Characterisation of bombesin-like immunoreactivity in human fetal lung

A sensitive radioimmunoassay for bombesin-like immunoreactivity (BLI) was developed and utilised in conjunction with G50 gel chromatography and reverse-phase HPLC, to study the content and molecular characteristics of bombesin-like peptides in acid extracts of human fetal lung. The antiserum, (B5), is directed towards the C-terminal region of the bombesin molecule and cross-reacts 70% with synthetic porcine GRP and the synthetic GRP fragment, GRP (14-27). Specimens of lung were collected from fetuses of gestational ages 15-22 weeks, following prostaglandin termination of pregnancy. The tissue was extracted into 0.1 N HCl at 90 degrees C. The mean BLI content was 50.2 pg/mg wet weight of tissue (range 15.5-136 pg/mg; n = 13). No correlation between gestational age and BLI content could be established. G50 gel chromatography of acid extracts, under dissociating conditions, revealed two peaks of BLI, one in the position of synthetic porcine GRP and the second, constituting greater than 90% of the immunoreactivity, eluting with synthetic amphibian bombesin. Reverse-phase ODS silica HPLC of this major G50 peak, utilising a methanol/trifluoroacetic acid gradient, indicated that this peptide was similar to the GRP C-terminal fragment, GRP (14-27). We have therefore (1) confirmed the presence and heterogeneity of BLI in human fetal lung, and (2) shown, for the first time, that the majority of this BLI more closely resembles a fragment of GRP than amphibian bombesin itself.     

Agonist-biased Trafficking of Somatostatin Receptor 2A in Enteric Neurons

Somatostatin (SST) 14 and SST 28 activate somatostatin 2A receptors (SSTR2A) on enteric neurons to control gut functions. SST analogs are treatments of neuroendocrine and bleeding disorders, cancer, and diarrhea, with gastrointestinal side effects of constipation, abdominal pain, and nausea. How endogenous agonists and drugs differentially regulate neuronal SSTR2A is unexplored. We evaluated SSTR2A trafficking in murine myenteric neurons and neuroendocrine AtT-20 cells by microscopy and determined whether agonist degradation by endosomal endothelin-converting enzyme 1 (ECE-1) controls SSTR2A trafficking and association with β-arrestins, key regulators of receptors. SST-14, SST-28, and peptide analogs (octreotide, lanreotide, and vapreotide) stimulated clathrin- and dynamin-mediated internalization of SSTR2A, which colocalized with ECE-1 in endosomes and the Golgi. After incubation with SST-14, SSTR2A recycled to the plasma membrane, which required active ECE-1 and an intact Golgi. SSTR2A activated by SST-28, octreotide, lanreotide, or vapreotide was retained within the Golgi and did not recycle. Although ECE-1 rapidly degraded SST-14, SST-28 was resistant to degradation, and ECE-1 did not degrade SST analogs. SST-14 and SST-28 induced transient interactions between SSTR2A and β-arrestins that were stabilized by an ECE-1 inhibitor. Octreotide induced sustained SSTR2A/β-arrestin interactions that were not regulated by ECE-1. Thus, when activated by SST-14, SSTR2A internalizes and recycles via the Golgi, which requires ECE-1 degradation of SST-14 and receptor dissociation from β-arrestins. After activation by ECE-1-resistant SST-28 and analogs, SSTR2A remains in endosomes because of sustained β-arrestin interactions. Therapeutic SST analogs are ECE-1-resistant and retain SSTR2A in endosomes, which may explain their long-lasting actions.     

Cortistatin modulates calcitonin gene-related peptide release from neuronal tissues of rat. Comparison with somatostatin

Cortistatin (CST) is an endogenous neuropeptide bearing strong structural and functional analogies with somatostatin (SST). Gene expression of CST and its putative receptor MrgX2 in dorsal root ganglia (DRG) neurons in man suggests the involvement of CST in pain transmission. In this study we have investigated the effects of CST and SST on calcitonin gene-related peptide (CGRP, the main neuropeptide mediator of pain transmission) from primary cultures of rat trigeminal neurons. Moreover, here for the first time we used organotypic cultures of rat brainstem to investigate the release of CGRP form nucleus caudalis as a model of pre-synaptic peptide release. In both experimental paradigm CGRP release was evaluated in the presence of CST or SST, with or without the addition of known secretagogues (namely high KCl concentrations, veratridine and capsaicin). We found that CST and SST do not modify basal CGRP secretion from trigeminal neurons, but both peptides were able to inhibit in a concentration-dependent manner the release of CGRP stimulated by KCl, veratridine or capsaicin. Likewise, in brainstem organotypic cultures CST and SST did not modify baseline CGRP secretion. Of the secretagogues used, capsaicin proved to be most effective compared to KCl and veratridine (8-fold vs 2-fold increase, respectively). Thereafter, CST and SST were tested on capsaicin-stimulated CGPR release only. Under these conditions, CST but not SST was able to inhibit in a significant manner pre-synaptic CGRP release from the brainstem, providing further evidence in support of a role for CST in pain transmission.     

Catabolism of rat growth hormone-releasing factor(1-29) amide in rat serum and liver.

Clinical and veterinary uses of growth hormone-releasing factor [GRF(1- 29)NH2] require the design of analogs that are resistant to proteolysis by serum and liver degrading enzymes. This study investigated rat GRF(1-29)NH2 processing in serum and liver homogenate by means of high pressure liquid chromatography (HPLC). Synthetic rGRF(1-29)NH2 (30 microM) was incubated (0-120 min, 37 degrees C) in serum (49 +/- 8 mg prot./ml). The rGRF(1-29)NH2 (10 microM) was also incubated (0-120 min, 37 degrees C) with liver homogenate (200 +/- 6 micrograms prot./ml). Time course studies of rGRF(1-29)NH2 disappearance showed apparent half-lives of 18 +/- 4 min and 13 +/- 3 min in serum and liver homogenate, respectively. This was accompanied by the appearance of degradation products that were all less hydrophobic than the native peptide. In the serum, two major metabolites were detected and isolated by preparative HPLC. Combined results of amino acid analysis, sequencing, and chromatography with synthetic homologs revealed the presence of rGRF(1-20)OH and (3-20)OH. A small amount of rGRF(12-29)NH2, coeluting with rGRF(3-20)OH, was also found by sequencing. In the liver, rGRF(1-18)OH, (3-18)OH, and (1-10)OH were identified. The peptide bond Ala2-Asp3 (DPP IV cleavage site) was hydrolyzed in both serum and liver. Other tissue-specific cleavage sites were Arg11-Arg12 and Arg20-Lys21 (trypsin-like cleavage site) in the serum, and Tyr10-Arg11 and Tyr18-Ala19 (chymotrypsin-like cleavage site) in the liver.(ABSTRACT TRUNCATED AT 250 WORDS)     

Changes in somatostatin-like immunoreactivity in lungs from perinatal guinea pigs and the effects of somatostatin-14 on lung liquid production.

Somatostatin-like immunoreactivity was measured by radioimmunoassay with a monoclonal antibody in lungs from perinatal guinea pigs (62 +/- 2 days of gestation). Fetuses delivered by Caesarean section and dissected before breathing showed 4748 +/- 758 pg/lung (n = 25). Fetuses allowed to breathe (neonates) showed marked increases in activity: 7629 +/- 1355 pg/lung (n = 12) after breathing 30 seconds, and 10729 +/- 1064 pg/lung (n = 6) after breathing 3 minutes (2.3-fold increase, P < 0.005). Values then declined (5203 +/- 1050 pg/lung (n = 9) at 30 minutes; 1458 +/- 105 pg/lung (n = 4) at 60 minutes). Changes were similar in pg/g wet tissue. HPLC characterized the immunoreactive peptides as somatostatin-14 (SS-14) and somatostatin-28 (SS-28) in both fetuses and neonates (n = 11). SS-28 made up only 13.7 +/- 1.7% of the activity; this percentage did not change with breathing. The effects of synthetic SS-14 on lung liquid production were investigated in in vitro lungs from 42 fetal guinea pigs. All 21 preparations immersed in 10(-5)-10(-7) M SS-14 during the middle hour of 3 h incubations reduced production, often approaching zero after treatment (rates, ml/kg body weight per h, succeeding hours: 10(-5) M (n = 9), 3.09 +/- 0.68, 0.93 +/- 0.39, -0.05 +/- 0.60 (fall significant during and after treatment, P < 0.025-0.005); 10(-6) M (n = 6), 3.06 +/- 0.68, 1.29 +/- 0.58, 0.36 +/- 0.38 (P < 0.05-0.005); 10(-7) M (n = 6), 1.96 +/- 0.66, 1.11 +/- 0.34, 0.64 +/- 0.28 (P < 0.05-0.025).(ABSTRACT TRUNCATED AT 250 WORDS)     

Cognitive screening test: short and long version

Three versions of the Cognitive Screening Test (CST), varying in length from seven to 20 items, are compared using the original data. The shortest version, the CST-7, is not inferior to the longer CST-14 and CST-20. Various psychometric properties are compared. According to the Mokken nonparametric IRT scaling technique, all three CST versions can be considered unidimensional. The Mokken scaling indices H and rho hardly differ in the three versions of the CST. The alpha indices from Classical Test Theory are also very comparable, as is the very good sensitivity and specificity (around 95%). Age and gender have no significant effect on the classification success of the CST-7. Cross-validation of the promising properties of this very short screening instrument is recommended.     

Sequence of plasmin proteolysis at the NH2-terminus of the b beta-chain of human fibrinogen

Employing high-performance liquid chromatography (HPLC), we have isolated and quantified the peptides that are released from the NH2-terminus of human fibrinogen B beta-chains by plasmin proteolysis. The peptides were identified by amino acid composition and by a radioimmunoassay developed for fibrinopeptide B detection. B beta 1-42 was the earliest fragment released during limited plasmin proteolysis. The level of this peptide reached a maximum and then began to decline during the course of the digestion. In addition, increasing levels of B beta 1-21 and of FPB followed the production of B beta 1-42. Using purified B beta 1-42 as a substrate, preferential cleavage was shown to occur at the 21-22 bond, with a minor cleavage at the 14-15 bond. Exhaustive digestion yielded two major components which were separated by HPLC: B beta 1-14 (FPB) and beta 22-42. The rate of cleavage at the 14-15 bond, which is the customary site of thrombin proteolysis, was not affected by the addition of hirudin indicating that this was not the result of trace contamination with thrombin. We have also examined plasmin proteolysis at the NH2-terminal region of the B beta-chains of a variety of fibrinogen derivatives and have found similar patterns of B beta 1-42 release. Using HPLC data, we have estimated the Km for plasmic cleavage of the beta 21-22 bond to be 1.8 X 10(-5) M and of the beta 14-15 bond to be 2.8 X 10(-5) M.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification by specific radioimmunoassay of two novel peptides derived from the C-terminus of porcine preprogastrin

A radioimmunoassay has been developed using antibodies to a synthetic analogue of the C-terminal hexapeptide sequence of the porcine gastrin precursor. Boiling water extracts of porcine antral mucosa contained immunoreactive material that diluted in parallel with standard peptide. Concentrations of immunoreactivity were 5.5 +/- 0.8 nmol X g-1 (mean +/- S.E.M.) in antral mucosa and were closely similar to those of C-terminal heptadecapeptide gastrin immunoreactivity (5.0 +/- 0.6 nmol X g-1). Approximately 30-fold lower concentrations were found in porcine duodenum. A similar distribution was found in ferret, but human, rat and chicken antrum did not contain significant quantities of immunoreactivity. Gel filtration of porcine antral extracts on Sephadex G-50 revealed a single peak of immunoreactivity eluting in a similar position to G17, but on anion-exchange chromatography two peaks of immunoreactive material were separated. These also differed in their retention time on reverse phase HPLC. Both peptides are probably derived by tryptic cleavage at the C-terminus of porcine preprogastrin. No evidence was found to suggest that there are significant quantities of unprocessed preprogastrin in hog antral mucosa. The precise chemical difference between the two immunoreactive peptides identified here remains to be established; together, however, they provide specific markers for progastrin synthesis.     

Synthetic peptides as substrates and inhibitors of human immune deficiency virus-1 protease

Retroviruses code for a virus-specific protease which is essential for polyprotein processing and viral infectivity. The human immune deficiency virus-1 protease is an aspartic protease of 9 kDa which was synthesized by recombinant DNA technology and arises by autocatalytic processing from a polyprotein precursor which has recently been demonstrated by use of a protease-specific monoclonal antibody. The protease was shown to form dimers. Here we demonstrate that synthetic peptides can be used as both model substrates as well as inhibitors for investigation of the protease. 14 synthetic peptides, 7-18 amino acids in length, containing putative protease cleavage sites of the viral polyprotein gag and pol precursors, have been analyzed with the partially purified protease by the use of high performance liquid chromatography. In seven cases, where cleavage was observed, the length of the peptides did not significantly influence the cleavage efficiencies, heptapeptides being large enough as model substrates. No cleavage was observed with a protein preparation purified in parallel from control bacteria not expressing the human immune deficiency virus-1 protease. The protease was not only able to cut next to a proline but also between other peptides indicating that the proline is not a prerequisite. Three peptides with either reduced bonds at the cleavage site or a substitution by statin were inhibitory while another uncleaved substrate was not. The usefulness of small model substrates for characterization of the protease is further demonstrated by determination of a kinetic optimum pH (3.5-5.5) and incubation temperature (37 degrees C).     

Novel activity of angiotensin-converting enzyme. Hydrolysis of cholecystokinin and gastrin analogues with release of the amidated C-terminal dipeptide.

ACE (angiotensin-converting enzyme; peptidyl dipeptidase A; EC 3.4.15.1), cleaves C-terminal dipeptides from active peptides containing a free C-terminus. We investigated the hydrolysis of cholecystokinin-8 [CCK-8; Asp-Tyr(SO3H)-Met-Gly-Trp-Met-Asp-Phe-NH2] and of various gastrin analogues by purified rabbit lung ACE. Although these peptides are amidated at their C-terminal end, they were metabolized by ACE to several peptide fragments. These fragments were analysed by h.p.l.c., isolated and identified by comparison with synthetic fragments, and by amino acid analysis. The initial and major site of hydrolysis was the penultimate peptide bond, which generated a major product, the C-terminal amidated dipeptide Asp-Phe-NH2. As a secondary cleavage, ACE subsequently released di- or tri-peptides from the C-terminal end of the remaining N-terminal fragments. The cleavage of CCK-8 and gastrin analogues was inhibited by ACE inhibitors (Captopril and EDTA), but not by other enzyme inhibitors (phosphoramidon, thiorphan, bestatin etc.). Hydrolysis of [Leu15]gastrin-(14-17)-peptide [Boc (t-butoxycarbonyl)-Trp-Leu-Asp-Phe-NH2] in the presence of ACE was found to be dependent on the chloride-ion concentration. Km values for the hydrolysis of CCK-8, [Leu15]gastrin-(11-17)-peptide and Boc-[Leu15]gastrin-(14-17)-peptide at an NaCl concentration of 300 mM were respectively 115, 420 and 3280 microM, and the catalytic constants were about 33, 115 and 885 min-1. The kcat/Km for the reactions at 37 degrees C was approx. 0.28 microM-1.min-1, which is approx. 35 times less than that reported for the cleavage of angiotensin I. These results suggest that ACE might be involved in the metabolism in vivo of CCK and gastrin short fragments.     

A conserved alpha-helix at the amino terminus of prosomatostatin serves as a sorting signal for the regulated secretory pathway

Mammalian prosomatostatin (PSST) contains the bioactive peptides SST-14 and SST-28 at the COOH-terminal end of the molecule and a putative sorting signal in the propeptide segment for targeting the precursor to the regulated secretory pathway. The NH(2)-terminal segment of PSST consists of an amphipathic alpha-helix, which has been totally conserved throughout vertebrate evolution. We have analyzed the PSST-(3--15) region for sorting function by alanine scanning and deletional mutagenesis. Mutants created were stably expressed in AtT-20 cells. Regulated secretion was studied by analyzing basal and stimulated release of SST-14 LI and by immunocytochemistry for staining of SST-14 LI in punctate granules. Deletion of the PSST-(3--15) segment blocked regulated secretion and rerouted PSST for constitutive secretion as unprocessed precursor. Alanine scanning mutagenesis identified the region Pro(5)--Gln(12) as being important in precursor targeting, with Leu(7) and Leu(11) being critical. Molecular modeling demonstrated that these two residues are located in close proximity on a hydrophobic surface of the alpha-helix. Disruption of the alpha-helix did not impair the ability of PSST to be processed at the COOH terminus to SST-14 and SST-28. Processing, however, was shifted to the early compartments of the secretory pathway rather than storage granules and was relatively inefficient.     

Dynamics of biosynthesis and release of crustacean hyperglycemic hormone isoforms in the X-organ-sinus gland complex of the crayfish Orconectes limosus.

The crustacean hyperglycemic hormone (CHH) is the major neuropeptide produced by the X-organ-sinus gland neurosecretory system of the crayfish, Orconectes limosus. This hormone is synthesized by two different cell types, as two isomers (CHH and D-Phe3-CHH) which display different activities The aim of this report is to analyze and compare the synthetic and secretory activities of these specialized cells. In vitro pulse-chase incubations and time-course experiments were conducted on isolated X-organ-sinus gland (XO-SG) complexes, followed by analysis of the labeled peptides. The different steps of the post-translational processing of the CHH precursor, including proteolytic cleavage of the propeptide, C-terminal amidation and N-terminal pyroglutamylation were characterized and the kinetics of CHHs maturation were estimated in the different parts of the neuroendocrine complex. Furthermore, synthesis of CHHs in XO-SG complexes and release in incubation media were investigated using combined HPLC/immunoassay. Under basal conditions, i.e. without stimulation, similar dynamics for both isomers were found and results indicate that newly synthesized CHHs are preferentially released.     

The Vaginal Microbiota over an 8- to 10-Year Period in a Cohort of HIV-Infected and HIV-Uninfected Women

BackgroundWe identified predominant vaginal microbiota communities, changes over time, and how this varied by HIV status and other factors in a cohort of 64 women.MethodsBacterial DNA was extracted from reposited cervicovaginal lavage samples collected annually over an 8–10 year period from Chicago Women’s Interagency HIV Study participants: 22 HIV-negative, 22 HIV-positive with stable infection, 20 HIV-positive with progressive infection. The vaginal microbiota was defined by pyrosequencing of the V1/V2 region of the 16S rRNA gene. Scheduled visits included Bacterial vaginsosis (BV) screening; clinically detected cases were referred for treatment. Hierarchical clustering identified bacterial community state types (CST). Multinomial mixed effects modeling determined trends over time in CST, by HIV status and other factors.ResultsThe median follow-up time was 8.1 years (range 5.5–15.3). Six CSTs were identified. The mean relative abundance (RA) of Lactobacillus spp. by CST (with median number of bacterial taxa) was: CST-1–25.7% (10), CST-2–27.1% (11), CST-3–34.6% (9), CST-4–46.8% (9), CST-5–57.9% (4), CST-6–69.4% (2). The two CSTs representing the highest RA of Lactobacillus and lowest diversity increased with each additional year of follow-up (CST-5, adjusted odds ratio (aOR) = 1.62 [95% CI: 1.34–1.94]; CST-6, aOR = 1.57 [95 CI: 1.31–1.89]), while the two CSTs representing lowest RA of Lactobacillus and higher diversity decreased with each additional year (CST-1, aOR = 0.89 [95% CI: 0.80–1.00]; CST-2, aOR = 0.86 [95% CI: 0.75–0.99]). There was no association between HIV status and CST at baseline or over time. CSTs representing lower RA of Lactobacillus were associated with current cigarette smoking.ConclusionsThe vaginal microbial community significantly improved over time in this cohort of women with HIV and at high risk for HIV who had regular detection and treatment referral for BV.     

Prothyrotropin-releasing hormone targets its processing products to different vesicles of the secretory pathway

Prothyrotropin-releasing hormone (pro-TRH) is initially cleaved by the prohormone convertase-1/3 (PC1/3) in the trans-Golgi network generating N- and C-terminal intermediate forms that are then packed into secretory vesicles. However, it is not known whether these peptides are differentially sorted within the secretory pathway. This is of key importance because the processing products of several prohormones fulfill different biological functions. Using AtT20 cells stably transfected with prepro-TRH cDNA, we found that two specific N- and C-terminal peptides were located in different vesicles. Furthermore, the C-terminal pro-TRH-derived peptides were more efficiently released in response to KCl and norepinephrine, a natural secretagogue of TRH. Similar sorting and secretion of N- and C-terminal peptides occurs in vivo. When we blocked the initial proteolytic processing by a mutagenic approach, the differential sorting and secretion of these peptides were prevented. In summary, our data show that pro-TRH-derived peptides are differentially sorted within the secretory pathway and that the initial cleavage in the trans-Golgi network is key to this process. This could be a common mechanism used by neuroendocrine cells to regulate independently the secretion of different bioactive peptides derived from the same gene product.     

Processing of proenkephalin-A in bovine chromaffin cells. Identification of natural derived fragments by N-terminal sequencing and matrix-assisted laser desorption ionization-time of flight mass spectrometry

A large variety of proenkephalin-A-derived peptides (PEAPs) are present in bovine adrenal medulla secretory granules that are cosecreted with catecholamines upon stimulation of chromaffin cells. In the present paper, after reverse phase high performance liquid chromatography of intragranular soluble material, PEAPs were immunodetected with antisera raised against specific proenkephalin-A (PEA) sequences (PEA63-70 and PEA224-237) and analyzed by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry. Thirty PEAPs were characterized in addition to enkephalins and whole PEA, indicating that preferential proteolytic attacks occurred at both N- and C-terminal regions. A similar approach was used to characterize PEA-derived fragments exocytotically released into the extracellular space that showed five additional minor PEAPs. Among all these naturally generated peptides, enkelytin, the antibacterial bisphos- phorylated C-terminal peptide (PEA209-237), was predominantly generated, as shown by MALDI-TOF mass spectrometry analysis, which constituted an efficient method for its identification. Finally, the data on PEA intragranular and extracellular processing in adrenal medulla are discussed in regard to the known enzymatic processing mechanisms. We note the high conservation of the cleavage points in evolutionarily diverse organisms, highlighting an important biological function for the released PEAPs.     

Presence and distribution of immunoreactive and bioactive FMRFamide-like peptides in the nervous system of the horseshoe crab, Limulus polyphemus

FMRFamide immunoreactivity was detected in all regions of the Limulus nervous system, including the brain (6.5 +/- 0.6 pg FMRFamide/mg), cardiac ganglion (2.06 +/- 0.67 pg FMRFamide/mg), and ventral nerve cord (5.8 +/- 0.7 pg FMRFamide/mg). The distribution of immunoreactive FMRFamide (irFMRFamide) was mapped by immunofluorescence and the distribution corresponded to regional RIA data. A good proportion of the CNS and cardiac ganglion neuropile contained irFMRFamide, and fluorescent cell bodies were observed in several areas. High performance liquid chromatography (HPLC) was employed to separate and characterize the FMRFamide-like peptides from extracts of Limulus brains. HPLC fractions were analyzed using coincidental radioimmunoassay and bioassay (the radula protractor muscle of Busycon contrarium). There appear to be at least three FMRFamide-like peptides in the Limulus brain, including one similar to clam FMRFamide. FMRFamide acts on Limulus heart in a biphasic manner at relatively high concentrations (10(-5)M), but has no effect on the activity of the isolated ventral nerve cord. These data suggest that in Limulus FMRFamide-like peptides are acting as neurotransmitters, or neuromodulators.     

Recombinant production of self-assembling SZ-structured peptides using SUMO as a fusion partner

ABSTRACT: BACKGROUND: Self-assembling peptides that form nanostructured hydrogels are important biomaterials for tissue engineering scaffolds. The P11-family of peptides includes, P11-4 (QQRFEWEFEQQ) and the complementary peptides P11-13 (EQEFEWEFEQE) and P11-14 (QQOrnFOrnWOrnFOrnQQ). These form self-supporting hydrogels under physiological conditions (pH 7.4, 140 mM NaCl) either alone (P11-4) or when mixed (P11-13 and P11-14). We report a SUMO-peptide expression strategy suitable for allowing release of native sequence peptide by SUMO protease cleavage. RESULTS: We have expressed SUMO-peptide fusion proteins from pET vectors by using autoinduction methods. Immobilised metal affinity chromatography was used to purify the fusion protein, followed by SUMO protease cleavage in water to release the peptides, which were recovered by reverse phase HPLC. The peptide samples were analysed by electrospray mass spectrometry and self-assembly was followed by circular dichroism and transmission electron microscopy. CONCLUSIONS: The fusion proteins were produced in high yields and the beta-structured peptides were efficiently released by SUMO protease resulting in peptides with no additional amino acid residues and with recoveries of 46% to 99%. The peptides behaved essentially the same as chemically synthesised and previously characterised recombinant peptides in self-assembly and biophysical assays.     

Cleavage of small peptides in vitro by human rhinovirus 14 3C protease expressed in Escherichia coli.

The 3C region of human rhinovirus 14 was expressed in Escherichia coli. The microbially synthesized protease was functional, since the expressed precursor underwent autoproteolytic processing to generate mature molecules of the expected molecular weight and antigenicity. Mutation of the putative active-site Cys-146 residue to an alanine resulted in the synthesis of unprocessed precursor molecules. Large quantities of the 20-kilodalton protease were purified by a simple purification protocol, and the resulting molecule was shown to be biologically active in vitro against synthetic peptides corresponding to the 2C-3A cleavage site. This site was cleaved with high efficiency and fidelity and was used to generate kinetic data on the 3C protease. The protease exhibited sensitivity to Zn2+, was capable of cleaving five of seven rhinovirus cleavage site peptides tested with variable efficiency, and could distinguish authentic substrate peptides from control peptides containing the dipeptide cleavage sequence pair Gln-Gly.