Disease-related amyloidogenic variants of human lysozyme trigger the unfolded protein response and disturb eye development in Drosophila melanogaster

We have created a Drosophila model of lysozyme amyloidosis to investigate the in vivo behavior of disease-associated variants. To achieve this objective, wild-type (WT) protein and the amyloidogenic variants F57I and D67H were expressed in Drosophila melanogaster using the UAS-gal4 system and both the ubiquitous and retinal expression drivers Act5C-gal4 and gmr-gal4. The nontransgenic w(1118) Drosophila line was used as a control throughout. We utilized ELISA experiments to probe lysozyme protein levels, scanning electron microscopy for eye phenotype classification, and immunohistochemistry to detect the unfolded protein response (UPR) activation. We observed that expressing the destabilized F57I and D67H lysozymes triggers UPR activation, resulting in degradation of these variants, whereas the WT lysozyme is secreted into the fly hemolymph. Indeed, the level of WT was up to 17 times more abundant than the variant proteins. In addition, the F57I variant gave rise to a significant disruption of the eye development, and this correlated to pronounced UPR activation. These results support the concept that the onset of familial amyloid disease is linked to an inability of the UPR to degrade completely the amyloidogenic lysozymes prior to secretion, resulting in secretion of these destabilized variants, thereby leading to deposition and associated organ damage.     

Expression, purification, and characterization of the recombinant calcium-binding equine lysozyme secreted by the filamentous fungus Aspergillus niger: comparisons with the production of hen and human lysozymes.

Equine lysozyme (EqL) has been expressed from a synthetic gene and secreted from a heterologous host, the filamentous fungus Aspergillus niger. By including 100 mM Ca2+ in the growth medium, secreted yields of more than 50 mg/liter could be achieved using polyvinylpyrrolidone (PVP) complete medium. In a soya medium yields of up to 150 mg/liter were achieved. The production of recombinant human lysozyme (HuL) from A. niger with yields of over 40 mg/liter was also achieved using PVP medium. Addition of Ca2+ to the growth medium reduced the yield of both HuL and hen egg white lysozyme (HEWL). Sequence differences between the three lysozymes, EqL, HuL, and HEWL, resulted in different susceptibilities to cleavage by A. niger proteases. An improved procedure for the purification of EqL and HuL from A. niger allowed separation of the proteins from pigments produced by the fungus. Detailed spectroscopic analysis, including 2D 1H NMR, for recombinant EqL and recombinant HuL confirm that both proteins possess their native structure and are purified to homogeneity.     

Analysis of unfolded protein response during single-chain antibody expression in Saccaromyces cerevisiae reveals different roles for BiP and PDI in folding

The production of recombinant proteins is a critical technology for biotechnology and biomedical research. Heterologous expression of secreted proteins can saturate the cell's capacity to properly fold protein, initiating the unfolded protein response (UPR), and resulting in a loss of protein expression. The overexpression of chaperone binding protein (BiP) and disulfide bond isomerase (PDI) in Saccaromyces cerevisiae can effectively increase protein production levels of single-chain antibody (scFv) 4-4-20. These studies show that overexpression of BiP did not reduce the UPR activated by heterologous protein expression; however, overexpression of PDI or co-overexpression of BiP and PDI could reduce the UPR. We observed that co-overexpression of BiP and PDI led to the greatest secretion of scFv from the cell, but BiP and PDI appear to interact with the newly synthesized scFv at different stages in the folding process, as determined by pulse-chase analysis. We propose that BiP acts primarily to facilitate translocation and retain unfolded or partially folded scFv, and PDI actively folds the scFv through its functions as a catalyst, and/or an isomerase, of disulfide bonds. Free BiP is released when scFv is folded, stabilizing Ire1p, and leading to the reduced UPR.     

Activation of the unfolded protein response in Pichia pastoris requires splicing of a HAC1 mRNA intron and retention of the C-terminal tail of Hac1p

We have shown that the unfolded protein response (UPR) in Pichia pastoris requires splicing of a non-conventional intron in the HAC1(u) mRNA in common with other eukaryotes. P. pastoris is a favoured yeast expression host for secreted production of heterologous proteins and the regulation of the UPR in P. pastoris may hold the key to its effective folding and secretion of proteins. We have also shown that the C-terminal region of the Hac1p from P. pastoris is required for functionality. Although the C-terminal regions of Hac1p from both S. cerevisiae and P. pastoris are rich in phenylalanine residues, the P. pastoris Hac1p lacks a C-terminal serine that is known to be important in the efficient functionality of Hac1p from S. cerevisiae.     

Construction of an expression system of insect lysozyme lacking thermal stability: the effect of selection of signal sequence on level of expression in the Pichia pastoris expression system.

Expression systems of human and silkworm lysozymes were constructed using the methylotrophic yeast Pichia pastoris as a host. The leader sequence and its prepro peptide of alpha-factor (a peptide pheromone derived from yeast) and the native signal sequences of these lysozymes, were used as secretion signals. When the alpha-factor leader is used as the signal sequence, human lysozyme is secreted at a much higher level than is silkworm lysozyme. On the other hand, silkworm lysozyme, when its native signal is used, is secreted more efficiently than human lysozyme. Therefore, we expected that human lysozyme cDNA with a silkworm native signal would be secreted more efficiently than human lysozyme with its native signal. However, its level of expression was not increased. This result indicates that the native signal of silkworm lysozyme does not promote the secretion of the lysozyme, but rather alpha-factor leader inhibits the secretion. Silkworm lysozyme with the alpha-factor leader is so unstable that it could be easily attacked by some proteases and our findings suggest that the level of expression of heterologous protein with signal peptides and its stability are greatly affected by the selection of the appropriate secretion signal sequence.     

A yeast platform for the production of single-chain antibody-green fluorescent protein fusions

Fusion proteins comprised of a binding domain and green fluorescent protein (GFP) have the potential to act as one-step binding reagents. In this study, eight single-chain antibodies (scFv) and one single-chain T-cell receptor (scTCR) were secreted as fusions to GFP using a Saccharomyces cerevisiae expression system. Fusion protein secretion levels ranged over 3 orders of magnitude, from 4 microg/liter to 4 mg/liter, and correlated well with the secretion levels of the unfused scFv/scTCR. Three fusion types with various linker lengths and fusion orientations were tested for each scFv/scTCR. Although the fusion protein secretion levels were not significantly affected by the nature of the fusion construct, the properties of the fusion protein were clearly influenced. The fluorescence yield per fusion molecule was increased by separating the scFv/scTCR and GFP with an extended (GGGGS)3 linker, and fusions with scFv/scTCR at the carboxy-terminus were more resistant to degradation. By evaluating leader sequence processing and using GFP fluorescence to track intracellular processing, it was determined that the majority of fusion protein synthesized by the yeast was not secreted and in most cases was accumulating in an immature, although active, endoplasmic-reticulum (ER)-processed form. This contrasted with unfused scFv, which accumulated in both immature ER-processed and mature post-Golgi forms. The results indicated that yeast can be used as an effective host for the secretion of scFv/scTCR-GFP fusion proteins and that as a result of intracellular secretory bottlenecks, there is considerable yeast secretory capacity remaining to be exploited.     

A Yeast Platform for the Production of Single-Chain Antibody-Green Fluorescent Protein Fusions

Fusion proteins comprised of a binding domain and green fluorescent protein (GFP) have the potential to act as one-step binding reagents. In this study, eight single-chain antibodies (scFv) and one single-chain T-cell receptor (scTCR) were secreted as fusions to GFP using a Saccharomyces cerevisiae expression system. Fusion protein secretion levels ranged over 3 orders of magnitude, from 4 μg/liter to 4 mg/liter, and correlated well with the secretion levels of the unfused scFv/scTCR. Three fusion types with various linker lengths and fusion orientations were tested for each scFv/scTCR. Although the fusion protein secretion levels were not significantly affected by the nature of the fusion construct, the properties of the fusion protein were clearly influenced. The fluorescence yield per fusion molecule was increased by separating the scFv/scTCR and GFP with an extended (GGGGS)₃ linker, and fusions with scFv/scTCR at the carboxy-terminus were more resistant to degradation. By evaluating leader sequence processing and using GFP fluorescence to track intracellular processing, it was determined that the majority of fusion protein synthesized by the yeast was not secreted and in most cases was accumulating in an immature, although active, endoplasmic-reticulum (ER)-processed form. This contrasted with unfused scFv, which accumulated in both immature ER-processed and mature post-Golgi forms. The results indicated that yeast can be used as an effective host for the secretion of scFv/scTCR-GFP fusion proteins and that as a result of intracellular secretory bottlenecks, there is considerable yeast secretory capacity remaining to be exploited.     

Reduced proteolysis of secreted gelatin and Yps1-mediated alpha-factor leader processing in a Pichia pastoris kex2 disruptant

Heterologous proteins secreted by yeast and fungal expression hosts are occasionally degraded at basic amino acids. We cloned Pichia pastoris homologs of the Saccharomyces cerevisiae basic residue-specific endoproteases Kex2 and Yps1 to evaluate their involvement in the degradation of a secreted mammalian gelatin. Disruption of the P. pastoris KEX2 gene prevented proteolysis of the foreign protein at specific monoarginylic sites. The S. cerevisiae alpha-factor preproleader used to direct high-level gelatin secretion was correctly processed at its dibasic site in the absence of the prototypical proprotein convertase Kex2. Disruption of the YPS1 gene had no effect on gelatin degradation or processing of the alpha-factor propeptide. When both the KEX2 and YPS1 genes were disrupted, correct precursor maturation no longer occurred. The different substrate specificities of both proteases and their mutual redundancy for propeptide processing indicate that P. pastoris kex2 and yps1 single-gene disruptants can be used for the alpha-factor leader-directed secretion of heterologous proteins otherwise degraded at basic residues.     

Decreased protein expression and intermittent recoveries in BiP levels result from cellular stress during heterologous protein expression in Saccharomyces cerevisiae

Cells are inherently robust to environmental perturbations and have evolved to recover readily from short-term exposure to heat, pH changes, and nutrient deprivation during times of stress. The stress of unfolded protein accumulation has been implicated previously in low protein yields during heterologous protein expression. Here we describe the dynamics of the response to this stress, termed the unfolded protein response (UPR), during the expression of the single chain antibody 4-4-20 (scFv) in Saccharomyces cerevisiae. Expression of scFv decreased the growth rate of yeast cells whether the scFv was expressed from single-copy plasmids or integrated into the chromosome. However, the growth rates recovered at longer expression times, and surprisingly, the recovery occurred more quickly in the high-copy integration strains. The presence of a functional UPR pathway was necessary for a recovery of normal growth rates. During the growth inhibition, the UPR pathway appeared to be activated, resulting in decreased intracellular scFv levels and intermittent recovery of the chaperone BiP within the endoplasmic reticulum. Intracellular scFv was observed primarily in the endoplasmic reticulum, consistent with activation of the UPR pathway. Although the intracellular scFv levels dropped over the course of the expression, this was not a result of scFv secretion. A functional UPR pathway was necessary for the drop in intracellular scFv, suggesting that the decrease was a direct response of UPR activation. Taken together, these results suggest that control of heterologous gene expression to avoid UPR activation will result in higher production levels.     

Expression,purification, and characterization of an unstable lysozyme mutant in Pichia pastoris

To investigate the expression and purification of an unstable heterologous protein in Pichia pastoris, the cDNA of H5-lysozyme, a hen egg lysozyme mutant with a hydrophobic pentapeptide (Phe-Phe-Val-Ala-Pro) fused to the carboxyl terminus, was integrated into the genome of P. pastoris. It was found that medium composition, induction time, and fermenter type were important factors for the expression of H5-lysozyme. Substantially active H5-lysozyme was secreted by induction with methanol when the prepro-sequence of alpha-factor was used as secretion signal sequence. The amount secreted was 422-fold greater than that observed with Saccharomyces cerevisiae. Recombinant H5-lysozyme was recovered and purified by cation-exchange chromatography directly from fermentation broth. The mutant lysozyme showed bactericidal activity against Gram-positive as well as Gram-negative bacteria.     

Human lysozyme secretion increased by alpha-factor pro-sequence in Pichia pastoris

To get high level secretion of human lysozyme in Pichia pastoris, the following three signal sequences and one prepro sequence were evaluated: chicken lysozyme signal peptide, leucine-rich artificial signal peptide, Saccharomyces invertase signal peptide, and Saccharomyces prepro sequence of alpha factor (MF-alpha Prepro). Transformants harboring a lysozyme gene with MF-alpha Prepro secreted 20-fold more lysozyme than those harboring the lysozyme gene with any one of the other three signal sequences. Three mutant leader sequences derived from MF-alpha Prepro were constructed to discover the function of the pro region. The secretion was dramatically decreased by eliminating the pro region of MF-alpha Prepro. In contrast, MF-alpha Prepro with the EAEAEA sequence directed the secretion of an equivalent level of lysozyme having the extra amino acids (EAEAEA) in its N-terminus. For the effective secretion of native human lysozyme, MF-alpha Prepro without any spacer sequences was most suitable. The secreted protein by MF-alpha Prepro construct was identical with the authentic human lysozyme, judging from N-terminal amino acid sequencing and molecular mass spectrometric and crystallographic analysis.     

High yield secretion of the sweet-tasting protein lysozyme from the yeast Pichia pastoris

Hen egg lysozyme (HEL) is one of the sweet-tasting proteins. To understand why lysozyme is sweet, the enzyme was synthesized at high yields by a recombinant method. The mature HEL gene was cloned from a Taq polymerase-amplified PCR product into the Pichia pastoris expression and secretion vector pPIC6alpha. This expression vector contains both the Saccharomyces cerevisiae pre-pro alpha-mating factor secretion signal and the blasticidin resistance gene (bsd) for selection of transformants in bacteria and yeast. Expression of HEL was carried out in fermenter cultures. Culture supernatants were concentrated by ultrafiltration and purified by CM-ion exchange chromatography. Approximately 400 mgL-1 of recombinant HEL was obtained. The high yield of recombinant lysozyme enabled us to perform a sensory analysis in humans. The purified recombinant lysozyme elicited as a sweet taste sensation as does the lysozyme purified directly from egg white, and showed full lytic activity against cells of Micrococcus luteus. These results demonstrate that the P. pastoris expression system with the blasticidin S selection system is useful in producing recombinant sweet-tasting protein in active form at a high yield.     

Secreted production of collagen-inspired gel-forming polymers with high thermal stability in Pichia pastoris

Previously, we have shown that gel-forming triblock proteins, consisting of random coil middle blocks and trimer-forming (Pro-Gly-Pro)(9) end blocks, are efficiently produced and secreted by the yeast Pichia pastoris. These end blocks had a melting temperature (T(m)) of ～41°C (at 1.1 mM of protein). The present work reveals that an increase of T(m) to ～74°C, obtained by extension of the end blocks to (Pro-Gly-Pro)(16), resulted in a five times lower yield and partial endoproteolytic degradation of the protein. A possible cause could be that the higher thermostability of the longer (Pro-Gly-Pro)(16) trimers leads to a higher incidence of trimers in the cell, and that this disturbs secretion of the protein. Alternatively, the increased length of the proline-rich (Pro-Gly-Pro)(n) domain may negatively influence ribosomal translation, or may result in, for example, hydrophobic aggregation or membrane-active behavior owing to the greater number of closely placed proline residues. To discriminate between these possibilities, we studied the production of molecules with randomized end blocks that are unable to form triple helices. The codon- and amino acid composition of the genes and proteins, respectively, remained unchanged. As these nontrimerizing molecules were secreted intact and at high yield, we conclude that the impaired secretion and partial degradation of the triblock with (Pro-Gly-Pro)(16) end blocks was triggered by the occurrence of intracellular triple helices. This degradation was overcome by using a yapsin 1 protease disruptant, and the intact secreted polymer was capable of forming self-supporting gels of high thermal stability.     

Oxidative protein folding and unfolded protein response elicit differing redox regulation in endoplasmic reticulum and cytosol of yeast

Oxidative protein folding can exceed the cellular secretion machinery, inducing the unfolded protein response (UPR). Sustained endoplasmic reticulum (ER) stress leads to cell stress and disease, as described for Alzheimer, Parkinson, and diabetes mellitus, among others. It is currently assumed that the redox state of the ER is optimally balanced for formation of disulfide bonds using glutathione as the main redox buffer and that UPR causes a reduction of this organelle. The direct effect of oxidative protein folding in the ER, however, has not yet been dissected from UPR regulation. To measure in vivo redox conditions in the ER and cytosol of the yeast model organism Pichia pastoris we targeted redox-sensitive roGFP variants to the respective organelles. Thereby, we clearly demonstrate that induction of the UPR causes reduction of the cytosol in addition to ER reduction. Similarly, a more reduced redox state of the cytosol, but not of the ER, is observed during oxidative protein folding in the ER without UPR induction, as demonstrated by overexpressing genes of disulfide bond-rich secretory proteins such as porcine trypsinogen or protein disulfide isomerase (PDI1) and ER oxidase (ERO1). Cytosolic reduction seems not to be caused by the action of glutathione reductase (GLR1) and could not be compensated for by overexpression of cytosolic glutathione peroxidase (GPX1). Overexpression of GPX1 and PDI1 oxidizes the ER and increases the secretion of correctly folded proteins, demonstrating that oxidative protein folding per se is enhanced by a more oxidized ER and is counterbalanced by a more reduced cytosol. As the total glutathione concentration of these strains does not change significantly, but the ratio of GSH to GSSG is altered, either transport or redox signaling between the glutathione pools of ER and cytosol is assumed. These data clearly demonstrate that protein folding and ER stress have a severe impact on the cytosolic redox balance, which may be a major factor during development of folding-related diseases.     

抗IV型胶原酶单链抗体在毕赤酵母中分泌表达*

利用毕赤酵母系统表达抗 IV型胶原酶人单链抗体。首先把目的基因克隆到毕赤酵母表达载体上 ,电击转化受体菌。在甲醇诱导下表达单链抗体。 SDS- PAGE和免疫印迹显示毕赤酵母分泌表达人单链抗体 ,表达量约 2 0 mg/ L酵母培养物。该表达系统与大肠杆菌相比 ,简化了表达产物的分离纯化程序。     

Single-chain Fv with Fc fragment of the human IgG1 tag: construction, Pichia pastoris expression and antigen binding characterization

We report two expression vectors in Pichia pastoris that direct the synthesis of recombinant single chain antibody variable region (scFv), derived from anti-Z-DNA monoclonal antibody Z22. The first vector codes for a scFv fused to the Ig binding domain of staphylococcal Protein A. The second vector codes for the scFv fused to the Fc fragment of the human IgG1. The fusion partner simplified the detection and purification of the secreted protein. These constructs yielded high level expression of an scFv with specific binding activity toward a Z form of DNA, with binding activity comparable to that of the scFv molecule produced in an Escherichia coli expression system and the original monoclonal antibody.     

Functional Production of a Soluble and Secreted Single-Chain Antibody by a Bacterial Secretion System

Single-chain variable fragments (scFvs) serve as an alternative to full-length monoclonal antibodies used in research and therapeutic and diagnostic applications. However, when recombinant scFvs are overexpressed in bacteria, they often form inclusion bodies and exhibit loss of function. To overcome this problem, we developed an scFv secretion system in which scFv was fused with osmotically inducible protein Y (osmY), a bacterial secretory carrier protein, for efficient protein secretion. Anti-EGFR scFv (αEGFR) was fused with osmY (N- and C-termini) and periplasmic leader sequence (pelB) to generate αEGFR-osmY, osmY-αEGFR, and pelB-αEGFR (control), respectively. In comparison with the control, both the osmY-fused αEGFR scFvs were soluble and secreted into the LB medium. Furthermore, the yield of soluble αEGFR-osmY was 20-fold higher, and the amount of secreted protein was 250-fold higher than that of osmY-αEGFR. In addition, the antigen-binding activity of both the osmY-fused αEGFRs was 2-fold higher than that of the refolded pelB-αEGFR from inclusion bodies. Similar results were observed with αTAG72-osmY and αHer2-osmY. These results suggest that the N-terminus of osmY fused with scFv produces a high yield of soluble, functional, and secreted scFv, and the osmY-based bacterial secretion system may be used for the large-scale industrial production of low-cost αEGFR protein.     

Contrasting secretory processing of simultaneously expressed heterologous proteins in Saccharomyces cerevisiae

In this study, secretory processing of cell-surface displayed Aga2p fusions to bovine pancreatic trypsin inhibitor (BPTI) and the single chain Fv (scFv) antibody fragment D1.3 are examined. BPTI is more efficiently processed than D1.3 both when secreted and surface-displayed, and D1.3 expression imparts a greater amount of secretory stress on the cell as assayed by a reporter of the unfolded protein response (UPR). Surprisingly, simultaneous expression of the two proteins in the same cell somewhat improves BPTI surface display while decreasing D1.3 surface display with minimal effect on UPR activation. Furthermore, co-expression leads to the accumulation of punctate vacuolar aggregates of D1.3 and increased secretion of the D1.3-Aga2p fusion into the supernatant. Overexpression of the folding chaperones protein disulfide isomerase (PDI) and BiP largely mitigates the D1.3 surface expression decrease, suggesting that changes in vacuolar and cell surface targeting may be due, in part, to folding inefficiency. Titration of constitutive UPR expression across a broad range progressively decreases surface display of both proteins as UPR increases. D1.3-Aga2p traffic through the late secretory pathway appears to be strongly affected by overall secretory load as well as folding conditions in the ER.