Limits...
Similarity of recombinant human perlecan domain 1 by alternative expression systems bioactive heterogenous recombinant human perlecan D1.

Ellis AL, Pan W, Yang G, Jones K, Chuang C, Whitelock JM, DeCarlo AA - BMC Biotechnol. (2010)

Bottom Line: Additionally, we demonstrate HS-mediated binding of both rhPln.D1 species to several other GFs.Finally, we corroborate the augmentation of FGF-mediated cell activation by rhPln.D1 and demonstrate mitogenic signalling through the FGFR1c receptor.With importance especially to the emerging field of DNA-based therapeutics, we have shown here that proteoglycan synthesis, in different cell lines where GAG profiles typically differ, can be directed by recombinant technology to produce populations of bioactive recombinants with highly similar GAG profiles.

View Article: PubMed Central - HTML - PubMed

Affiliation: Agenta Biotechnologies, Inc., Innovation Depot, Birmingham, AL 35203, USA.

ABSTRACT

Background: Heparan sulfate glycosaminoglycans are diverse components of certain proteoglycans and are known to interact with growth factors as a co-receptor necessary to induce signalling and growth factor activity. In this report we characterize heterogeneously glycosylated recombinant human perlecan domain 1 (HSPG2 abbreviated as rhPln.D1) synthesized in either HEK 293 cells or HUVECs by transient gene delivery using either adenoviral or expression plasmid technology.

Results: By SDS-PAGE analysis following anion exchange chromatography, the recombinant proteoglycans appeared to possess glycosaminoglycan chains ranging, in total, from 6 kDa to >90 kDa per recombinant. Immunoblot analysis of enzyme-digested high Mr rhPln.D1 demonstrated that the rhPln.D1 was synthesized as either a chondroitin sulfate or heparan sulfate proteoglycan, in an approximately 2:1 ratio, with negligible hybrids. Secondary structure analysis suggested helices and sheets in both recombinant species. rhPln.D1 demonstrated binding to rhFGF-2 with an apparent kD of 2 ± 0.2 nM with almost complete susceptibility to digestion by heparinase III in ligand blot analysis but not to chondroitinase digestion. Additionally, we demonstrate HS-mediated binding of both rhPln.D1 species to several other GFs. Finally, we corroborate the augmentation of FGF-mediated cell activation by rhPln.D1 and demonstrate mitogenic signalling through the FGFR1c receptor.

Conclusions: With importance especially to the emerging field of DNA-based therapeutics, we have shown here that proteoglycan synthesis, in different cell lines where GAG profiles typically differ, can be directed by recombinant technology to produce populations of bioactive recombinants with highly similar GAG profiles.

Show MeSH
Tryptic peptides of purified rhPln.198 by mass spectrometry. Purified rhPln.198 was subjected to trypsin digest and fragments were analyzed by mass spectrometry. Red underline signifies fragments identified in this analysis of enriched rhPln.198.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2944331&req=5

Figure 3: Tryptic peptides of purified rhPln.198 by mass spectrometry. Purified rhPln.198 was subjected to trypsin digest and fragments were analyzed by mass spectrometry. Red underline signifies fragments identified in this analysis of enriched rhPln.198.

Mentions: Mass spectrometry analysis of trypsin-digested purified rhPln.198 resulted in the identification of several Pln.D1 fragments (Figure 3) but no other perlecan domains were detected. However, analysis of western blots with gels developed with silver or Stains-All suggested that the higher Mr fractions selected for pooling and analysis had minor levels of contaminating proteins (Figure 1). By mass spectrometry of enriched rhPln.198 or rhPln.247, only three other minor protein contaminants were identified, presumably from the use of fetal bovine serum in the growth medium: bovine lumican, bovine hemoglobin, and BSA (data not shown).


Similarity of recombinant human perlecan domain 1 by alternative expression systems bioactive heterogenous recombinant human perlecan D1.

Ellis AL, Pan W, Yang G, Jones K, Chuang C, Whitelock JM, DeCarlo AA - BMC Biotechnol. (2010)

Tryptic peptides of purified rhPln.198 by mass spectrometry. Purified rhPln.198 was subjected to trypsin digest and fragments were analyzed by mass spectrometry. Red underline signifies fragments identified in this analysis of enriched rhPln.198.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2944331&req=5

Figure 3: Tryptic peptides of purified rhPln.198 by mass spectrometry. Purified rhPln.198 was subjected to trypsin digest and fragments were analyzed by mass spectrometry. Red underline signifies fragments identified in this analysis of enriched rhPln.198.
Mentions: Mass spectrometry analysis of trypsin-digested purified rhPln.198 resulted in the identification of several Pln.D1 fragments (Figure 3) but no other perlecan domains were detected. However, analysis of western blots with gels developed with silver or Stains-All suggested that the higher Mr fractions selected for pooling and analysis had minor levels of contaminating proteins (Figure 1). By mass spectrometry of enriched rhPln.198 or rhPln.247, only three other minor protein contaminants were identified, presumably from the use of fetal bovine serum in the growth medium: bovine lumican, bovine hemoglobin, and BSA (data not shown).

Bottom Line: Additionally, we demonstrate HS-mediated binding of both rhPln.D1 species to several other GFs.Finally, we corroborate the augmentation of FGF-mediated cell activation by rhPln.D1 and demonstrate mitogenic signalling through the FGFR1c receptor.With importance especially to the emerging field of DNA-based therapeutics, we have shown here that proteoglycan synthesis, in different cell lines where GAG profiles typically differ, can be directed by recombinant technology to produce populations of bioactive recombinants with highly similar GAG profiles.

View Article: PubMed Central - HTML - PubMed

Affiliation: Agenta Biotechnologies, Inc., Innovation Depot, Birmingham, AL 35203, USA.

ABSTRACT

Background: Heparan sulfate glycosaminoglycans are diverse components of certain proteoglycans and are known to interact with growth factors as a co-receptor necessary to induce signalling and growth factor activity. In this report we characterize heterogeneously glycosylated recombinant human perlecan domain 1 (HSPG2 abbreviated as rhPln.D1) synthesized in either HEK 293 cells or HUVECs by transient gene delivery using either adenoviral or expression plasmid technology.

Results: By SDS-PAGE analysis following anion exchange chromatography, the recombinant proteoglycans appeared to possess glycosaminoglycan chains ranging, in total, from 6 kDa to >90 kDa per recombinant. Immunoblot analysis of enzyme-digested high Mr rhPln.D1 demonstrated that the rhPln.D1 was synthesized as either a chondroitin sulfate or heparan sulfate proteoglycan, in an approximately 2:1 ratio, with negligible hybrids. Secondary structure analysis suggested helices and sheets in both recombinant species. rhPln.D1 demonstrated binding to rhFGF-2 with an apparent kD of 2 ± 0.2 nM with almost complete susceptibility to digestion by heparinase III in ligand blot analysis but not to chondroitinase digestion. Additionally, we demonstrate HS-mediated binding of both rhPln.D1 species to several other GFs. Finally, we corroborate the augmentation of FGF-mediated cell activation by rhPln.D1 and demonstrate mitogenic signalling through the FGFR1c receptor.

Conclusions: With importance especially to the emerging field of DNA-based therapeutics, we have shown here that proteoglycan synthesis, in different cell lines where GAG profiles typically differ, can be directed by recombinant technology to produce populations of bioactive recombinants with highly similar GAG profiles.

Show MeSH