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Determination of supplier-to-supplier and lot-to-lot variability in glycation of recombinant human serum albumin expressed in Oryza sativa.

ABSTRACT: The use of different expression systems to produce the same recombinant human protein can result in expression-dependent chemical modifications (CMs) leading to variability of structure, stability and immunogenicity. Of particular interest are recombinant human proteins expressed in plant-based systems, which have shown particularly high CM variability. In studies presented here, recombinant human serum albumins (rHSA) produced in Oryza sativa (Asian rice) (OsrHSA) from a number of suppliers have been extensively characterized and compared to plasma-derived HSA (pHSA) and rHSA expressed in yeast (Pichia pastoris and Saccharomyces cerevisiae). The heterogeneity of each sample was evaluated using size exclusion chromatography (SEC), reversed-phase high-performance liquid chromatography (RP-HPLC) and capillary electrophoresis (CE). Modifications of the samples were identified by liquid chromatography-mass spectrometry (LC-MS). The secondary and tertiary structure of the albumin samples were assessed with far U/V circular dichroism spectropolarimetry (far U/V CD) and fluorescence spectroscopy, respectively. Far U/V CD and fluorescence analyses were also used to assess thermal stability and drug binding. High molecular weight aggregates in OsrHSA samples were detected with SEC and supplier-to-supplier variability and, more critically, lot-to-lot variability in one manufactures supplied products were identified. LC-MS analysis identified a greater number of hexose-glycated arginine and lysine residues on OsrHSA compared to pHSA or rHSA expressed in yeast. This analysis also showed supplier-to-supplier and lot-to-lot variability in the degree of glycation at specific lysine and arginine residues for OsrHSA. Both the number of glycated residues and the degree of glycation correlated positively with the quantity of non-monomeric species and the chromatographic profiles of the samples. Tertiary structural changes were observed for most OsrHSA samples which correlated well with the degree of arginine/lysine glycation. The extensive glycation of OsrHSA from multiple suppliers may have further implications for the use of OsrHSA as a therapeutic product.

SUBMITTER: Frahm GE

PROVIDER: S-EPMC4192584 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Determination of supplier-to-supplier and lot-to-lot variability in glycation of recombinant human serum albumin expressed in Oryza sativa.

Frahm Grant E GE Smith Daryl G S DG Kane Anita A Lorbetskie Barry B Cyr Terry D TD Girard Michel M Johnston Michael J W MJ

PloS one 20141009 10

The use of different expression systems to produce the same recombinant human protein can result in expression-dependent chemical modifications (CMs) leading to variability of structure, stability and immunogenicity. Of particular interest are recombinant human proteins expressed in plant-based systems, which have shown particularly high CM variability. In studies presented here, recombinant human serum albumins (rHSA) produced in Oryza sativa (Asian rice) (OsrHSA) from a number of suppliers hav ...[more]

PMID: 25299339

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Project description:BackgroundDirect seeding is an efficient cultivation technique in rice. However, poor low temperature germinability (LTG) of modern rice cultivars limits its application. Identifying the genes associated with LTG and performing molecular breeding is the fundamental way to address this issue. However, few LTG QTLs have been fine mapped and cloned so far.ResultsIn the present study, the LTG evaluation of 375 rice accessions selected from the Rice Diversity Panel 2 showed that there were large LTG variations within the population, and the LTG of Indica group was significantly higher than that of Japonica and Aus groups (p < 0.01). In total, eleven QTLs for LTG were identified through genome-wide association study (GWAS). Among them, qLTG_sRDP2-3/qLTG_JAP-3, qLTG_AUS-3 and qLTG_sRDP2-12 are first reported in the present study. The QTL on chromosome 10, qLTG_sRDP2-10a had the largest contribution to LTG variations in 375 rice accessions, and was further validated using single segment substitution line (SSSL). The presence of qLTG_sRDP2-10a could result in 59.8% increase in LTG under 15 °C low temperature. The expression analysis of the genes within qLTG_sRDP2-10a region indicated that LOC_Os10g22520 and LOC_Os10g22484 exhibited differential expression between the high and low LTG lines. Further sequence comparisons revealed that there were insertion and deletion sequence differences in the promoter and intron region of LOC_Os10g22520, and an about 6 kb variation at the 3' end of LOC_Os10g22484 between the high and low LTG lines, suggesting that the sequence variations of the two genes could be the cause for their differential expression in high and low LTG lines.ConclusionAmong the 11 QTLs identified in this study, qLTG_sRDP2-10a could also be detected in other three studies using different germplasm under different cold environments. Its large effect and stable expression make qLTG_sRDP2-10a particularly valuable in rice breeding. The two genes, LOC_Os10g22484 and LOC_Os10g22520, were considered as the candidate genes underlying qLTG_sRDP2-10a. Our results suggest that integrating GWAS and SSSL can facilitate identification of QTL for complex traits in rice. The identification of qLTG_sRDP2-10a and its candidate genes provide a promising source for gene cloning of LTG and molecular breeding for LTG in rice.

Dataset Information

Determination of supplier-to-supplier and lot-to-lot variability in glycation of recombinant human serum albumin expressed in Oryza sativa.

Publications

Determination of supplier-to-supplier and lot-to-lot variability in glycation of recombinant human serum albumin expressed in Oryza sativa.

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