Acid protease from Trichoderma reesei: limited proteolysis of fungal carbohydrases

Mechanisms regulating post-secretory limited proteolysis, carried out by the acid protease from Trichoderma reesei, were studied by following the release of α-galactosidase and multiple forms of cellobiohydrolase from this species. Both the rate of the proteolysis and the mode of action of the protease were affected by the pH of the culture medium, and only weakly depended on the amount of the enzyme. At pH between 2.7 and 3.5 the proteolytic reaction was limited, while at lower pH proteins were completely digested. Proteolysis depended on the degree of glycosylation of secreted enzymes. Inhibition of post-secretory deglycosylation decreased the rate of limited proteolysis in the culture medium in the course of fungal growth. Glucose and cellobiose, the main products of cellulose degradation carried out by the fungal cellulolytic complex, inhibited the proteolysis of the cellobiohydrolase in a concentration-dependent manner. A 32-kDa aspartic protease (EC 3.4.23.18) secreted by T. reesei was purified to homogeneity. The acid protease cleaved α-galactosidase and cellobiohydrolase into the same proteolytic fragments that had been isolated from the culture medium. Received: 4 December 1998 / Received revision: 22 February 1999 / Accepted: 5 March 1999     

Production of human serum albumin by sugar starvation induced promoter and rice cell culture

Human serum albumin (HSA) is the most widely used clinical serum protein. Currently, commercial HSA can only be obtained from human plasma, due to lack of commercially feasible recombinant protein expression systems. In this study, inducible expression and secretion of HSA by transformed rice suspension cell culture was established. Mature form of HSA was expressed under the control of the sucrose starvation-inducible rice α Amy3 promoter, and secretion of HSA into the culture medium was achieved by using the α Amy3 signal sequence. High concentrations of HSA were secreted into culture medium in a short time (2–4 days) by sucrose depletion after cell concentrations had reached a peak density in culture medium containing sucrose. The recombinant HSA had the same electrophoretic mobility as commercial HSA and was stable and free from apparent proteolysis in the culture medium. In a flask scale culture with repeated sucrose provision-depletion cycles, HSA was stably produced with yields up to 11.5% of total medium proteins or 15 mg/L per cycle after each sucrose provision-depletion cycle. A bubble column type bioreactor was designed for production of HSA. In the bioreactor scale culture, HSA was produced with yields up to 76.4 mg/L 4 days after sucrose depletion. HSA was purified from the culture medium to high purity by a simple purification scheme. Enrichment of HSA in culture medium simplifies downstream purification, minimizes protease degradation, and may reduce production cost. The combination of a DNA construct containing the α Amy3 promoter and signal sequence, and the use of a rice suspension cell culture can provide an effective system for the production of recombinant pharmaceutical proteins.     

Process development for high-level secretory production of carboxypeptidase Y by Saccharomyces cerevisiae

In order to develop a production process for carboxypeptidase Y (CPY, yeast vacuolar protease) secreted by Saccharomyces cerevisiae KS58-2D, medium composition, culture conditions, and expression systems were investigated. We found that the addition of histidine to thiamine-free medium, in which CPY production was almost negligible, raised the intracellular thiamine level, resulting in the increase of CPY production. On the basis of the choice of an expression system that uses an inducible GAL10 promoter, reassessment of histidine concentration in the medium, and optimization of the pH level during cultivation (pH 6.5), active CPY was secreted in a quantity of over 400 mg/l, which was more than tenfold that higher than that previously reported. The process developed could be easily scaled-up to industrial-scale fermentation. Received: 16 January 1998 / Received revision: 16 February 1998 / Accepted: 27 February 1998     

Improved process for production of recombinant yeast-derived monomeric human G-CSF

The human granulocyte colony-stimulating factor (hG-CSF) was efficiently secreted at high levels in fed-batch cultures of recombinant Saccharomyces cerevisiae. However, the secreted recombinant hG-CSF (rhG-CSF) was shown to exist as large multimers in the culture broth due to strong hydrophobic interaction. It was hardly monomerized even by urea at high concentration. This multimer has been reported to diminish specific receptor-binding activity of hG-CSF and causes undesirable problems in the downstream process. When the rhG-CSF was secreted to extracellular broth in the presence of a non-ionic surfactant (Tween 80) in the culture media, the multimerization of the secreted rhG-CSF was efficiently prevented in the fed-batch cultures. Also, the monomer fraction and secretion efficiency of rhG-CSF were significantly increased at the higher culture pH (6.5). Without using any denaturing agents, the secreted rhG-CSF monomer was easily purified with high recovery yield and purity via a simple purification process under acidic conditions, consisting of diafiltration, cation exchange, and gel filtration chromatography. A lyophilization process devoid of intermonomer aggregation was also designed using effective stabilizing agents. Received: 2 March 1999 / Received revision: 16 April 1999 / Accepted: 23 April 1999     

Identification, gene cloning and expression of serine proteases in the extracellular medium of Nicotiana tabacum cells

Recombinant proteins secreted from plant suspension cells into the medium are susceptible to degradation by host proteases secreted during growth. Some degradation phenomena are inhibited in the presence of various protease inhibitors, such as EDTA or AEBSF/PMSF, suggesting the presence of different classes of proteases in the medium. Here, we report the results of a proteomic analysis of the extracellular medium of a Nicotiana tabacum bright yellow 2 culture. Several serine proteases belonging to a Solanaceae-specific subtilase subfamily were identified and the genes for four cloned. Their expression at the RNA level during culture growth varied depending on the gene. An in-gel protease assay (zymography) demonstrated serine protease activity in the extracellular medium from cultures. This was confirmed by testing the degradation of an antibody added to the culture medium. This particular subtilase subfamily, therefore, represents an interesting target for gene silencing to improve recombinant protein production. Key message The extracellular medium of Nicotiana tabacum suspension cells contains serine proteases that degrade antibodies.     

Glucose- and K+-induced acidification in different yeast species

The process of acidification of the external medium after addition of glucose and subsequently of KCl to a suspension of yeast cells varies substantially from species to species. After glucose it is most pronounced inSaccharomyces cerevisiae andSchizosaccharomyces pombe but is very much lower inLodderomyces elongisporus, Dipodascus magnusii andRhodotorula gracilis. Both the buffering capacity and the varied effects of vanadate, suloctidil and erythrosin B indicate that the acidification is by about one-half due to the activity of plasma membrane H⁺-ATPase and by about one-half to the extrusion of acidic metabolites from cells. This is supported by the finding that a respiratory quotient greater than one (in various strains ofS. cerevisiae and inS. pombe) is indicative of a greater buffering capacity and overall acidification of the medium. Taking into account the virtually negligible buffering capacity of the medium in the pH range where the effect of K⁺ is observed, the effect of K⁺ is generally of a similar magnitude as that of adding glucose. It is clearly dependent on (anaerobic) production of metabolic energy, quite distinct from the dependence of the H⁺-ATPase-caused acidification.     

Purification and partial characterization of an elastinolytic proteinase from Aspergillus flavus culture filtrates

 A 23-kDa protein with elastinolytic activity was purified from Aspergillus flavus (NRRL 18543) culture filtrates by gel-filtration chromatography. Severe inhibition of the elastinolytic activity by 1,10-phenanthrolene (5 mM) and EDTA (0.8 mM) indicated that the protein belongs to the metallo class of proteases. The isoelectric point was 9.0. Natural substrates susceptible to cleavage by this protease, in addition to elastin, included cottonseed storage protein, collagen, ovalbumin and bovine serum albumin. The 23-kDa protein was thermostable to 70°C and retained its elastinolytic activity in concentrated form at 4°C for 6 months. Elastinolytic activity was initially secreted into the culture medium as a 35-kDa protein, which was subsequently converted to a 23-kDa protein, presumably through autolysis. This putative proteolytic degradation product appears to be identical to the 23-kDa protein recovered from the gel-filtration column. The 23-kDa protease may confer selective advantage to the fungus in the extracellular environment because of its temperature and pH stability and wide range of potential natural protein substrates. Received: 24 October 1995/Received last revision: 27 March 1996/Accepted: 30 March 1996     

Secretion by Saccharomyces cerevisiae of rat apolipoprotein E as a fusion to Mucor rennin

As the first step for production of rat apolipoprotein E (rApoE) in Saccharomyces cerevisiae, the rApoE cDNA was cloned and its nucleotide sequence was determined. When the intact rApoE gene including the presequence-encoding region was expressed under the control of the yeast GAL7 promoter, no protein immunoreactive with anti-rApoE antibody was detected either in the culture medium or inside the cells. For the purpose of the extracellular production of rApoE, three fusion genes were constructed in which the mature rApoE-encoding sequence was connected after the pre, prepro, and whole regions of the gene encoding a fungal aspartic proteinase, Mucor pusillus rennin (MPP), since MPP is efficiently secreted from recombinant S. cerevisiae containing the MPP gene. When these three fusion genes were expressed under the control of the GAL7 promoter, only one, encoding the mature rApoE connected to the whole MPP sequence, directed efficient secretion of the fused protein. The maximum yield of the fused protein secreted into the medium reached 11.8 mg/l and the calculated rApoE part was 5.3 mg in the fused protein. The excreted fusion protein was glycosylated at the original two sites in the MPP part. The fused protein was gradually degraded in the medium probably by proteases of the host cell, because no such degradation occured in a yeast pep4mutant strain.     

Secretory and extracellular production of recombinant proteins using<Emphasis Type="Italic"> Escherichia coli</Emphasis>

Escherichia coli is one of the most widely used hosts for the production of recombinant proteins. However, there are often problems in recovering substantial yields of correctly folded proteins. One approach to solve these problems is to have recombinant proteins secreted into the periplasmic space or culture medium. The secretory production of recombinant proteins has several advantages, such as simplicity of purification, avoidance of protease attack and N-terminal Met extension, and a better chance of correct protein folding. In addition to the well-established Sec system, the twin-arginine translocation (TAT) system has recently been employed for the efficient secretion of folded proteins. Various strategies for the extracellular production of recombinant proteins have also been developed. For the secretory production of complex proteins, periplasmic chaperones and protease can be manipulated to improve the yields of secreted proteins. This review discusses recent advances in secretory and extracellular production of recombinant proteins using E. coli.     

Yeast secretory expression of insulin precursors

Since the 1980s, recombinant human insulin for the treatment of diabetes mellitus has been produced using either the yeast Saccharomyces cerevisiae or the prokaryote Escherichia coli. Here, development of the insulin secretory expression system in S. cerevisiae and its subsequent optimisation is described. Expression of proinsulin in S. cerevisiae does not result in efficient secretion of proinsulin or insulin. However, expression of a cDNA encoding a proinsulin-like molecule with deletion of threonine^B30 as a fusion protein with the S. cerevisiaeα-factor prepro-peptide (leader), followed either by replacement of the human proinsulin C-peptide with a small C-peptide (e.g. AAK), or by direct fusion of lysine^B29 to glycine^A1, results in the efficient secretion of folded single-chain proinsulin-like molecules to the culture supernatant. The secreted single-chain insulin precursor can then be purified and subsequently converted to human insulin by tryptic transpeptidation in organic–aqueous medium in the presence of a threonine ester. The leader confers secretory competence to the insulin precursor, and constructed (synthetic) leaders have been developed for efficient secretory expression of the insulin precursor in the yeasts S. cerevisiae and Pichia pastories. The Kex2 endoprotease, specific for dibasic sites, cleaves the leader-insulin precursor fusion protein in the late secretory pathway and the folded insulin precursor is secreted to the culture supernatant. However, the Kex2 endoprotease processing of the pro-peptide-insulin precursor fusion protein is incomplete and a significant part of the pro-peptide-insulin precursor fusion protein is secreted to the culture supernatant in a hyperglycosylated form. A spacer peptide localised between the leader and the insulin precursor has been developed to optimise Kex2 endoprotease processing and insulin precursor fermentation yield. Received: 8 February 2000 / Received revision: 2 May 2000 / Accepted: 2 May 2000     

The Bacillus subtilis menCD promoter is responsive to extracellular pH

Activation kinetics of a Bacillus subtilis menaquinone biosynthetic gene promoter (the menCD promoter) were measured during growth and sporulation, with the aid of a menCD-lacZ translational gene fusion. Transient maximal activation was seen shortly after the end of exponential growth in unbuffered complex medium containing a low glucose concentration. These activation kinetics were correlated with transient acidification of the medium under conditions permitting TCA cycle function during the post-exponential period, while mutations that blocked TCA cycle function (cit mutants) were associated with sustained acidification and promoter activation during this period. In cit ⁺ strains, buffering of the medium to pH 5.7 caused sustained maximal activation, while buffering to pH 7.2 prevented enhancement of activation. The menCD promoter appears to be responsive to extracellular acidic pH.     

Characteristics of glycosylated streptokinase secreted from Pichia pastoris: enhanced resistance of SK to proteolysis by glycosylation

Degradation of streptokinase (SK) has been frequently observed during large-scale protein production. An enhanced susceptibility of SK to degradation has been correlated with its existence in a partially unfolded state. The influence of the carbohydrate moiety on the stability and functional characteristics of SK has been examined by obtaining the glycoform of SK following its secretion through the methylotrophic yeast Pichia pastoris. Secretion of the protein product was achieved by replacing the native secretion signal codons of SK with those from α-factor leader peptide and expressing the fusion construct under the control of the methanol-inducible alcohol oxidase (ox) promoter of P. pastoris after its integration into the host chromosome. Western blot and zymographic analysis of proteins secreted from the recombinant P. pastoris indicated that SK was glycosylated by the host cells, which resulted in the appearance of a SK species migrating slowly, corresponding to a 55-kDa protein product as compared to the 47-kDa native SK. The glycosylated SK retained a plasminogen activation capability identical to that of its unglycosylated counterpart. Glycoform SK exhibited an enhanced stability profile at 25 °C and 37 °C and improved resistance towards protease treatment compared to unglycosylated SK secreted through P. pastoris after tunicamycin treatment or that secreted from the recombinant Escherichia coli. The results presented thus illustrate that N-linked glycosylation of SK results in 30–40% enhancement of the protein stability and resistance towards degradation but does not interfere with its fibrinolytic function. Received: 1 March 1999 / Received last revision: 5 October 1999 / Accepted: 10 October 1999     

Expression of the Arxula adeninivorans glucoamylase gene in Kluyveromyces lactis

 The glucoamylase gene of the yeast Arxula adeninivorans was expressed in Kluyveromyces lactis by using the GAP promoter from Saccharomyces cerevisiae and a multicopy plasmid vector. The transformants secreted 90.1% of the synthesized glucoamylase into the culture medium. The secreted glucoamylase activities are about 20 times higher in comparison to those of Saccharomyces cerevisiae transformants using the same promoter. Secreted glucoamylase possesses identical N-terminal amino acid sequences to those secreted by A. adeninivorans showing that cleavage of the N-terminal signal peptide takes place at the same site. Biochemical characteristics of glucoamylase expressed by K. lactis and A. adeninivorans are very similar. Received: 12 June 1995/Received revision: 17 July 1995/Accepted: 26 July 1995     

Extracellular protease of Natrialba magadii: purification and biochemical characterization

A serine protease secreted by the haloalkaliphilic archaeon Natrialba magadii at the end of the exponential growth phase was isolated. This enzyme was purified 83 fold with a total yield of 25% by ethanol precipitation, affinity chromatography, and gel filtration. The native molecular mass of the enzyme determined by gel filtration was 45 kDa. Na. magadii extracellular protease was dependent on high salt concentrations for activity and stability, and it had an optimum temperature of 60°C in the presence of 1.5 M NaCl. The enzyme was stable and had a broad pH profile (6–12) with an optimum pH of 8–10 for azocasein hydrolysis. The protease was strongly inhibited by diisopropyl fluorophosphate (DFP), phenylmethyl sulfonylfluoride (PMSF), and chymostatin, indicating that it is a serine protease. It was sensitive to denaturing agents such as SDS, urea, and guanidine HCl and activated by thiol-containing reducing agents such as dithiotreitol (DTT) and 2-mercaptoethanol. This protease degraded casein and gelatin and showed substrate specificity for synthetic peptides containing Phe, Tyr, and Leu at the carboxyl terminus, showing that it has chymotrypsin-like activity. Na. magadii protease presented no cross-reactivity with polyclonal antibodies raised against the extracellular protease of Natronococcus occultus, suggesting that although these proteases share several biochemical traits, they might be antigenically unrelated. Received: October 1, 1999 / Accepted: February 1, 2000     

Expression and Secretion of Scytalidopepsin B,an Acid Protease from Scytalidium lignicolum,in Yeast

An expression and secretion system for scytalidopepsin B, an acid protease from Scytalidium lignicolum, was constructed in yeast. Saccharomyces cerevisiae AH22 was transformed with an yeast-E. coli shuttle vector, pAM82, in which an yeast invertase signal segment and the cDNA encoding the pro- and mature enzyme regions were inserted. The transformant was found to secret a pepstatin-insensitive acid protease, when cultured aerobically in a low phosphate (Pi) medium. Amino terminal amino acid sequencing analysis indicated that the recombinant acid protease was accurately processed and secreted as a mature form.     

Secretion and localization of invertase and inulinase in recombinant Saccharomyces cerevisiae

Summary Secretion of invertase and inulinase produced by recombinant Saccharomyces cerevisiae cells were investigated under derepression conditions of GALI promoter. Secreted invertase mainly localized in the periplasmic space, but most of inulinase was found in the extracellular culture medium. This high level of extracellular secretion of inulinase was not dependent on the growth phase in which derepression of GALI promoter occurs. Our results indicate that the inulinase polypeptide itself may have a function for the protein secretion into the culture medium.     

Effect of N-Linked Glycosylation on Secretion, Activity, and Stability of α-Amylase from Aspergillus oryzae

The effect of N-linked glycosylation on secretion, activity, and stability of α-amylase from Aspergillus oryzae grown as dispersed filaments was studied. In the presence of tunicamycin the fungus grew either as dispersed filaments or as one large pellet, whereas growth was as dispersed filaments in all control cultures. The presence of tunicamycin affected neither biomass, level of secreted α-amylase, nor total amount of secreted protein in cultures growing as dispersed filaments. In these cultures both glycosylated and nonglycosylated α-amylase appeared in the culture medium as well as in the cells, whereas in control cultures only the glycosylated form of α-amylase was found in the medium and in the cells. The presence of nonglycosylated α-amylase in the medium seemed to result from active secretion rather than from autolysis of the mycelium or extracellular deglycosylation. Deglycosylation with Endo H of crude α-amylase in culture filtrate did not affect its stability towards heat, acid pH, or proteolytic degradation. Received: 22 December 1997 / Accepted: 24 February 1998     

Production of human caseinomacropeptide in recombinant Saccharomyces cerevisiae and Pichia pastoris

Caseinomacropeptide is a polypeptide of 64 amino acid residues (106–169) derived from the C-terminal part of the mammalian milk k-casein. This macropeptide has various biological activities and is used as a functional food ingredient as well as a pharmaceutical compound. The gene encoding the human caseinomacropeptide (hCMP) was synthesized and expressed with an α-factor secretion signal in the two yeast strains, Saccharomyces cerevisiae and Pichia pastoris. The complete polypeptide of the recombinant hCMP was produced and secreted in a culture medium by both the strains, but the highest production was observed in S. cerevisiae with a galactose-inducible promoter. In a fed-batch bioreactor culture, 2.5 g/l of the recombinant hCMP was obtained from the S. cerevisiae at 97 h.     

Expression of a catalytic domain of a Neocallimastix frontalis endoxylanase gene (xyn3) in Kluyveromyces lactis and Penicillium roqueforti

A cDNA fragment encoding the A catalytic domain of the Neocallimastix frontalis endoxylanase XYN3 was amplified and cloned by the polymerase chain reaction technique. The xyn3A DNA fragment was inserted between the Saccharomyces cerevisiae phosphoglycerate kinase gene promoter and terminator sequences on a multicopy episomal plasmid for Kluyveromyces lactis. The XYN3A domain was successfully expressed in K. lactis and functional endoxylanase was secreted by the yeast cells with the K. lactis killer toxin secretion signal. The XYN3A domain was also expressed in a strain of Penicillium roqueforti as a fusion protein (ShBLE::XYN3A) of the phleomycin-resistance gene product and the endoxylanase. Active endoxylanase was efficiently secreted from the fungal cells with the Trichoderma viride cellobiohydrolase (CBH1) secretion signal and processed by a related KEX2 endoprotease of the secretion pathway. Several differently glycosylated forms of the recombinant enzymes were secreted by the yeast and the filamentous fungus. Received: 10 November 1998 / Received revision: 8 March 1999 / Accepted: 14 March 1999     

Biosynthesis of Inositol by an Enzyme System in the Rice Seeds

As the first step for production of human apolipoprotein E (hApoE) in Saccharomyces cerevisiae, the hApoE cDNA was cloned in Escherichia coli, on the basis of the nucleotide sequence reported previously. When the hApoE cDNA including its pre-sequence-encoding region was expressed under the control of the GAL7 promoter, no protein immunoreactive with anti-hApoE antibody was detected either in the culture medium or inside the cells. For efficient production and secretion of hApoE in S. cerevisiae, the mature hApoE-encoding region was fused to the prepro-sequence region of Rhizomucor rennin (MPR) and to the whole MPR gene including its prepro- and mature-MPR regions. When the fusion gene consisting of the prepro-sequence-encoding region and hApoE regions was expressed in S. cerevisiae, no protein reactive with the anti-hApoE antibody was detected in any fraction of the yeast cells, probably due to rapid degradation of the hApoE protein by yeast proteases. On the othe hand, when hApoE was expressed as a fusion to the whole MPR protein, a considerable amount of the fused protein was secreted into the medium. The preprosequence of MPR was correctly processed from the fused protein in the medium by autocatalytic activity of MPR and by a protease(s) of the host cell. Integration of the fusion gene into the chromosome at a copy number of eight led to secretion of the fused protein in a larger amount than the case when the fusion gene was carried on a 2-µm plasmid with its copy number of a few hundreds, because the 2-µm derived plasmid containing the fusion gene was very unstable in the yeast cells. The secretion level was also improved by changing g the culture conditions. A maximum yield of hApoE part in the secreted fused protein was estimated to be 23.7 mg per liter and the amount of the fused protein was calculated to be 53.0 mg per liter.