A single mutation in the activation site of bovine trypsinogen enhances its accumulation in the fermentation broth of the yeast Pichia pastoris

We produced bovine trypsinogen in the yeast Pichia pastoris. Little or no trypsinogen was detected when the gene with its native leader sequence was expressed under the control of the strong aox1 promoter, suggesting that expression of the wild-type bovine trypsinogen was toxic to the cells. We altered the trypsinogen native propeptide sequence by replacing the lysine at position 6 with an aspartic acid, thus destroying the site in the propeptide cleaved by enterokinase and by trypsin. This mutant accumulated up to 10 mg of trypsinogen per liter in shake flask cultures and about 40 mg/liter in 6-liter fermentors. Trypsinogen could be activated in vitro with a dipeptidyl-aminopeptidase, which selectively removed the modified trypsinogen propeptide; the resulting trypsin was fully active and showed evidence of glycosylation. Thus, we have developed a novel protein production scheme that can be used for the expression of proteins, such as proteases, that are deleterious to the producing organism. This system relies on the expression of a zymogen that cannot be activated in vivo coupled with its in vitro purification and activation.     

Overexpression in Escherichia coli and functional reconstitution of anchovy trypsinogen from the bacterial inclusion body

We have synthesized and optimized a high-yielding Escherichia coli expression system to produce trypsinogen from anchovy Engraulis japonicus and have developed conditions for its successful refolding. Recombinant anchovy trypsinogen precipitated in E. coli Rosetta (DE3) placI strain as inclusion bodies was denatured by 6 M guanidine-HCl followed by refolding with drop wise addition to a large excess of a folding buffer containing 0.5 M non-detergent sulfobetaine (NDSB-251) and a redox potential of oxidized and reduced glutathiones. The folded trypsinogen was autocatalytically activated to its mature form, trypsin, and purified with a MonoQ ion-exchange column. NH₂-terminal amino acid sequencings revealed that E. coli efficiently processed NH₂-terminal methionine residue from the expressed trypsinogen and that trypsinogen was activated at the correct site to generate active trypsin. The recombinant enzyme showed kinetic properties comparable to those of the native enzyme and demonstrated a typical cleavage preference for arginine over lysine residue against a protein substrate. The optimized expression and folding procedures yielded 12 mg of purified, active trypsin from 1 L of bacterial culture or 45 g wet weight cells, which is quite enough for various analytical and semipreparative purposes.     

Functional expression of trypsin from Streptomyces griseus by Pichia pastoris

In the present study, the genes encoding trypsinogen and active trypsin from Streptomyces griseus were both cloned and expressed in the methylotrophic yeast Pichia pastoris with the α-factor secretion signal under the control of the alcohol oxidase promoter. The mature trypsin was successfully accumulated extracellularly in soluble form with a maximum amidase activity of 6.6?U?ml^?1 (batch cultivation with flask cultivation) or 14.4?U?ml^?1 (fed-batch cultivation with a 3-l fermentor). In contrast, the recombinant trypsinogen formed inclusion bodies and no activity was detected. Replacement of the trypsin propeptide Ala-Pro-Asn-Pro confirmed that its physiological function was as a repressor of activity. More importantly, our results proved that the propeptide inhibited the activity of trypsinogen after its successful folding.     

Functional expression and purification of bovine enterokinase light chain in recombinant Escherichia coli

Enterokinase (EC 3.4.21.9) is a serine proteinase of the intestinal brush border that exhibits specificity for the sequence (Asp)(4)-Lys and converts trypsinogen into its active form, trypsin. A codon optimized sequence coding light chain (catalytic subunit) of bovine enterokinase gene (sBEKLC) was synthesized, and it was fused with DsbA to construct the expression vector (pET39-sBEKLC). Then, the plasmid was transformed into E. coli BL21 (DE3) for expression. Under optimal conditions, the volumetric productivity of fusion protein reached 151.2 mg L(-1), i.e., 80.6 mg sBEKLC L(-1). The cold osmotic shock technique was successfully used to extract sBEKLC from periplasmic space, and nickel affinity chromatography was employed to obtain mature sBEKLC. Finally, about 6.8 mg of bioactive sBEKLC was purified from 1 liter fermentation broth and could be used to cleave one tested fusion protein with an inter-domain enteropeptidase recognition site. This work will be helpful for large-scale production of this increasingly demanded enterokinase.     

Refolding of reduced, denatured trypsinogen and trypsin immobilized on Agarose beads.

The reoxidation of fully reduced and denatured bovine trypsinogen and the regeneration of the native structure can be accomplished if the protein is initially attached to Agarose beads. Reoxidation was performed under aerobic conditions, in the presence of mercaptoethanol and dehydroascorbate or with a mixture of reduced and oxidized glutathione. In 24 hours, the yields of regenerated trypsinogen were 60 to 70% with 0.2 to 0.6 mg of protein bound/ml of gel but 30% or less if greater than 1.7 mg of protein were bound. Rapid reoxidation, with dehydroascorbate as catalyst, gave molecules which could not be converted to active trypsin. However, if the incorrectly folded structures were placed in a mixture of reduced and oxidized glutathione, the molecules underwent disulfide interchange and could continue to refold. The rapidly reoxidized molecules regained their native structure with the same rate and to the same extent as they did initially in the absence of rapid reoxidation. Therefore, the rate-limiting step in the refolding of trypsinogen was disulfide interchange. The regenerated Agarose-bound trypsinogen displayed the usual properties of the native molecule in (a) its conversion to active trypsin by a process of limited proteolysis, (b) the kinetic constants of the activated product toward typical trypsin substrates, and (c) the limited cleavage of 1 disulfide bond with sodium borohydride. Refoldind of immobilized trypsin was also observed with an overall yield of 50%. Trypsin can fold spontaneously to its native structure even though it lacks the NH2-terminal hexapeptide of its precursor.     

Rational Design of a Novel Propeptide for Improving Active Production of Streptomyces griseus Trypsin in Pichia pastoris

Applying in silico simulations and in vitro experiments, the amino acid proline was proved to have a profound influence on Streptomyces griseus trypsinogen, and the hydrogen bond between H⁵⁷ and D¹⁰² was found to be crucial for trypsin activity. By introducing an artificial propeptide, IVEF, the titer of trypsin was increased 6.71-fold.     

Axial arrangement of crossbridges in thick filaments of vertebrate skeletal muscle.

X-ray intensity data to 1.8 Å resolution were collected from native trigonal crystals of bovine trypsinogen. The orientation and position of the trypsinogen molecules within their crystal cells were determined by Patterson search techniques using the refined model of bovine trypsin (Bode &; Schwager, 1975), and by subsequent R factor refinement. The translation functions allowed discrimination between the enantiomorphic space groups P3₂21 and P3₁21. After one constrained crystallographic refinement cycle, which reduced the crystallographic reliability factor (R) from 35% to 31%, a preliminary difference Fourier map showed several interesting details. Several refinement cycles reduced the value of R to 23%. The overall chain folding is very similar to trypsin. The chain segments, including residues 184 to 193² and 217 to 223, which form the specificity pocket in trypsin, are flexible in trypsinogen. The autolysis loop is partially mobile between residues 142 and 152. There is no continuing electron density for the N terminal residues preceding Tyr20. This indicates that the N terminus may be only weakly fixed to the rest of the molecule or may even float freely in solution.     

Refolding of sepharose-bound trypsinogen with disulfide 179 to 203 reduced and carboxymethylated

Disulfide 179 to 203 of native bovine trypsin was reduced with sodium borohydride and converted to the S-carboxymethyl derivative. The modified zymogen was attached to CNBr-activated Sepharose, and the resulting immobilized protein was used in refolding studies. The fully reduced protein was kept at 35°, at pH 8.5, under aerobic conditions, in a mixture of reduced and oxidized glutathione, until the sulfhydryl groups were reoxidized. A maximum yield of 55% was found for the regeneration of S-(carboxymethyl)₂-trypsinogen, and the activated product, S-(carboxymethyl)₂-trypsin, reacted with an active site reagent and gave the expected specific activity toward a typical trypsin substrate. Apparently, the refolding of immobilized S-(carboxymethyl)₂-trypsinogen regenerated the native structure of trypsinogen even though one of the six disulfides could no longer be formed.     

Effect of manganese(II) ion on trypsinogen activation

The effect of Mn²⁺ and Ca²⁺ on the kinetics of the tryptic activation of bovine trypsinogen was studied at pH 7.3 and 36.5°C. For comparison, the rate constants of autolysis and esterolytic activity of trypsin were also determined. It can be concluded that Mn²⁺ increases the conversion rate of trypsinogen into trypsin in a 25–40% larger extent than Ca²⁺. The manganese(II) ion bond to trypsinogen is supposed to keep the N-terminal part of the zymogen in a better conformation for binding at the primary and secondary binding sites of trypsin.     

A Nine-Residue Synthetic Propeptide Enhances Secretion Efficiency of Heterologous Proteins in Lactococcus lactis

Lactococcus lactis, a gram-positive organism widely used in the food industry, is a potential candidate for the secretion of biologically useful proteins. We examined the secretion efficiency and capacity of L. lactis by using the Staphylococcus aureus nuclease (Nuc) as a heterologous model protein. When expressed in L. lactis from an efficient lactococcal promoter and its native signal peptide, only ~60% of total Nuc was present in a secreted form at ~5 mg per liter. The remaining 40% was found in a cell-associated precursor form. The secretion efficiency was reduced further to ~30% by the deletion of 17 residues of the Nuc native propeptide (resulting in NucT). We identified a modification which improved secretion efficiency of both native Nuc and NucT. A 9-residue synthetic propeptide, LEISSTCDA, which adds two negative charges at the +2 and +8 positions, was fused immediately after the signal peptide cleavage site. In the case of Nuc, secretion efficiency was increased to ~80% by LEISSTCDA insertion without altering the signal peptide cleavage site, and the yield was increased two- to fourfold (up to ~20 mg per liter). The improvement of NucT secretion efficiency was even more marked and rose from 30 to 90%. Similarly, the secretion efficiency of a third protein, the α-amylase of Bacillus stearothermophilus, was also improved by LEISSTCDA. These data indicate that the LEISSTCDA synthetic propeptide improves secretion of different heterologous proteins in L. lactis.     

Refolding of the mixed disulfide of bovine trypsinogen and glutathione.

The mixed disulfide of bovine trypsinogen and glutathione refolded with high yields at protein concentrations of 20 microgram/ml or less, at 4-25 degrees C, pH 8.0 to 8.7, in the presence of 3 to 6 mM cysteine under anaerobic conditions. The regenerated protein behaved as native trypsinogen as judged by gel exclusion chromatography, isoelectric focusing, and activation with bovine enterokinase or trypsin. However, refolded samples that were quenched with iodoacetate and analyzed by disc gel electrophoresis formed two components corresponding to trypsinogen and S-(carboxymethylcysteine)2-(179-203)-trypsinogen. The use of cysteine as a disulfide interchange catalyst caused reduction of the 179 to 203 disulfide bond, and quenching of the refolding mixture with iodoacetate produced the carboxymethylated derivative. The overall yield of the regenerated product was 70% and the half-time at 4 degrees C was 55 min.     

Purification, characterization and cDNA cloning of a trypsin from the hepatopancreas of snakehead (Channa argus)

A trypsin was purified from the hepatopancreas of snakehead (Channa argus) by ammonium sulfate fractionation and a series of column chromatographies including DEAE-Sepharose, Sephacryl S-200 HR and Hi-Trap Capto-Q. The molecular mass of the purified trypsin was about 22 kDa, as estimated by SDS-PAGE. The optimum pH and temperature of the purified trypsin were 9.0 and 40 °C, respectively. The trypsin was stable in the pH range of 7.5-9.5 and below 45 °C. The enzymatic activity was strongly inhibited by serine proteinase inhibitors, such as MBTI, Pefabloc SC, PMSF, LBTI and benzamidine. Peptide mass fingerprinting (PMF) of the purified protein obtained 2 peptide fragments with 25 amino acid residues and were 100% identical to the trypsinogen from pufferfish (Takifugu rubripes). The activation energy (Ea) of this enzyme was 24.65 kJ·M^− 1. Apparent K_m was 1.02 μM and k_cat was 148 S^− 1 for fluorogenic substrate Boc-Phe-Ser-Arg-MCA. A trypsinogen gene encoding 247 amino acid residues was further cloned on the basis of the sequence obtained from PMF and the conserved site peptide of trypsinogen together with 5′-RACE and 3′-RACE. The deduced amino acid sequence contains a signal peptide of 15 residues and an activation peptide of 9 amino acid residues with a mature protein of 223 residues. The catalytic triad His-64, Asp-107, Ser-201 and 12 Cys residues which may form 6 disulfide bonds were conserved. Compared with the PMF data, only 2 amino acid residues difference were identified, suggesting the cloned trypsinogen is quite possibly the precursor of the purified trypsin.     

Do-it-yourself histidine-tagged bovine enterokinase: A handy member of the protein engineer's toolbox

Enterokinase, a two-chain duodenal serine protease, activates trypsinogen by removing its N-terminal propeptide. Due to a clean cut after the non-primed site recognition sequence, the enterokinase light chain is frequently employed in biotechnology to separate N-terminal affinity tags from target proteins with authentic N-termini. In order to obtain large quantities of this protease, we adapted an in vitro folding protocol for a pentahistidine-tagged triple mutant of the bovine enterokinase light chain. The purified, highly active enzyme successfully processed recombinant target proteins, while the pentahistidine-tag facilitated post-cleavage removal. Hence, we conclude that producing enterokinase in one's own laboratory is an efficient alternative to the commercial enzyme.     

A simple method for isolating trypsin from trichloroacetic acid extracts of bovine pancreas

Trichloroacetic acid was used to isolate trypsin and trypsinogen from bovine pancreas. Trypsinogen, which is at first soluble in trichloroacetic acid, slowly forms a sediment. In alkaline medium and in the presence of calcium chloride, proenzyme is activated to enzyme which has high proteolytic, esterase, and amidase activity and is homogenous in polyacrylamide gel electrophoresis. It is suggested that the gradual reduction of trypsinogen solubility in trichloroacetic acid is associated with the presence of basic pancreatic trypsin inhibitor in trichloroacetic acid extracts from a bovine pancreas.     

The transition of bovine trypsinogen to a trypsin-like state upon strong ligand binding. The refined crystal structures of the bovine trypsinogen-pancreatic trypsin inhibitor complex and of its ternary complex with Ile-Val at 1.9 A resolution

The complex formed by bovine trypsinogen and the pancreatic trypsin inhibitor crystallizes in large crystals isomorphous with trypsin-PTI² complex crystals Rühlmann et al. 1973. X-ray diffraction data to 1.9 Å resolution were collected in the absence and presence of Ile-Val dipeptide. Both trypsinogen complex structures have been crystallographically refined, using the refined trypsin-PTI complex Huber et al. 1974a as a starting model. The final R values are 0.25 and 0.26, respectively. The mean main-chain atom deviations between the three complex structures are about 0.15 Å. In contrast, the mean deviation between the complexed and the free trypsinogen Fehlhammer et al. 1977 is 0.28 Å, reflecting the influence of crystal packing and complexation. The trypsinogen component adopts a trypsin-like conformation upon PTI binding: The Asp194 side-chain turns around and the activation domain becomes rigid, forming the specificity pocket and the Ile16 binding cleft. The specific interactions between PTI and trypsin are also observed in the trypsinogen complex. As in free trypsinogen, the N-terminus including residues Val10 to Gly18 is mobile and sticks out into solution. Apart from the different arrangement of the N-termini in the two complexes, the only significant, but minor structural difference is the enhanced thermal mobility of the autolysis loop in the trypsinogen complex. Upon binding of the Ile-Val dipeptide, the autolysis loop becomes fixed as in the trypsin complex. The Ile-Val position is identical in the ternary and the trypsin complex.     

Determination of trypsin by its accelerating effect on the onset of trypsinogen activation

A method for the determination of trypsin activity is described, based on the kinetics of trypsinogen to trypsin conversion. The time of onset of the autocatalytic conversion, followed in vitro, was found to depend on the amount of exogenous trypsin added. The time for half-maximal trypsinogen conversion was proportionally shortened from 240 to 20 min within the range of 0–1 μg trypsin added to an incubation system containing a large excess of trypsinogen. Optimum conditions for the assay, in terms of temperature dependence and trypsinogen concentration, were determined. The method can be used for the measurement of trypsin in purified preparations and in biological specimens devoid of other activators of trypsinogen. The amount of performed trypsin in pancreatic extracts from rats in some pathological conditions may also be estimated.     

Comparison of anionic and cationic trypsinogens: the anionic activation domain is more flexible in solution and differs in its mode of BPTI binding in the crystal structure

Unlike bovine cationic trypsin, rat anionic trypsin retains activity at high pH. This alkaline stability has been attributed to stabilization of the salt bridge between the N-terminal Ile16 and Asp194 by the surface negative charge (Soman K, Yang A-S, Honig B, Fletterick R., 1989, Biochemistry 28:9918-9926). The formation of this salt bridge controls the conformation of the activation domain in trypsin. In this work we probe the structure of rat trypsinogen to determine the effects of the surface negative charge on the activation domain in the absence of the Ile16-Asp194 salt bridge. We determined the crystal structures of the rat trypsin-BPTI complex and the rat trypsinogen-BPTI complex at 1.8 and 2.2 A, respectively. The BPTI complex of rat trypsinogen resembles that of rat trypsin. Surprisingly, the side chain of Ile16 is found in a similar position in both the rat trypsin and trypsinogen complexes, although it is not the N-terminal residue and cannot form the salt bridge in trypsinogen. The resulting position of the activation peptide alters the conformation of the adjacent autolysis loop (residues 142-153). While bovine trypsinogen and trypsin have similar CD spectra, the CD spectrum of rat trypsinogen has only 60% of the intensity of rat trypsin. This lower intensity most likely results from increased flexibility around two conserved tryptophans, which are adjacent to the activation domain. The NMR spectrum of rat trypsinogen contains high field methyl signals as observed in bovine trypsinogen. It is concluded that the activation domain of rat trypsinogen is more flexible than that of bovine trypsinogen, but does not extend further into the protein core.     

Binding of the bovine pancreatic secretory trypsin inhibitor (Kazal) to bovine serine (pro)enzymes

The effect of temperature and pH on the association equilibrium constant (K_a) for the binding of the bovine pancreatic secretory trypsin inhibitor (bovine PSTI, type I; Kazal inhibitor) to bovine β-trypsin, bovine α-chymotrypsin and bovine trypsinogen has been investigated. The results suggest that serine (pro)enzyme inhibitor interaction involves both rigorous spatial configuration and molecular flexibility.     

Isolation and nucleotide sequence of cDNA clone for bovine pancreatic anionic trypsinogen. Structural identity within the trypsin family.

A cDNA clone encoding an anionic form of bovine trypsinogen was isolated from a pancreatic cDNA library. The corresponding 855-nucleotide mRNA contains a short 5' noncoding region of 8 nucleotides and a long 3' noncoding region of 56 nucleotides in addition to a poly(A) tail of at least 50 nucleotides. The deduced amino acid sequence for the anionic pretrypsinogen (247 residues) includes the N-terminal 15-amino-acid signal peptide followed by an 8-amino-acid activation peptide. The zymogen (232 residues) contains an additional C-terminal serine, compared with the amino acid sequence of bovine cationic trypsinogen. The identity between the anionic and cationic forms of bovine trypsinogen (65%) is lower than that existing between the anionic protein and other mammalian anionic trypsinogens (73-85%), suggesting that trypsin gene duplication in mammals occurred prior to the evolutionary events responsible for the species divergence. Bovine pancreatic anionic trypsin possesses all the key amino acids characteristic of the serine protease family.     

Functional and Structural Characterization of Vibrio cholerae Extracellular Serine Protease B,VesB

The chymotrypsin subfamily A of serine proteases consists primarily of eukaryotic proteases, including only a few proteases of bacterial origin. VesB, a newly identified serine protease that is secreted by the type II secretion system in Vibrio cholerae, belongs to this subfamily. VesB is likely produced as a zymogen because sequence alignment with trypsinogen identified a putative cleavage site for activation and a catalytic triad, His-Asp-Ser. Using synthetic peptides, VesB efficiently cleaved a trypsin substrate, but not chymotrypsin and elastase substrates. The reversible serine protease inhibitor, benzamidine, inhibited VesB and served as an immobilized ligand for VesB affinity purification, further indicating its relationship with trypsin-like enzymes. Consistent with this family of serine proteases, N-terminal sequencing implied that the propeptide is removed in the secreted form of VesB. Separate mutagenesis of the activation site and catalytic serine rendered VesB inactive, confirming the importance of these features for activity, but not for secretion. Similar to trypsin but, in contrast to thrombin and other coagulation factors, Na⁺ did not stimulate the activity of VesB, despite containing the Tyr²⁵⁰ signature. The crystal structure of catalytically inactive pro-VesB revealed that the protease domain is structurally similar to trypsinogen. The C-terminal domain of VesB was found to adopt an immunoglobulin (Ig)-fold that is structurally homologous to Ig-folds of other extracellular Vibrio proteins. Possible roles of the Ig-fold domain in stability, substrate specificity, cell surface association, and type II secretion of VesB, the first bacterial multidomain trypsin-like protease with known structure, are discussed.