Identification of the dipeptidyl aminopeptidase responsible for N-terminal clipping of recombinant Exendin-4 precursor expressed in Pichia pastoris

Prabha, Lakshmi; Govindappa, Nagaraj; Adhikary, Laxmi; Melarkode, Ramakrishnan; Sastry, Kedarnath N.

doi:10.1016/j.pep.2008.10.021

Cited by 10 publications

(7 citation statements)

References 12 publications

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“…The gene coding for protein mannosyltransferases have been inactivated by insertional inactivation method [18,19]. The inactivation was confirmed by genomic PCR.…”

Section: Discussionmentioning

confidence: 99%

“…The linearized vector was transformed into the IP clone#1 by electroporation and transformants were selected on YPDS plates containing 100 lg/ml Zeocin. The transformants were screened by genomic PCR using primers specifically designed in such way that the PCR amplicon will be obtained only when the disruption occurs at the PMT1 gene locus [19]. The primer combinations used for screening were P1 (binds in the PMT1 genomic locus) and P2 (binds in the disruption vector region).…”

Section: Inactivation Of Pmt1 Gene Coding Sequence In Ip Clone #1mentioning

confidence: 99%

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PMT1 gene plays a major role in O-mannosylation of insulin precursor in Pichia pastoris

Govindappa

Hanumanthappa

Krishna

et al. 2013

Protein Expression and Purification

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“…The gene coding for protein mannosyltransferases have been inactivated by insertional inactivation method [18,19]. The inactivation was confirmed by genomic PCR.…”

Section: Discussionmentioning

confidence: 99%

Section: Inactivation Of Pmt1 Gene Coding Sequence In Ip Clone #1mentioning

confidence: 99%

PMT1 gene plays a major role in O-mannosylation of insulin precursor in Pichia pastoris

Govindappa

Hanumanthappa

Krishna

et al. 2013

Protein Expression and Purification

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“…Protein polymers containing N-terminal Xaa-Ala or Xaa-Pro dipeptides can be protected simply by adding one or more N-terminal shielding residues, such that these motifs are no longer at the N-terminus. Alternatively, P. pastoris strains disrupted in the STE13 gene homolog are likely useful (Hopkins et al, 2014; Prabha et al, 2009).…”

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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“…However, Indeed, elastin-like protein polymers produced in P. pastoris contained 80% of species lacking the first four residues GPVP [15]. A variant of Plasmodium falciparum circumsporozoite protein similarly suffered deletion in P. pastoris of no less than 30 Asn-Ala and Asn-Pro dipeptides from its N-terminus [303], undoubtedly caused by DPAPase A. Interestingly, Prabha et al [258] showed that the protease may also attack other than the canonical Xaa-Ala and Xaa-Pro dipeptides, as in their case an N-terminal His-Gly dipeptide was removed. This proteolysis was successfully abolished upon disruption of the P. pastoris STE13…”

Section: Kex1 Proteasementioning

confidence: 99%

“…gene homolog [258]. The vacuolar dipeptidyl aminopeptidase B (a serine protease encoded by the DAP2 gene), which has been shown a weak suppressor of ste13 defects in S. cerevisiae with similar substrate specificity [156], did not seem to be involved in this degradation.…”

Section: Kex1 Proteasementioning

confidence: 99%

Production of protein‐based polymers in Pichia pastoris

Werten

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A long-sought objective in material science is the development of polymers with controlled monomer sequence [1, 2]. Although progress has been made in synthetic chemistry [3], the level of control evident in natural sequential polymers such as DNA and proteins is unparalleled. These biological macromolecules feature a defined molecular size, as well as a controlled sequence of the nucleotide or amino acid monomers. Proteins fold into a three-dimensional structure defined by their primary sequence, resulting in unique properties. From 20 different amino acid monomers, nature has created an awe-inspiring wealth of different proteins, including enzymes, antibodies, and peptide hormones, as well as nonbioactive proteins with a structure-forming, rheological, or colloidal function. The latter category includes proteins such as collagen and elastin that fulfill a major role in the structure of various tissues, and silks used in animal architecture such as silkworm cocoons and spider webs (for more information see Section 9.1 of the General discussion). These proteins typically feature highly repetitive sequences with biased amino acid composition, and can often reversibly self-assemble into supramolecular structures through the formation of noncovalent bonds. The natural materials derived from them display remarkable toughness, elasticity, and other properties that have inspired material scientists to mimic them using modern protein engineering. These so-called protein-based polymers, or protein polymers for short, are produced as heterologous proteins in a suitable host, just like enzymes and other proteins, although their highly repetitive sequence, biased amino acid composition, and physicochemical properties do present additional difficulty. The genes encoding natural protein polymers are sometimes used, but more often genes are synthesized that encode simplified mimics, or even completely de novo-designed protein polymers [4-6]. Multifunctional block copolymers can be prepared by combining different polymer types into one molecule [1, 2, 7-9]. Ever since the pioneering work by Cappello and Ferrari of Protein Polymer Technologies [10, 11], Escherichia coli has by far been the most widely used production host for protein polymers. This workhorse of protein engineering is genetically very well accessible, and offers fast growth and efficient low-cost production [12]. Several other hosts have been used for the production of protein polymers, including plants, insect cells, transgenic animals, Aspergillus nidulans, Saccharomyces cerevisiae,

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Identification of the dipeptidyl aminopeptidase responsible for N-terminal clipping of recombinant Exendin-4 precursor expressed in Pichia pastoris

Cited by 10 publications

References 12 publications

PMT1 gene plays a major role in O-mannosylation of insulin precursor in Pichia pastoris

PMT1 gene plays a major role in O-mannosylation of insulin precursor in Pichia pastoris

Production of protein-based polymers in Pichia pastoris

Production of protein‐based polymers in Pichia pastoris

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