Construction and expression of two-copy engineered yeast of feruloyl esterase

Background Aspergillus niger has the ability to secrete feruloyl esterase. However, for economically viable industrial applications, it is necessary to increase their catalytic activities and/or protein yields to satisfy the increasing needs for feruloyl esterases. Results The gene AnFaeA that encodes a type A feruloyl esterase was successfully expressed in Pichia pastoris by a two-copy engineered yeast. After a screen in shaker flask, a one-copy strain GSKFA3 having the highest feruloyl esterase activity of 2.4 U/mL was obtained. Then, the pPICZaA-AnFaeA plasmid was transformed into GSKFA3 and the transformants were grown on YPDS plates with antibiotic Zeocin. After cultivation, a two-copy strain GSKZaFA20 with the highest feruloyl esterase activity of 15.49 U/mL was obtained. The expressed protein (recombinant AnFaeA) may be a glycoprotein with an apparent molecular weight of 40 kDa. It displayed the maximum activity at pH 6.0 and 50°C, and was stable at a pH range of 4.0-6.5 and at below 45°C. Its activity was not significantly affected by K+, Ca2 +, Mg2 +, Cu2 +, Zn2 +, Mn2 +, Na+ and EDTA, but activated by Fe2 +. The Km and Vmax toward 4-nitrophenyl ferulate were 5.5 mM and 69.0 U/mg, respectively. Conclusions The two-copy strain GSKZaFA20 showed a 4.4-fold increase in extracellular enzyme activity compared with the one-copy strain GSKFA3. Construction of two-copy strain improved secretion of recombinant AnFaeA in P. pastoris.

PCR assembly of synthetic human erythropoietin gene

Human erythropoietin (huEPO) is a glycoprotein with important physiological functions, such as erythropoiesis, angiogenesis, and wound healing. A therapeutic protein, huEPO is commonly used to treat patients suffering from renal and non-renal anemia. Recombinant human erythropoietin (rhuEPO) and endogenous huEPO are similar with respect to their biological and chemical properties. In this study, we describe the construction of synthetic huEPO gene to produce rhuEPO. The synthetic huEPO gene was constructed by overlapping oligonucleotides assembly and amplified by polymerase chain reaction (PCR). Twenty oligonucleotide sets, covering the huEPO gene sequence and two newly introduced restriction enzyme sites, were pulled together and amplified using Pfu DNA polymerase to produce the expected DNA products with sizes of ~500bp and ~600bp. The PCR products were ligated into pGEM-T plasmid vector to facilitate DNA sequencing process of the constructed huEPO gene and downstream cloning manipulation. DNA sequence analysis showed correctly assembled oligonucleotide sets, representing the huEPO gene sequence albeit with minor base mutations. Hence, oligonucleotides assembly and PCR amplification provide a convenient and speedy method for the synthesis of huEPO gene without depending on mRNA isolation and reverse transcription or the need to have a genomic library.

Bases moleculares do efeito do pH na atividade catalítica de duas lisozimas digestivas de Musca domestica (Diptera) / Molecular basis of the pH effect on the catalytic activity of two digestive lysozymes from Musca dosmetica (Diptera)

Lisozimas são enzimas que fazem parte do mecanismo de defesa contra bactérias, no entanto lisozimas com função digestiva também são encontradas no trato digestivo de vertebrados e no intestino médio de insetos. As Iisozimas digestivas de insetos são do tipo "c" e assim compartilham semelhanças estruturais e mecanísticas com a lisozima da clara de ovo de galinha (HEWL). Entretanto, para desempenhar sua função digestiva, as lisozimas de insetos apresentam algumas propriedades particulares entre as quais se destaca um pH ótimo mais ácido em relação às lisozimas não-digestivas. Para elucidar as bases moleculares dessa diferença no pH ótimo, duas lisozimas digestivas (lisozima 1 — AAQ20048 e lisozima 2 — AAQ20047) da larva de Musca domestica (mosca — Diptera Cyclorrhapha), clonadas em Pichia pastoris e purificadas, foram caracterizadas estruturalmente e cineticamente com o substrato sintético (MUQ3) e natural (cápsulas de Micrococcus lysodeikticus). Foi observado que o efeito do pH na atividade das lisozimas 1 e 2 sobre o MUQ3 é uma curva com formato de sino e pH ótimo mais ácido que o da HEWL. Essas curvas foram reflexos da diminuição simultânea dos valores de p’K IND.a’s do nucleófilo e do doador de prótons...

High expression of human carboxypeptidase M in Pichia pastoris: Purification and partial characterization

Carboxypeptidase M (CPM) is an extracellular glycosylphosphatidyl-inositol-anchored membrane glycoprotein, which removes the C-terminal basic residues, lysine and arginine, from peptides and proteins at neutral pH. CPM plays an important role in the control of peptide hormones and growth factor activity on the cell surface. The present study was carried out to clone and express human CPM in the yeast Pichia pastoris in order to evaluate the importance of this enzyme in physiological and pathological processes. The cDNA for the enzyme was amplified from total placental RNA by RT-PCR and cloned in the vector pPIC9, which uses the methanol oxidase promoter and drives the expression of high levels of heterologous proteins in P. pastoris. The cpm gene, after cloning and transfection, was integrated into the yeast genome, which produced the active protein. The recombinant protein was secreted into the medium and the enzymatic activity was measured using the fluorescent substrate dansyl-Ala-Arg. The enzyme was purified by a two-step protocol including gel filtration and ion-exchange chromatography, resulting in a 1753-fold purified active protein (16474 RFU mg protein-1 min-1). This purification protocol permitted us to obtain 410 mg of the purified protein per liter of fermentation medium. SDS-PAGE showed that recombinant CPM migrated as a single band with a molecular mass similar to that of native placental enzyme (62 kDa), suggesting that the expression of a glycosylated protein had occurred. These results demonstrate for the first time the establishment of a method using P. pastoris to express human CPM necessary to the development of specific antibodies and antagonists, and the analysis of the involvement of this peptidase in different physiological and pathological processes.