Secretion of human growth hormone by the food-grade bacterium Staphylococcus carnosus requires a propeptide irrespective of the signal peptide used

Staphylococcal exoproteins can be divided into two groups. One group comprises proteins bearing only a signal peptide, the other group requires an additional propeptide for secretion. The secretion signals of the propeptide-requiring lipase from Staphylococcus hyicus (Lip) have been frequently used to produce recombinant secretory proteins in the food-grade species Staphylococcus carnosus. However, it has been unclear whether recombinant proteins can be secreted using signal peptides of staphylococcal proteins without propeptide. The human growth hormone protein (hGH) was fused to various staphylococcal secretion signals of proteins without propeptide (Seb, SceA, and SceB) and of proteins requiring a propeptide (lipase, lysostaphin, and glycerol ester hydrolase). Secretory hGH was efficiently produced by S. carnosus after fusion with any propeptide-containing secretion signal, whereas precursor proteins were retained in the cells when only a signal peptide was used. Addition of the first six amino acid residues of mature SceA to the signal peptide did also not lead to secretion of hGH. It was concluded that the properties of the mature protein domains determine whether a propeptide is required for secretion or not. The Lip propeptide could be efficiently removed from hGH after introduction of an enterokinase cleavage site between the two protein domains.     

Feature-based reappraisal of the Bacillus subtilis exoproteome

Proteomics-based verification of computer-assisted predictions on bacterial protein export have indicated that problems occur with the distinction between (Sec-type) signal peptides that govern protein secretion, and lipoprotein signal peptides or amino-terminal membrane anchors that cause protein retention in the membrane. Therefore, the main aim of this study was to investigate whether feature-based predictions by the SecretomeP (SecP) algorithm will aid the proteomics-based analysis of protein export in Bacillus subtilis. The SecP algorithm is trained to recognize features such as secondary structure and disordered regions, which are generally present in secreted proteins. The results showed that membrane-retained proteins receive, in general, high SecP scores, similar to the scores of secretory proteins. Importantly, the SecP algorithm aided in the re-evaluation of a class of previously identified proteins that remain attached to the membrane despite the presence of an apparent Sec-type signal peptide. These so-called 'Sec-attached' proteins receive on average a lower SecP score, and several of these proteins could be unmasked as transmembrane proteins by combined SecP and signal peptide analyses. Finally, the present study suggests that feature-based outlier analysis may provide leads towards the discovery of novel special-purpose pathways for bacterial protein export.     

SPRED: A machine learning approach for the identification of classical and non-classical secretory proteins in mammalian genomes

Eukaryotic protein secretion generally occurs via the classical secretory pathway that traverses the ER and Golgi apparatus. Secreted proteins usually contain a signal sequence with all the essential information required to target them for secretion. However, some proteins like fibroblast growth factors (FGF-1, FGF-2), interleukins (IL-1 alpha, IL-1 beta), galectins and thioredoxin are exported by an alternative pathway. This is known as leaderless or non-classical secretion and works without a signal sequence. Most computational methods for the identification of secretory proteins use the signal peptide as indicator and are therefore not able to identify substrates of non-classical secretion. In this work, we report a random forest method, SPRED, to identify secretory proteins from protein sequences irrespective of N-terminal signal peptides, thus allowing also correct classification of non-classical secretory proteins. Training was performed on a dataset containing 600 extracellular proteins and 600 cytoplasmic and/or nuclear proteins. The algorithm was tested on 180 extracellular proteins and 1380 cytoplasmic and/or nuclear proteins. We obtained 85.92% accuracy from training and 82.18% accuracy from testing. Since SPRED does not use N-terminal signals, it can detect non-classical secreted proteins by filtering those secreted proteins with an N-terminal signal by using SignalP. SPRED predicted 15 out of 19 experimentally verified non-classical secretory proteins. By scanning the entire human proteome we identified 566 protein sequences potentially undergoing non-classical secretion. The dataset and standalone version of the SPRED software is available at http://www.inb.uni-luebeck.de/tools-demos/spred/spred.     

Secretion of recombinant Bacillus hydrolytic enzymes using Escherichia coli expression systems

Bacillus spp. are Gram-positive bacteria that secrete a large number of extracellular proteins of industrial relevance. In this report, three Bacillus extracellular hydrolytic enzymes, i.e., alpha-amylase, mannanase and chitinase, were cloned and over-expressed in Gram-negative Escherichia coli. We found that both the native signal peptides and that of E. coli outer membrane protein, OmpA, could be used to direct the secretion of the recombinant enzymes. The expressed enzymes were observed as clearing zones on agar plates or in zymograms. Determination of enzyme activities in different cell compartments suggested that the ability of the enzymes to be secreted out into the culture medium depends on the time of induction, the type of the signal peptides and the molecular mass of the enzymes. After overnight induction, most of the enzyme activities (85-96%) could be harvested from the culture supernatant. Our results suggest that various signal peptides of Bacillus spp. can be recognized by the E. coli secretion machinery. It seems possible that other enzymes with similar signal peptide could be secreted equally well in E. coli expression systems. Thus, our finding should be able to apply for cloning and extracellular production of other Bacillus hydrolytic enzymes as well as other proteins.     

MMP-9信号肽高效诱导PEX重组蛋白在COS7细胞中分泌表达

为了便于收集和纯化, 重组蛋白常需要引导至真核细胞外。蛋白能否分泌主要取决于其是否含有信号肽, 由于不同信号肽诱导蛋白分泌的效率不同,高效信号肽的筛选已成为生物工程领域提高重组蛋白产量的重要策略之一。为了筛选诱导MMP-2 C末端PEX在COS7细胞中高效分泌表达的信号肽,在PEX的N末端分别融合大鼠生长激素(rGH)、小鼠IgG κ链和人基质金属蛋白酶-9（matrix metalloproteinase 9, MMP-9）的信号肽并比较三种信号肽引导PEX分泌表达的效率。Western免疫印迹和ELISA蛋白定量检测表明MMP-9的信号肽引导PEX蛋白分泌的效率约为其它两种信号肽的两倍。利用Ni-NTA亲和柱对细胞培养基中的PEX进行纯化,蛋白产量约为1mg/L,纯化的PEX重组蛋白具有抑制鸡尿囊膜（chorioallantoic membrane,CAM）血管发生的作用。以上结果提示MMP-9的信号肽有效诱导具有生物活性的PEX重组蛋白在COS7细胞中分泌表达。     

The Canonical Twin-Arginine Translocase Components Are Not Required for Secretion of Folded Green Fluorescent Protein from the Ancestral Strain of Bacillus subtilis

Cellular processes, such as the digestion of macromolecules, phosphate acquisition, and cell motility, require bacterial secretion systems. In Bacillus subtilis, the predominant protein export pathways are Sec (generalized secretory pathway) and Tat (twin-arginine translocase). Unlike Sec, which secretes unfolded proteins, the Tat machinery secretes fully folded proteins across the plasma membrane and into the medium. Proteins are directed for Tat-dependent export by N-terminal signal peptides that contain a conserved twin-arginine motif. Thus, utilizing the Tat secretion system by fusing a Tat signal peptide is an attractive strategy for the production and export of heterologous proteins. As a proof of concept, we expressed green fluorescent protein (GFP) fused to the PhoD Tat signal peptide in the laboratory and ancestral strains of B. subtilis. Secretion of the Tat-GFP construct, as well as secretion of proteins in general, was substantially increased in the ancestral strain. Furthermore, our results show that secreted, fluorescent GFP could be purified directly from the extracellular medium. Nonetheless, export was not dependent on the known Tat secretion components or the signal peptide twin-arginine motif. We propose that the ancestral strain contains additional Tat components and/or secretion regulators that were abrogated following domestication.     

Engineering Signal Peptides for Enhanced Protein Secretion from Lactococcus lactis

Lactococcus lactis is an attractive vehicle for biotechnological production of proteins and clinical delivery of therapeutics. In many such applications using this host, it is desirable to maximize secretion of recombinant proteins into the extracellular space, which is typically achieved by using the native signal peptide from a major secreted lactococcal protein, Usp45. In order to further increase protein secretion from L. lactis, inherent limitations of the Usp45 signal peptide (Usp45sp) must be elucidated. Here, we performed extensive mutagenesis on Usp45sp to probe the effects of both the mRNA sequence (silent mutations) and the peptide sequence (amino acid substitutions) on secretion. We screened signal peptides based on their resulting secretion levels of Staphylococcus aureus nuclease and further evaluated them for secretion of Bacillus subtilis α-amylase. Silent mutations alone gave an increase of up to 16% in the secretion of α-amylase through a mechanism consistent with relaxed mRNA folding around the ribosome binding site and enhanced translation. Targeted amino acid mutagenesis in Usp45sp, combined with additional silent mutations from the best clone in the initial screen, yielded an increase of up to 51% in maximum secretion of α-amylase while maintaining secretion at lower induction levels. The best sequence from our screen preserves the tripartite structure of the native signal peptide but increases the positive charge of the n-region. Our study presents the first example of an engineered L. lactis signal peptide with a higher secretion yield than Usp45sp and, more generally, provides strategies for further enhancing protein secretion in bacterial hosts.     

Colon tumour secretopeptidome: Insights into endogenous proteolytic cleavage events in the colon tumour microenvironment

The secretopeptidome comprises endogenous peptides derived from proteins secreted into the tumour microenvironment through classical and non-classical secretion. This study characterised the low-M_r (< 3 kDa) component of the human colon tumour (LIM1215, LIM1863) secretopeptidome, as a first step towards gaining insights into extracellular proteolytic cleavage events in the tumour microenvironment. Based on two biological replicates, this secretopeptidome isolation strategy utilised differential centrifugal ultrafiltration in combination with analytical RP-HPLC and nanoLC-MS/MS. Secreted peptides were identified using a combination of Mascot and post-processing analyses including MSPro re-scoring, extended feature sets and Percolator, resulting in 474 protein identifications from 1228 peptides (≤ 1% q-value, ≤ 5% PEP) — a 36% increase in peptide identifications when compared with conventional Mascot (homology ionscore thresholding). In both colon tumour models, 122 identified peptides were derived from 41 cell surface protein ectodomains, 23 peptides (12 proteins) from regulated intramembrane proteolysis (RIP), and 12 peptides (9 proteins) generated from intracellular domain proteolysis. Further analyses using the protease/substrate database MEROPS, (http://merops.sanger.ac.uk/), revealed 335 (71%) proteins classified as originating from classical/non-classical secretion, or the cell membrane. Of these, peptides were identified from 42 substrates in MEROPS with defined protease cleavage sites, while peptides generated from a further 205 substrates were fragmented by hitherto unknown proteases. A salient finding was the identification of peptides from 88 classical/non-classical secreted substrates in MEROPS, implicated in tumour progression and angiogenesis (FGFBP1, PLXDC2), cell–cell recognition and signalling (DDR1, GPA33), and tumour invasiveness and metastasis (MACC1, SMAGP); the nature of the proteases responsible for these proteolytic events is unknown. To confirm reproducibility of peptide fragment abundance in this study, we report the identification of a specific cleaved peptide fragment in the secretopeptidome from the colon-specific GPA33 antigen in 4/14 human CRC models. This improved secretopeptidome isolation and characterisation strategy has extended our understanding of endogenous peptides generated through proteolysis of classical/non-classical secreted proteins, extracellular proteolytic processing of cell surface membrane proteins, and peptides generated through RIP. The novel peptide cleavage site information in this study provides a useful first step in detailing proteolytic cleavage associated with tumourigenesis and the extracellular environment. This article is part of a Special Issue entitled: An Updated Secretome.     

How are the Non-classically Secreted Bacterial Proteins Released into the Extracellular Milieu?

Most bacterial proteins that are destined to leave the cytoplasm are exported across the cell membrane to their sites of function. These proteins are generally exported via the classical secretion pathway, in which the signal peptide plays a central role. However, some bacterial proteins have been found in the extracellular milieu without any apparent signal peptide. As none of the classical secretion systems is involved in their secretion, this occurrence is termed non-classical protein secretion. The mechanism or mechanisms responsible for non-classical secretion are contentious. This review compiles evidence from the debate over whether the release of the non-classically secreted proteins is the result of cell lysis and discusses how these proteins are exported to the exterior of the cell.     

非经典的蛋白质分泌途径

蛋白质分泌是细胞间进行信息传递的重要方式之一。一般的分泌蛋白在其N端都具有信号肽序列,可以通过经典的内质网-高尔基体途径进行运输,并最终分泌到细胞外。近来发现细胞内存在另一类分泌蛋白,它们能够分泌到细胞外发挥功能,但却没有典型的信号肽序列,被称为非经典分泌蛋白。越来越多的证据表明,这类蛋白质的分泌有其独特的机制。本综述了这类蛋白质的各种分泌机制及可能的生理意义。     

A Phytase-Based Reporter System for Identification of Functional Secretion Signals in Bifidobacteria

Health-promoting effects have been attributed to a number of Bifidobacterium sp. strains. These effects as well as the ability to colonise the host depend on secreted proteins. Moreover, rational design of protein secretion systems bears the potential for the generation of novel probiotic bifidobacteria with improved health-promoting or therapeutic properties. To date, there is only very limited data on secretion signals of bifidobacteria available. Using in silico analysis, we demonstrate that all bifidobacteria encode the major components of Sec-dependent secretion machineries but only B. longum strains harbour Tat protein translocation systems. A reporter plasmid for secretion signals in bifidobacteria was established by fusing the coding sequence of the signal peptide of a sialidase of Bifidobacterium bifidum S17 to the phytase gene appA of E. coli. The recombinant strain showed increased phytase activity in spent culture supernatants and reduced phytase levels in crude extracts compared to the control indicating efficient phytase secretion. The reporter plasmid was used to screen seven predicted signal peptides in B. bifidum S17 and B. longum E18. The tested signal peptides differed substantially in their efficacy to mediate protein secretion in different host strains. An efficient signal peptide was used for expression and secretion of a therapeutically relevant protein in B. bifidum S17. Expression of a secreted cytosine deaminase led to a 100-fold reduced sensitivity of B. bifidum S17 to 5-fluorocytosine compared to the non-secreted cytosine deaminase suggesting efficient conversion of 5-fluorocytosine to the cytotoxic cancer drug 5-fluorouracil by cytosine deaminase occurred outside the bacterial cell. Selection of appropriate signal peptides for defined protein secretion might improve therapeutic efficacy as well as probiotic properties of bifidobacteria.     

The Twin-Arginine Signal Peptide of Bacillus subtilis YwbN Can Direct either Tat- or Sec-Dependent Secretion of Different Cargo Proteins: Secretion of Active Subtilisin via the B. subtilis Tat Pathway

Proteins that are produced for commercial purposes in Bacillus subtilis are commonly secreted via the Sec pathway. Despite its high secretion capacity, the secretion of heterologous proteins via the Sec pathway is often unsuccessful. Alternative secretion routes, like the Tat pathway, are therefore of interest. Two parallel Tat pathways with distinct specificities have previously been discovered in B. subtilis. To explore the application potential of these Tat pathways, several commercially relevant or heterologous model proteins were fused to the signal peptides of the known B. subtilis Tat substrates YwbN and PhoD. Remarkably, the YwbN signal peptide directed secretion of active subtilisin, a typical Sec substrate, via the B. subtilis TatAyCy route. In contrast, the same signal peptide directed Tat-independent secretion of the Bacillus licheniformis α-amylase (AmyL). Moreover, the YwbN signal peptide directed secretion of SufI, an Escherichia coli Tat substrate, in a Tat-independent manner, most likely via Sec. Our results suggest that cytoplasmic protein folding prior to translocation is probably a major determinant of Tat-dependent protein secretion in B. subtilis, as is the case with E. coli. We conclude that future applications for the Tat system of B. subtilis will most likely involve commercially interesting proteins that are Sec incompatible.     

Sec-Mediated Secretion of Bacteriocin Enterocin P by Lactococcus lactis

Most lactic acid bacterium bacteriocins utilize specific leader peptides and dedicated machineries for secretion. In contrast, the enterococcal bacteriocin enterocin P (EntP) contains a typical signal peptide that directs its secretion when heterologously expressed in Lactococcus lactis. Signal peptide mutations and the SecA inhibitor azide blocked secretion. These observations demonstrate that EntP is secreted by the Sec translocase.     

Proteomics-based consensus prediction of protein retention in a bacterial membrane

The availability of complete bacterial genome sequences allows proteome-wide predictions of exported proteins that are potentially retained in the cytoplasmic membranes of the corresponding organisms. In practice, however, major problems are encountered with the computer-assisted distinction between (Sec-type) signal peptides that direct exported proteins into the growth medium and lipoprotein signal peptides or amino-terminal membrane anchors that cause protein retention in the membrane. In the present studies, which were aimed at improving methods to predict protein retention in the bacterial cytoplasmic membrane, we have compared sets of membrane-attached and extracellular proteins of Bacillus subtilis that were recently identified through proteomics approaches. The results showed that three classes of membrane-attached proteins can be distinguished. Two classes include 43 lipoproteins and 48 proteins with an amino-terminal transmembrane segment, respectively. Remarkably, a third class includes 31 proteins that remain membrane-retained despite the presence of typical Sec-type signal peptides with consensus signal peptidase recognition sites. This unprecedented finding indicates that unknown mechanisms are involved in membrane retention of this class of proteins. A further novelty is a consensus sequence indicative for release of certain lipoproteins from the membrane by proteolytic shaving. Finally, using non-overlapping sets of secreted and membrane-retained proteins, the accuracy of different signal peptide prediction algorithms was assessed. Accuracy for the prediction of protein retention in the membrane was increased to 82% using a majority-vote approach. Our findings provide important leads for future identification of surface proteins from pathogenic bacteria, which are attractive candidate infection markers and potential targets for drugs or vaccines.     

Type I signal peptidase and protein secretion in Staphylococcus aureus

Staphylococcus aureus is an important human pathogen whose virulence relies on the secretion of many different proteins. In general, the secretion of most proteins in S. aureus, as well as other bacteria, is dependent on the type I signal peptidase (SPase)-mediated cleavage of the N-terminal signal peptide that targets a protein to the general secretory pathway. The arylomycins are a class of natural product antibiotics that inhibit SPase, suggesting that they may be useful chemical biology tools for characterizing the secretome. While wild-type S. aureus (NCTC 8325) is naturally resistant to the arylomycins, sensitivity is conferred via a point mutation in its SPase. Here, we use a synthetic arylomycin along with a sensitized strain of S. aureus and multidimensional protein identification technology (MudPIT) mass spectrometry to identify 46 proteins whose extracellular accumulation requires SPase activity. Forty-four possess identifiable Sec-type signal peptides and thus are likely canonically secreted proteins, while four also appear to possess cell wall retention signals. We also identified the soluble C-terminal domains of two transmembrane proteins, lipoteichoic acid synthase, LtaS, and O-acyteltransferase, OatA, both of which appear to have noncanonical, internal SPase cleavage sites. Lastly, we identified three proteins, HtrA, PrsA, and SAOUHSC_01761, whose secretion is induced by arylomycin treatment. In addition to elucidating fundamental aspects of the physiology and pathology of S. aureus, the data suggest that an arylomycin-based therapeutic would reduce virulence while simultaneously eradicating an infection.     

High-Salinity Growth Conditions Promote Tat-Independent Secretion of Tat Substrates in Bacillus subtilis

The Gram-positive bacterium Bacillus subtilis contains two Tat translocases, which can facilitate transport of folded proteins across the plasma membrane. Previous research has shown that Tat-dependent protein secretion in B. subtilis is a highly selective process and that heterologous proteins, such as the green fluorescent protein (GFP), are poor Tat substrates in this organism. Nevertheless, when expressed in Escherichia coli, both B. subtilis Tat translocases facilitated exclusively Tat-dependent export of folded GFP when the twin-arginine (RR) signal peptides of the E. coli AmiA, DmsA, or MdoD proteins were attached. Therefore, the present studies were aimed at determining whether the same RR signal peptide-GFP precursors would also be exported Tat dependently in B. subtilis. In addition, we investigated the secretion of GFP fused to the full-length YwbN protein, a strict Tat substrate in B. subtilis. Several investigated GFP fusion proteins were indeed secreted in B. subtilis, but this secretion was shown to be completely Tat independent. At high-salinity growth conditions, the Tat-independent secretion of GFP as directed by the RR signal peptides from the E. coli AmiA, DmsA, or MdoD proteins was significantly enhanced, and this effect was strongest in strains lacking the TatAy-TatCy translocase. This implies that high environmental salinity has a negative influence on the avoidance of Tat-independent secretion of AmiA-GFP, DmsA-GFP, and MdoD-GFP. We conclude that as-yet-unidentified control mechanisms reject the investigated GFP fusion proteins for translocation by the B. subtilis Tat machinery and, at the same time, set limits to their Tat-independent secretion, presumably via the Sec pathway.     

Secretion of cartilage oligomeric matrix protein is affected by the signal peptide

Cartilage oligomeric matrix protein (COMP) is a secreted glycoprotein found in the extracellular matrices of skeletal tissues. Mutations associated with two human skeletal dysplasias, pseudoachondroplasia and multiple epiphyseal dysplasia, disturb COMP secretion leading to intracellular accumulation of mutant COMP, especially in chondrocytes. Here we show that the manifestation of this secretory defect is dramatically influenced by the signal peptide that targets COMP for secretion. The comparison of wild type and mutant COMP secretion directed by the COMP or BM40 signal peptide in HEK-293 cells and rat chondrosarcoma cells revealed that the BM40 signal peptide substantially enhances secretion of mutant COMP that accumulates in endoplasmic reticulum-like structures when targeted by its own signal peptide. Additionally, we demonstrate that mutant COMP forms mixed pentamers with wild type COMP. Our findings suggest that the secretory defect in pseudoachondroplasia and multiple epiphyseal dysplasia is not specific for chondrocytes, nor does it require interaction of mutant COMP with other matrix proteins prior to transport from the cell. They also imply a previously unappreciated role for the signal peptide in the regulation of protein secretion beyond targeting to the endoplasmic reticulum.     

Secretion, processing and activation of bacterial extracellular proteases

Many different bacteria secrete proteases into the culture medium. Extracellular proteases produced by Gram-positive bacteria are secreted by a signal-peptide-dependent pathway and have a propeptide located between the signal peptide and the mature protein. Many extracellular proteases synthesized by Gram-negative bacteria are also produced as precursors with a signal peptide. However, at least two species of Gram-negative bacteria secrete one or more proteases via a novel signal-peptide-independent route. Most proteases secreted by Gram-negative bacteria also have a propeptide whose length and location vary according to the protease. Specific features of protease secretion pathways and the mechanisms of protease activation are discussed with particular reference to some of the best-characterized extracellular proteases produced by Gram-positive and Gram-negative bacteria.     

Proteome-based identification of signal peptides for improved secretion of recombinant cyclomaltodextrin glucanotransferase in Escherichia coli

Secretion of recombinant proteins in heterologous host has drawn attention for its simpler purification and downstream processes. Searching for secretion aid molecules to improve protein secretion can be done through synthetic biology, screening of genome data and proteome-based approach. In the present study, the extracellular proteome on starch-containing medium of Bacillus lehensis G1 was analyzed to identify naturally secreted proteins with signal peptide. A total of 87 protein spots were identified by mass spectrometry, which were categorized mostly in the metabolism of carbohydrates and related molecules (20%). Over-expression and secretion studies were performed for all the 14 selected signal peptides fused to a reporter protein, cyclomaltodextrin glucanotransferase (CGTase). All clones were found to allow CGTase to be excreted into the medium, as observed and measured from the iodine plate assay and enzyme activity assay. Compared to native signal peptide (G1) of CGTase, signal peptide of GlcNAc-binding protein A (GAP) significantly improved CGTase activities by 735% and 205% in extracellular and periplasmic compartment, respectively, with an increase of only ∼1.7 fold the amount of β-galactosidase (cell lysis) in the medium. GAP has the highest secretion rate of 45.6 U/ml/h among all clones, where physicochemical characteristics of signal peptide play significant role.     

Recombinant Protein Secretion in Pseudozyma flocculosa and Pseudozyma antarctica with a Novel Signal Peptide

Secretion of recombinant proteins aims to reproduce the correct posttranslational modifications of the expressed protein while simplifying its recovery. In this study, secretion signal sequences from an abundantly secreted 34-kDa protein (P34) from Pseudozyma flocculosa were cloned. The efficiency of these sequences in the secretion of recombinant green fluorescent protein (GFP) was investigated in two Pseudozyma species and compared with other secretion signal sequences, from S. cerevisiae and Pseudozyma spp. The results indicate that various secretion signal sequences were functional and that the P34 signal peptide was the most effective secretion signal sequence in both P. flocculosa and P. antarctica. The cells correctly processed the secretion signal sequences, including P34 signal peptide, and mature GFP was recovered from the culture medium. This is the first report of functional secretion signal sequences in P. flocculosa. These sequences can be used to test the secretion of other recombinant proteins and for studying the secretion pathway in P. flocculosa and P. antarctica.