Specificity characterization of the α-mating factor hormone by Kex2 protease

Manfredi, Marcella Araujo; Antunes, Alyne Alexandrino; Jesus, Larissa de Oliveira Passos; Juliano, María A.; Juliano, Luiz; Júdice, Wagner Alves de Souza

doi:10.1016/j.biochi.2016.10.003

Cited by 9 publications

(3 citation statements)

References 54 publications

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“…Much insight into the sequence specificity of Kex2 can be gleaned from the studies by Bevan et al (1998) and Suzuki et al (2000), in which the effect on cleavage was investigated of substitutions at substrate positions P2 and P3/P4, respectively. Although Kex2 is thought to positively recognize only the region N-terminal to the cleaved bond (Bourbonnais et al, 1988; Rockwell et al, 2002; Rockwell et al, 1997), possibly also the P1’ residue should be taken into account, as efficient substrates tend to have small or acidic residues at this position (Bader et al, 2008; Manfredi et al, 2016; Rholam et al, 1995). Pro at P1’ appears to disturb Kex2 processing (Guisez et al, 1991; Xie et al, 2007).…”

Section: Challenges and Possible Solutionsmentioning

confidence: 99%

Production of protein-based polymers in Pichia pastoris

Werten

Eggink

Stuart

et al. 2019

Biotechnology Advances

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Materials science and genetic engineering have joined forces over the last three decades in the development of so-called protein-based polymers. These are proteins, typically with repetitive amino acid sequences, that have such physical properties that they can be used as functional materials. Well-known natural examples are collagen, silk, and elastin, but also artificial sequences have been devised. These proteins can be produced in a suitable host via recombinant DNA technology, and it is this inherent control over monomer sequence and molecular size that renders this class of polymers of particular interest to the fields of nanomaterials and biomedical research. Traditionally, Escherichia coli has been the main workhorse for the production of these polymers, but the methylotrophic yeast Pichia pastoris is finding increased use in view of the often high yields and potential bioprocessing benefits. We here provide an overview of protein-based polymers produced in P. pastoris . We summarize their physicochemical properties, briefly note possible applications, and detail their biosynthesis. Some challenges that may be faced when using P. pastoris for polymer production are identified: (i) low yields and poor process control in shake flask cultures; i.e. , the need for bioreactors, (ii) proteolytic degradation, and (iii) self-assembly in vivo . Strategies to overcome these challenges are discussed, which we anticipate will be of interest also to readers involved in protein expression in P. pastoris in general.

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Section: Challenges and Possible Solutionsmentioning

confidence: 99%

Production of protein-based polymers in Pichia pastoris

Werten

Eggink

Stuart

et al. 2019

Biotechnology Advances

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“…It was reported that the modification of the P1’ site significantly improves the production of exogenous secreted proteins [ 35 ]. However, a low preference of selectivity for most residues at the P1’ site by Kex2 depends on the residue at the P3’ position and its binding to the S3 subsite [ 36 ]. In this study, NZ2114 was chosen as the object peptide, and the P1’ site amino acid with optimal cutting efficiency was screened; both expression and druggability of the candidate F-NZ2114 (short for FNZ) of the AMP derivative were further studied.…”

Section: Introductionmentioning

confidence: 99%

Molecular Modification of Kex2 P1’ Site Enhances Expression and Druggability of Fungal Defensin

et al. 2023

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Pichia pastoris is the widely used expression system for producing recombinant secretory proteins. It is known that Kex2 protease plays a vital role in the process of protein secretion, in which the P1’ site affects its cleavage efficiency. To enhance the expression level of fungal defensin-derived peptide NZ2114, this work attempts to optimize the P1’ site of Kex2 by replacing it with 20 amino acids in turn. The results showed that when the amino acid of the P1’ site was changed to Phe (F), the yield of target peptide significantly increased from 2.39 g/L to 4.81 g/L. Additionally, the novel peptide F-NZ2114 (short for FNZ) showed strong antimicrobial activity against Gram-positive (G+) bacteria, especially for Staphylococcus aureus and Streptococcus agalactiae (MIC: 4–8 μg/mL). The FNZ was very stable and retained high activity in various conditions; in addition, a low cytotoxicity and no hemolysis were observed even at a high concentration of 128 μg/mL, and a longer postantibiotic effect was reached. The above results indicate that this engineering strategy provided a feasible optimization scheme for enhancing the expression level and druggability of this antimicrobial peptide from fungal defensin and other similar targets by this updated recombinant yeast.

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“…Kex2 is also found in Pichia pastoris (UniPort Q5J881) in one family, S8 and it has a similarity of 46.42% with S. cerevisiae Kex2. The roles both of S. cerevisiae Kex2 and P. pastoris Kex2 are to process mating-α factors, remove peptides signal from pre-proteins and secrete mature proteins (Mizuno et al 1988;Manfredi et al 2016).…”

Section: Introductionmentioning

confidence: 99%

Construction of Saccharomyces cerevisiae KEX2-650 gene expression vector and its introduction into Escherichia coli DH5?

ADIBAH

FUAD²,

Budiarti³

et al. 2022

Biodiversitas

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Abstract. Adibah DI, Fuad AM, Budiarti S, Pratiwi RD. 2022. Construction of Saccharomyces cerevisiae KEX2-650 gene expression vector and its introduction into Escherichia coli DH5?. Biodiversitas 23: 4289-4296. Kex2 (EC 3.4.21.61) is a serine protease that binds Ca2+ molecules and naturally found intracellularly as a transmembrane protein. Kex2 has a unique function, the conversion process of recombinant protein precursor into mature protein. Kex2 from Saccharomyces cerevisiae has similar functions also with furin in mammals (about 47% of sequence similarity). To gain advantage, extracellular Kex2 would be highly favorable for this process. This study aimed to construct recombinant Kex2 that could be produced extracellularly in Pichia pastoris host through pD902-KEX2-699 vector (synthetic) with FLAG-tag and 6 His-tag by removing most of C-terminal region, including transmembrane domain (TMD) from KEX2 gene sequence. Constructed KEX2 is the KEX2-650 variant with TMD deletion and cytoplasmic domain. The recombinant plasmid was constructed through site-directed mutagenesis using FP-Kex2-699 and RP-Kex2-650 primers, including the BamHI site for plasmid religation. PCR site-directed mutagenesis produces an amplicon DNA with an expected length of 5551 bp. After restriction (BamHI) and religation, the plasmid was reintroduced into Escherichia coli DH5? and obtained 16 colonies. Verification PCR target gene showed that clones number 9 produced an amplicon of expected length (646 bp). DNA sequencing analysis confirmed that TMD was removed from the gene construct to form the KEX2-650 construct.

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Specificity characterization of the α-mating factor hormone by Kex2 protease

Cited by 9 publications

References 54 publications

Production of protein-based polymers in Pichia pastoris

Production of protein-based polymers in Pichia pastoris

Molecular Modification of Kex2 P1’ Site Enhances Expression and Druggability of Fungal Defensin

Construction of Saccharomyces cerevisiae KEX2-650 gene expression vector and its introduction into Escherichia coli DH5?

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