High sensitive detection method for protein by combining the magnetic separation with cation exchange based signal amplification.

PSA is a member of low abundance proteins and serves as a critical indicator of the development and therapy efficacy for prostate cancer. In this study, a facile and high sensitive method was developed for serum PSA detection by integrating the immunomagnetic separation and cation exchange based signal amplification. On the basis of nanoparticle preparation and immunoprobe construction, PSA in serum was captured, separated by the immunomagnetic probe and then interacted with the quantum dots (QDs) based immunofluorescence probe; Zn2+ inside QDs was replaced by Ag+ within seconds, after which fluorescence signal was amplified by Fluozin-3, the Zn2+ responsive dye. Under optimized conditions, low detection limit (1.56pg/mL), wide linear range (1.56-25ng/mL) and good repeatability (intra-coefficient variation=3.18%) were achieved, which is superior to commercialized ELISA kit. These results demonstrated the potential of our high sensitive method for PSA detection in clinical.

Purification of a Human Prostate Specific Antigen.

Rabbit antiserum raised against the crude extract of normal human prostatic tissue contained antibodies to a prostatic tissue-specific antigen as shown by immunoprecipitation techniques. Using this antiserum a prostate antigen was detected in normal, benign hypertrophic, and malignant prostatic tissues, but not in other human tissues. The prostate antigen was purified to homogeneity from prostatic tissues and showed a single protein band on analytical polyacrylamide gel electrophoresis and isoelectric focusing. This report thus presents the first demonstration of the purification of a prostate-specific antigen that does not represent prostatic acid phosphatase.

Analysis of Urinary Prostate-Specific Antigen Glycoforms in Samples of Prostate Cancer and Benign Prostate Hyperplasia.

Glycans of prostate-specific antigen (PSA) in prostate cancer were found to be different from that in benign disease. It is difficult to analyze heterogeneous PSA glycoforms in each individual specimen because of low protein abundance and the limitation of detection sensitivity. We developed a method for prostate cancer diagnosis based on PSA glycoforms. Specific glycoforms were screened in each clinical sample based on liquid chromatography-tandem mass spectrometry with ion accumulation. To look for potential biomarkers, normalized abundance of each glycoform in benign prostate hyperplasia (BPH) and in prostate cancer was evaluated. The PSA glycoform, Hex5HexNAc4NeuAc1dHex1, and monosialylated, sialylated, and unfucosylated glycoforms differed significantly between the prostate cancer and BPH samples. The detection sensitivity (87.5%) and specificity (60%) for prostate cancer identification are higher than those of the serum PSA marker. As low as 100 amol PSA could be detected with the ion accumulation method which has not been reported before. The improved detection specificity can help reduce unnecessary examinations.

An efficient method for native protein purification in the selected range from prostate cancer tissue digests.

BACKGROUND: Prostate cancer (CP) cells differ from their normal counterpart in gene expression. Genes encoding secreted or extracellular proteins with increased expression in CP may serve as potential biomarkers. For their detection and quantification, assays based on monoclonal antibodies are best suited for development in the clinical setting. One approach to obtain antibodies is to use recombinant proteins as immunogen. However, the synthesis of recombinant protein for each identified candidate is time-consuming and expensive. It is also not practical to generate high quality antibodies to all identified candidates individually. Furthermore, non-native forms (e.g., recombinant) of proteins may not always lead to useful antibodies. Our approach was to purify a subset of proteins from CP tissue specimens for use as immunogen. METHODS: In the present investigation, ten cancer specimens obtained from cases scored Gleason 3+3, 3+4 and 4+3 were digested by collagenase to single cells in serum-free tissue culture media. Cells were pelleted after collagenase digestion, and the cell-free supernatant from each specimen were pooled and used for isolation of proteins in the 10-30 kDa molecular weight range using a combination of sonication, dialysis and Amicon ultrafiltration. Western blotting and mass spectrometry (MS) proteomics were performed to identify the proteins in the selected size fraction. RESULTS: The presence of cancer-specific anterior gradient 2 (AGR2) and absence of prostate-specific antigen (PSA)/KLK3 were confirmed by Western blotting. Proteomics also detected AGR2 among many other proteins, some outside the selected molecular weight range, as well. CONCLUSIONS: Using this approach, the potentially harmful (to the mouse host) exogenously added collagenase was removed as well as other abundant prostatic proteins like ACPP/PAP and AZGP1 to preclude the generation of antibodies against these species. The paper presents an optimized scheme for convenient and rapid isolation of native proteins in any desired size range with minor modifications.

DNA extraction from filter-paper spots of vaginal samples collected after sexual violence.

OBJECTIVE: To detect the presence of male DNA in vaginal samples collected from survivors of sexual violence and stored on filter paper. METHODS: A pilot study was conducted to evaluate 10 vaginal samples spotted on sterile filter paper: 6 collected at random in April 2009 and 4 in October 2010. Time between sexual assault and sample collection was 4-48hours. After drying at room temperature, the samples were placed in a sterile envelope and stored for 2-3years until processing. DNA extraction was confirmed by polymerase chain reaction for human ß-globin, and the presence of prostate-specific antigen (PSA) was quantified. The presence of the Y chromosome was detected using primers for sequences in the TSPY (Y7/Y8 and DYS14) and SRY genes. RESULTS: ß-Globin was detected in all 10 samples, while 2 samples were positive for PSA. Half of the samples amplified the Y7/Y8 and DYS14 sequences of the TSPY gene and 30% amplified the SRY gene sequence of the Y chromosome. Four male samples and 1 female sample served as controls. CONCLUSION: Filter-paper spots stored for periods of up to 3years proved adequate for preserving genetic material from vaginal samples collected following sexual violence.

Evaluation of molecular species of prostate-specific antigen complexed with immunoglobulin M in prostate cancer and benign prostatic hyperplasia.

This study was aimed at defining molecular species of prostate-specific antigen (PSA) in immune complexes with immunoglobulin M (IgM). Having in mind the oligoreactivity of IgM and its preference for carbohydrate antigens, there is the possibility that it can selectively recognize known PSA glycoisoforms. PSA-IgM complexes and free PSA fractions were separated from the sera of subjects with prostate cancer (PCa) and benign prostatic hyperplasia (BPH) by gel filtration and subjected to on-chip immunoaffinity and ion-exchange chromatography. PSA-immunoreactive species were detected using surface-enhanced laser desorption/ionization time of flight mass spectrometry. The obtained spectra were analyzed for protein and glycan composition. The general pattern of the molecular species of PCa PSA and BPH PSA found in complexes with IgM was similar. It comprised major peaks at 17 kDa and minor peaks at 28 kDa, corresponding to the entire mature glycosylated PSA. The main difference was the presence of incompletely glycosylated 26.8 kDa species, having putative paucimannosidic structures, observed in PCa PSA-IgM, but not in BPH PSA-IgM. Characteristic PCa PSA-IgM glycoforms pose the question of the possible role of glycosylation as a framework for immune surveillance and may be of interest in light of recent data indicating mannose-containing glycans as cancer biomarker.

MMP20 and KLK4 activation and inactivation interactions in vitro.

OBJECTIVE: Enamelysin (MMP20) and kallikrein 4 (KLK4) are believed to be necessary to clear proteins from the enamel matrix of developing teeth. MMP20 is expressed by secretory stage ameloblasts, while KLK4 is expressed from the transition stage throughout the maturation stage. The aim of this study is to investigate the activation of KLK4 by MMP20 and the inactivation of MMP20 by KLK4. DESIGN: Native pig MMP20 (pMMP20) and KLK4 (pKLK4) were isolated directly from enamel scrapings from developing molars. Recombinant human proKLK4 (rh-proKLK4) was activated by incubation with pMMP20 or recombinant human MMP20 (rhMMP20), and the resulting KLK4 activity was detected by zymography. Reaction products were isolated by reverse-phase high performance liquid chromatography (RP-HPLC), and their N-termini characterized by Edman degradation. The pMMP20 was incubated with pKLK4 under mildly acidic or under physiologic conditions, and enzyme activity was analyzed by zymography. The catalytic domain of rhMMP20 was incubated with pKLK4 or recombinant human KLK4 (rhKLK4) and the digestion products were characterized by zymography and Edman degradation. RESULTS: Both pMMP20 and rhMMP20 activated rh-proKLK4 by cleaving at the propeptide-enzyme junction used in vivo. The pMMP20 was inactivated by pKLK4 under physiologic conditions, but not under mildly acidic conditions. Both pKLK4 and rhKLK4 cleaved MMP20 principally at two sites in the catalytic domain of MMP20. CONCLUSIONS: MMP20 activates proKLK4 and KLK4 inactivates MMP20 in vitro, and these actions are likely to occur during enamel formation in vivo.

Epithelial-mesenchymal interactions induce enamel matrix proteins and proteases in the epithelial cells of the rests of Malassez in vitro.

Epithelial-mesenchymal interactions influence morphogenesis and cell differentiation in periodontal tissue regeneration. The current study examined the expression of amelogenin, ameloblastin, matrix metallopeptidase-20 (MMP-20), and kallikrein-4 (KLK-4) and their effects on the interactions between the epithelial cells of Malassez and periodontal ligament fibroblasts. Explants of human periodontal ligament tissues produced outgrowths containing both the epithelial cells of Malassez and periodontal ligament fibroblasts after incubation in a modified serum-free medium. Both the epithelial cells and fibroblasts were co-cultured in the same dish. The distribution and expression of all four factors were evaluated using immunohistochemistry, in-situ hybridization and RT-PCR analysis. The epithelial cells of Malassez were cultured separately and were used as the control. Immunohistochemical analysis revealed weak expression of amelogenin, ameloblastin, MMP-20 and KLK-4 in epithelial cells of Malassez co-cultured with periodontal ligament fibroblasts. in-situ hybridization and RT-PCR confirmed significant mRNA expression of these factors in co-cultured cells compared with control cells. MMP20 mRNA was not expressed in control cells. These results suggest that the epithelial-mesenchymal interactions promote differentiation of human epithelial cells of Malassez and that the induction of enamel matrix proteases facilitates the degradation of enamel matrix proteins.

Human salivary cystatin SA exhibits antimicrobial effect against Aggregatibacter actinomycetemcomitans.

BACKGROUND AND OBJECTIVE: Healthy subjects who do not have Aggregatibacter actinomycetemcomitans in their oral cavity may possess factors in saliva that might demonstrate antibacterial activity against the bacterium. The aim of this study was to identify and purify proteins from saliva of healthy subjects that might demonstrate antibacterial activity against A. actinomycetemcomitans and test the same against the bacteria. MATERIAL AND METHODS: Saliva from 10 healthy volunteers was tested individually for its anti-A. actinomycetemcomitans activity. Among the 10 subjects, eight demonstrated anti-A. actinomycetemcomitans activity. Saliva was collected from one healthy volunteer who demonstrated the highest antimicrobial activity against A. actinomycetemcomitans. After clarifying the saliva, it was subjected to an affinity chromatography column with A. actinomycetemcomitans. The proteins bound to A. actinomycetemcomitans were eluted from the column and identified using mass spectrometry (MALDI-TOF/TOF MS). Among other proteins that bound to A. actinomycetemcomitans, which included lactoferrin, immunoglobulin A and kallikrein, cystatin SA was observed in significantly higher concentrations, and this was purified from the eluate. The purified cystatin SA was tested at different concentrations for its ability to kill A. actinomycetemcomitans in a 2 h cell killing assay. The bacteria were also treated with a proteinase inhibitor, leupeptin, to clarify whether the antimicrobial effect of cystatin SA was related to its protease inhibitory function. Cystatin SA was also tested for its ability to prevent binding of A. actinomycetemcomitans to buccal epithelial cells (BECs) in an A. actinomycetemcomitans-BEC binding assay. RESULTS: Cystatin SA (0.1 mg/mL) demonstrated a statistically significant antimicrobial activity against A. actinomycetemcomitans. The effect of cystatin SA decreased with lower concentrations, with 0.01 mg/mL showing no effect. The addition of monoclonal cystatin SA antibodies to the purified sample completely negated the antimicrobial effect. Treatment of A. actinomycetemcomitans with leupeptin resulted in no antimicrobial effect, suggesting that the antimicrobial activity of cystatin SA is independent of its protease inhibitory function. A. actinomycetemcomitans pretreated with cystatin SA showed reduced binding to BECs, suggesting a potential role for cystatin SA in decreasing the colonization of A. actinomycetemcomitans. CONCLUSION: The present study shows that cystatin SA demonstrates antimicrobial activity against the periodontopathogen A. actinomycetemcomitans, and future studies determining the mechanism of action are necessary. The study also shows the ability of cystatin SA to reduce significantly the binding of A. actinomycetemcomitans to BECs.

Establishment and optimization of a wheat germ cell-free protein synthesis system and its application in venom kallikrein.

Wheat germ cell-free protein synthesis systems have the potential to synthesize functional proteins safely and with high accuracy, but the poor energy supply and the instability of mRNA templates reduce the productivity of this system, which restricts its applications. In this report, phosphocreatine and pyruvate were added to the system to supply ATP as a secondary energy source. After comparing the protein yield, we found that phosphocreatine is more suitable for use in the wheat germ cell-free protein synthesis system. To stabilize the mRNA template, the plasmid vector, SP6 RNA polymerase, and Cu(2+) were optimized, and a wheat germ cell-free protein synthesis system with high yield and speed was established. When plasmid vector (30 ng/µl), SP6 RNA polymerase (15 U), phosphocreatine (25 mM), and Cu(2+) (5 mM) were added to the system and incubated at 26°C for 16 h, the yield of venom kallikrein increased from 0.13 to 0.74 mg/ml. The specific activity of the recombinant protein was 1.3 U/mg, which is only slightly lower than the crude venom kallikrein (1.74 U/mg) due to the lack of the sugar chain. In this study, the yield of venom kallikrein was improved by optimizing the system, and a good foundation has been laid for industrial applications and for further studies.

Isolation and characterisation of a kallikrein-like enzyme from Agkistrodon halys pallas snake venom.

BACKGROUND: Viper snake venoms contain a great variety of toxic proteins. These components mediate their toxicity by either stimulating or inhibiting the haemostatic system of human victims or experimental animals, resulting in common clinical complications of blood clotting or uncontrolled haemorrhage. Therefore it is deemed important to isolate the active component(s) from snake venom with kallikrein-like activity. RESULTS: A kallikrein-like proteinase of Agkistrodon halys pallas snake venom, designated AHP-Ka, was purified by anion exchange chromatography and affinity chromatography. Physicochemical studies showed that the purified enzyme was a 34 kDa monomeric glycoprotein, the molecular weight of which decreased to 26 kDa after deglycosylation with peptide N-glycosidase F (PNGase F). Sequence studies on the NH(2) -terminal region of the protein indicated that AHP-Ka shared a high degree of sequence homology with other serine proteinases from snake venoms. AHP-Ka showed high catalytic activity and kallikrein-like activity on substrates such as arginine esterase BAEE and chromogenic H-D-Pro-Phe-Arg-pNA·2HCl (S-2302) and was inhibited by protease inhibitor phenylmethylsulfonyl fluoride (PMSF). CONCLUSION: The results showed that AHP-Ka isolated from A. halys pallas snake venom and purified by anion exchange chromatography and affinity chromatography is in fact a kallikrein-like enzyme.

Kallikrein-related peptidase-8 (KLK8) is an active serine protease in human epidermis and sweat and is involved in a skin barrier proteolytic cascade.

Kallikrein-related peptidase-8 (KLK8) is a relatively uncharacterized epidermal protease. Although proposed to regulate skin-barrier desquamation and recovery, the catalytic activity of KLK8 was never demonstrated in human epidermis, and its regulators and targets remain unknown. Herein, we elucidated for the first time KLK8 activity in human non-palmoplantar stratum corneum and sweat ex vivo. The majority of stratum corneum and sweat KLK8 was catalytically active, displaying optimal activity at pH 8.5 and considerable activity at pH 5. We also showed that KLK8 is a keratinocyte-specific protease, not secreted by human melanocytes or dermal fibroblasts. KLK8 secretion increased significantly upon calcium induction of terminal keratinocyte differentiation, suggesting an active role for this protease in upper epidermis. Potential activators, regulators, and targets of KLK8 activity were identified by in vitro kinetic assays using pro-KLK8 and mature KLK8 recombinant proteins produced in Pichia pastoris. Mature KLK8 activity was enhanced by calcium and magnesium ions and attenuated by zinc ions and by autocleavage after Arg(164). Upon screening KLK8 cleavage of a library of FRET-quenched peptides, trypsin-like specificity was observed with the highest preference for (R/K)(S/T)(A/V) at P1-P1'-P2'. We also demonstrated that KLK5 and lysyl endopeptidase activate latent pro-KLK8, whereas active KLK8 targets pro-KLK11, pro-KLK1, and LL-37 antimicrobial peptide activation in vitro. Together, our data identify KLK8 as a new active serine protease in human stratum corneum and sweat, and we propose regulators and targets that augment its involvement in a skin barrier proteolytic cascade. The implications of KLK8 elevation and hyperactivity in desquamatory and inflammatory skin disease conditions remain to be studied.

A new model of experimental autoimmune keratoconjunctivitis sicca (KCS) induced in Lewis rat by the autoantigen Klk1b22.

PURPOSE: This study was designed to generate an inducible autoimmune model of keratoconjunctivitis sicca (KCS) for study of pathogenesis of the disease. METHODS: Lewis rats were immunized with a mixture of lacrimal and salivary gland extract or recombinant mouse protein kallikrein 1b22 (Klk1b22) emulsified in complete Freund's adjuvant (CFA). For disease induction by adoptive transfer of primed cells, donor rats were received with T-cell blasts. KCS were observed by either clinical signs or histology. RESULTS: The autoantigen Klk1b22, isolated from the lacrimal and salivary glands, readily induced Sjögren's syndrome (SS)-like KCS in the recipients. The diseased animals presented the clinical and pathologic symptoms that resemble related human disease. Most immunized rats showed an increase, then a decrease in tear volume, together with corneal opacity and ocular lesions. Histologic examination revealed that the rats displayed the cardinal signs of primary SS-like KCS, including marked lymphocytic infiltration of the lacrimal and salivary glands and destruction of the acinar cells. Immunofluorescence studies showed that both CD8(+) and CD4(+) T cells were heavily infiltrated, with the former cells predominant in the damaged ducts. Finally, adoptive transfer of Klk1b22-reactive T cells induced more severe disease with earlier onset. CONCLUSIONS: Klk1b22 is an autoantigen for inducing an experimental SS-like KCS in Lewis rats. The availability of this new and reproducible rat model should provide a new and needed tool for studying the pathogenesis of SS.

Immunofluorometric activity-based probe analysis of active KLK6 in biological fluids.

Immunoassay measurements of human kallikrein-related peptidases (KLKs) such as prostate-specific antigen (KLK3) are of great value as diagnostic indices of cancer. Despite extensive knowledge of the abundance of immunoreactive KLKs in normal and cancer-related settings, there is little information available about the proportion of immunoreactive KLK that represents active enzyme in such samples. Using KLK6 as a prototype enzyme, we have developed an assay using a serine proteinase-targeted activity-based probe coupled to antibody capture. By employing activity-based labeling, we were able to quantify the proportion of enzymatically active relative to total immunoreactive KLK6 in crude cerebrospinal fluid from routine analyses and ascites fluid from ovarian cancer patients, as well as in supernatants from cancer cell lines. Our approach allowed monitoring of pro-KLK6 conversion to its active enzyme species and demonstrated that up to 5% of immunoreactive KLK6 detected in clinical samples represents active enzyme. We suggest that this new activity-based probe assay will prove of value as a complement to routine KLK immunoassay measurements for validating KLKs as cancer biomarkers.

Purification and characterisation of proteases A and D from Debaryomyces hansenii.

The proteases A (PrA; EC. 3.4.23.25) and D (PrD; EC. 3.4.24.37) of Debaryomyces hansenii CECT 12487 were characterised after their isolation by fractionation with protamine sulfate followed by three chromatographic separations, which included two anion exchange and one gel filtration chromatographic steps. The whole procedures for PrA and PrD resulted in 1349 and 2560 purification-fold with a recovery yield of 1.4 and 1.3%, respectively. PrA was active at acidic-neutral pH with an optimum pH between 5.0 and 6.0. PrD was active at neutral-basic pH with an optimum pH between 7.0 and 8.0. The molecular mass of the native PrA was 55 kDa and (being) 42 kDa in denaturing conditions. Polyclonal-antibodies raised against PrA from Saccharomyces cerevisiae cross-reacted with the corresponding PrA from D. hansenii. PrD showed a native molecular mass of 68 kDa and 65 kDa in denaturing conditions. PrA was an aspartic protease effectively inhibited by pesptatin A while PrD was classified as a metallo protease inhibited by 1,10-phenantroline and affected by some divalent cations such as zinc, cadmium and magnesium. The homology of the PrA to the lisosomal cathepsin D suggests its possible participation in the ripening of fermented meat products.

[Cloning and expression of the gene of chymotrypsin-like protease of human kallikrein-7 in Escherichia coli and isolation of recombinant protein].

A sample of the human cDNA was used to amplify the segment encoding biosynthesis of chymotrypsin-like protease of kallikrein-7 and its cloning into the expressing plasmid pET23a(+). Biosynthesis of KLK-7 in transformed E. coil BL21(DE3) cells was accompanied by formation of insoluble inclusion bodies. The recombinant KLK-7 was extracted from the inclusion bodies using 7 M urea in the presence of 2-mercaptoethanol. The extracted recombinant KLK-7 was purified using methods of metal-chelate and ion-exchange chromatography, converted into a soluble form, and used for preparing monospecific antiserum.

Kallikrein-mediated proteolysis regulates the antimicrobial effects of cathelicidins in skin.

The presence of cathelicidin antimicrobial peptides provides an important mechanism for prevention of infection against a wide variety of microbial pathogens. The activity of cathelicidin is controlled by enzymatic processing of the proform (hCAP18 in humans) to a mature peptide (LL-37 in human neutrophils). In this study, elements important to the processing of cathelicidin in the skin were examined. Unique cathelicidin peptides distinct from LL-37 were identified in normal skin. Through the use of selective inhibitors, SELDI-TOF-MS, Western blot, and siRNA, the serine proteases stratum corneum tryptic enzyme (SCTE, kallikrein 5) and stratum corneum chymotryptic protease (SCCE, kallikrein 7) were shown to control activation of the human cathelicidin precursor protein hCAP18 and also influence further processing to smaller peptides with alternate biological activity. The importance of this serine protease activity to antimicrobial activity in vivo was illustrated in SPINK5-deficient mice that lack the serine protease inhibitor LEKTI. Epidermal extracts of these animals show a significant increase in antimicrobial activity compared with controls, and immunoabsorption of cathelicidin diminished antimicrobial activity. These observations demonstrate that the balance of proteolytic activity at an epithelial interface will control innate immune defense.

Crystal structures of human tissue kallikrein 4: activity modulation by a specific zinc binding site.

Human tissue kallikrein 4 (hK4) belongs to a 15-member family of closely related serine proteinases. hK4 is predominantly expressed in prostate, activates hK3/PSA, and is up-regulated in prostate and ovarian cancer. We have identified active monomers of recombinant hK4 besides inactive oligomers in solution. hK4 crystallised in the presence of zinc, nickel, and cobalt ions in three crystal forms containing cyclic tetramers and octamers. These structures display a novel metal site between His25 and Glu77 that links the 70-80 loop with the N-terminal segment. Micromolar zinc as present in prostatic fluid inhibits the enzymatic activity of hK4 against fluorogenic substrates. In our measurements, wild-type hK4 exhibited a zinc inhibition constant (IC50) of 16 microM including a permanent residual activity, in contrast to the zinc-independent mutants H25A and E77A. Since the Ile16 N terminus of wild-type hK4 becomes more accessible for acetylating agents in the presence of zinc, we propose that zinc affects the hK4 active site via the salt-bridge formed between the N terminus and Asp194 required for a functional active site. hK4 possesses an unusual 99-loop that creates a groove-like acidic S2 subsite. These findings explain the observed specificity of hK4 for the P1 to P4 substrate residues. Moreover, hK4 shows a negatively charged surface patch, which may represent an exosite for prime-side substrate recognition.

Kallikrein-related peptidase 14 may be a major contributor to trypsin-like proteolytic activity in human stratum corneum.

We have previously presented evidence that two human kallikrein-related peptidases, KLK5 (hK5, stratum corneum tryptic enzyme, SCTE) and KLK7 (hK7, stratum corneum chymotryptic enzyme, SCCE), which are abundant in the stratum corneum, may be involved in desquamation. Since we had noted that not all trypsin-like activity in the plantar stratum corneum could be ascribed to KLK5, we set out to identify other skin proteases with similar primary substrate specificity. Here we describe purification of a protease identified as KLK14 from plantar stratum corneum, and show that this enzyme may be responsible for as much as 50% of the total trypsin-like activity in this tissue, measured as activity towards a chromogenic substrate cleaved by a wide variety of enzymes with trypsin-like specificity. This was in spite of very low levels of KLK14 protein compared to KLK5 and KLK7. KLK14 could be detected by immunoblotting in normal superficial stratum corneum of all individuals examined. The majority of KLK14 in the plantar stratum corneum is present in its catalytically active form. KLK14 could be immunohistochemically detected in sweat ducts, preferentially in the intraepidermal parts (the acrosyringium), and in sweat glands. The role played by this very efficient protease under normal and disease conditions in the skin remains to be elucidated.

Affinity chromatography matures as bioinformatic and combinatorial tools develop.

Affinity chromatography has the reputation of a more expensive and less robust than other types of liquid chromatography. Furthermore, the technique is considered to stand a modest chance of large-scale purification of proteinaceous pharmaceuticals. This perception is changing because of the pressure for quality protein therapeutics, and the realization that higher returns can be expected when ensuring fewer purification steps and increased product recovery. These developments necessitated a rethinking of the protein purification processes and restored the interest for affinity chromatography. This liquid chromatography technique is designed to offer high specificity, being able to safely guide protein manufactures to successfully cope with the aforementioned challenges. Affinity ligands are distinguished into synthetic and biological. These can be generated by rational design or selected from ligand libraries. Synthetic ligands are generated by three methods. The rational method features the functional approach and the structural template approach. The combinatorial method relies on the selection of ligands from a library of synthetic ligands synthesized randomly. The combined method employs both methods, that is, the ligand is selected from an intentionally biased library based on a rationally designed ligand. Biological ligands are selected by employing high-throughput biological techniques, e.g. phage- and ribosome-display for peptide and microprotein ligands, in addition to SELEX for oligonucleotide ligands. Synthetic mimodyes and chimaeric dye-ligands are usually designed by rational approaches and comprise a chloro-triazinlyl scaffold. The latter substituted with various amino acids, carbocyclic, and heterocyclic groups, generates libraries from which synthetic ligands can be selected. A 'lead' compound may help to generating a 'focused' or 'biased' library. This can be designed by various approaches, e.g.: (i) using a natural ligand-protein complex as a template; (ii) applying the principle of complementarity to exposed residues of the protein structure; and (iii) mimicking directly a natural biological recognition interaction. Affinity ligands, based on the peptide structure, can be peptides, peptide-mimetic derivatives (<30 monomers) and microproteins (e.g. 25-200 monomers). Microprotein ligands are selected from biological libraries constructed of variegated protein domains, e.g. minibody, Kunitz, tendamist, cellulose-binding domain, scFv, Cytb562, zinc-finger, SpA-analogue (Z-domain).