58-kDa fragment which was highly toxic to the insect and a 49-kDa fragment which was not toxic. In the midgut juice, the protoxin was processed into a 58-kDa toxin within 1 min, but after digestion for 1 h, the 58-kDa fragment was further cleaved within domain I, resulting in the protease-resistant 49-kDa fragment. Both the 58-kDa and nontoxic 49-kDa fragments were also found in vivo when (125)I-labeled toxin was fed to the insects. N-terminal sequencing revealed that the protease cleavage sites are at the C termini of Tyr49 and Leu144 for the active fragment and the smaller fragment, respectively. To prevent the production of the nontoxic fragment during midgut processing, five mutant proteins were constructed by replacing Leu144 of the toxin with Asp (L144D), Ala (L144A), Gly (L144G), His (L144H), or Val (L144V) by using a pair of complementary mutagenic oligonucleotides in PCR. All of the mutant proteins were highly resistant to the midgut proteases and chymotrypsin. Digestion of the mutant proteins by insect midgut extract and chymotrypsin produced only the active 58-kDa fragment, except that L144H was partially cleaved at residue 144.     

Production of Chymotrypsin-Resistant Bacillus thuringiensis Cry2Aa1 δ-Endotoxin by Protein Engineering

Cleavage of the Cry2Aa1 protoxin (molecular mass, 63 kDa) from Bacillus thuringiensis by midgut juice of gypsy moth (Lymantria dispar) larvae resulted in two major protein fragments: a 58-kDa fragment which was highly toxic to the insect and a 49-kDa fragment which was not toxic. In the midgut juice, the protoxin was processed into a 58-kDa toxin within 1 min, but after digestion for 1 h, the 58-kDa fragment was further cleaved within domain I, resulting in the protease-resistant 49-kDa fragment. Both the 58-kDa and nontoxic 49-kDa fragments were also found in vivo when ¹²⁵I-labeled toxin was fed to the insects. N-terminal sequencing revealed that the protease cleavage sites are at the C termini of Tyr49 and Leu144 for the active fragment and the smaller fragment, respectively. To prevent the production of the nontoxic fragment during midgut processing, five mutant proteins were constructed by replacing Leu144 of the toxin with Asp (L144D), Ala (L144A), Gly (L144G), His (L144H), or Val (L144V) by using a pair of complementary mutagenic oligonucleotides in PCR. All of the mutant proteins were highly resistant to the midgut proteases and chymotrypsin. Digestion of the mutant proteins by insect midgut extract and chymotrypsin produced only the active 58-kDa fragment, except that L144H was partially cleaved at residue 144.