RESUMO
Bovine pancreatic trypsin inhibitor (BPTI) was secreted by Aspergillus niger at yields of up to 23 mg l-1 using a protein fusion strategy. BPTI was linked to part of the fungal glucoamylase protein (GAM) with a dibasic amino acid (KEX2) processing site at the fusion junction. Electrospray ionisation mass spectrometry and N-terminal protein sequencing revealed that, although biologically active in vitro, the purified products from a number of independent transformants consisted of a mixture of BPTI molecules differing at the N-terminus. Approximately 35-60% of this mixture was processed correctly. Aberrant processing of the GAM-BPTI fusion protein by the A. niger KEX2-like endoprotease was the most likely cause of this variation although the involvement of other fungal endoproteases could not be ruled out. In vitro studies have highlighted a weak interaction between BPTI and the Saccharomyces cerevisiae KEX2 endoprotease, suggesting that BPTI is not a potent inhibitor of KEX2p. A small proportion of the recombinant BPTI (10%) showed 'nicking' of the K15-A16 bond, indicating an interaction with a fungal trypsin-like enzyme. Mutant BPTI homologues designed to have anti-elastase activity, BPTI(K15V), BPTI(K15V,P13I) and BPTI(K15V,G12A), have also been expressed and secreted by A. niger. They also showed a similar spectrum of aberrant N-terminal processing but no 'nicking' of the K15-V16 bond was observed. Comparison of A. niger with other expression systems showed that it is an effective system for producing BPTI and its homologues, although not all molecules were correctly processed. This variation in processing efficiency may be useful in understanding the important determinants of protein processing in this fungus.
Assuntos
Aprotinina/genética , Aprotinina/metabolismo , Aspergillus niger/genética , Aspergillus niger/metabolismo , Pró-Proteína Convertases , Proteínas de Saccharomyces cerevisiae , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação/genética , Biotecnologia , Bovinos , Primers do DNA/genética , Técnicas In Vitro , Dados de Sequência Molecular , Mutação , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/enzimologia , Subtilisinas/metabolismo , Transformação GenéticaRESUMO
The binding of BPTI and SBTI with trypsin has been investigated by ESI MS, using the mutant K15V-BPTI and the chemically modified RcamBPTI as controls. Although high cone voltages (+80 V) produce sharp spectra of BPTI, RcamBPTI, SBTI and trypsin alone, the complexes of BPTI, RcamBPTI and SBTI with trypsin undergo partial dissociation due to collisional activation. At lower cone voltages (+40 V) these non-covalent complexes are stable. The charge distribution on the trypsin and the inhibitors produced by gas phase dissociation of the complexes are markedly different from those of the components alone, indicating that ESI MS provides a novel probe for exploring the ionic interactions at the contact surface of proteins. Moreover, by determining the cone voltage at which the gas phase dissociation of complexes occurs it may be possible to use ESI MS to compare the binding energies of closely related complexes.
Assuntos
Aprotinina/metabolismo , Espectrometria de Massas/métodos , Inibidor da Tripsina de Soja de Kunitz/metabolismo , Inibidores da Tripsina/metabolismo , Tripsina/metabolismo , Acetatos/química , Aprotinina/química , Aprotinina/genética , Dissulfetos/química , Eletroquímica/métodos , Íons , Mutação , Desnaturação Proteica , Soluções , Tripsina/química , Inibidor da Tripsina de Soja de Kunitz/química , Inibidores da Tripsina/químicaRESUMO
The interactions of three BPTI homologues with human leukocyte elastase and porcine pancreatic elastase have been investigated. The principal mutation in determining the specificity of inhibition was the Lys15-Val mutation at the P1 position. An additional mutation at P3, i.e., BPTI (Lys15-Val, Pro13-Ile), increased the inhibition of HLE to a Ki = 2.5 x 10(-10) M, but decreased the inhibition of PPE, showing this to be a useful site for improving selectivity. Kinetic evidence suggests that the inhibition of HLE by BPTI homologues probably takes place by a two-step mechanism in which an isomerization step occurs after initial binding. 1H NMR spectroscopy of the BPTI (Lys15-Val) and BPTI (Lys15-Val, Pro13-Ile) mutants indicates that small conformational changes are associated with the mutations, but these are localized in the immediate vicinity of the mutation in the outer binding loop and in the inner loop connected to it through the Cys14-Cys38 disulfide bridge.
Assuntos
Aprotinina/farmacologia , Elastase Pancreática/antagonistas & inibidores , Inibidores de Serina Proteinase/farmacologia , Sequência de Aminoácidos , Animais , Aprotinina/química , Aprotinina/genética , Aprotinina/metabolismo , Bovinos , Humanos , Cinética , Elastase de Leucócito , Espectroscopia de Ressonância Magnética , Modelos Químicos , Dados de Sequência Molecular , Mutação , Elastase Pancreática/metabolismo , Ligação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacologia , Inibidores de Serina Proteinase/química , SuínosRESUMO
Biologically active bovine pancreatic trypsin inhibitor (BPTI) was produced in Escherichia coli using an OmpA leader-peptide fusion-protein system, and BPTI homologues were generated by cassette mutagenesis. Amino acids in the reactive loop of alpha 1-proteinase inhibitor (alpha 1-PI) were incorporated into the reactive loop of BPTI in a stepwise approach such that the contribution of individual amino acids could be assessed. The introduction of mutations into BPTI diminished the yield of heterologous protein relative to wild-type BPTI. However, for three BPTI homologues sufficient material was isolated to allow characterization of the proteins by electrospray MS and N-terminal peptide sequencing.
