Purification and characterization of recombinant collagens/gelatins from transgenic corn seeds

Abstract

<p>Corn offers a number of advantages as production host for low- and intermediate-value protein-based products that could be produced in large volume as co-products of corn based biorefining. We selected one such potential product, a recombinant human 100 kDa type I collagen alpha 1 (CIalpha1) and a 44 kDa single chain fragment (a gelatin). The equivalency to the intended product was established by characterizing the immunorecognition, molecular weight, amino acid composition and sequence, and post-translational modifications.;Purification for characterization of the recombinant proteins from transgenic corn grain began with protein extraction in 0.1M phosphoric acid buffer. The use of low pH minimized the extraction of host components while allowing efficient recovery of both 44 kDa and 100 kDa CIalpha1s. The 44 kDa CIalpha1 was purified by a two-step process consisting of ion-exchange chromatography followed by a gel filtration chromatography, resulting in 70% purity and 60% yield. The N-terminal sequence, amino acid composition and immunoreactivity of the purified 44 kDa CIalpha1 was similar to that of an analogous 44 kDa CIalpha1 fragment produced by yeast Pichia. The corn-derived 44 kDa CIalpha1 fragment had an intact protein mass of 44,088 kDa that was within 0.2% of the mass calculated from the intended sequence. 78% coverage of the primary sequence of the corn-derived 44 kDa CIalpha1 was confirmed with tandem mass spectrometry. Glycoprotein staining of the 44 kDa CIalpha1 revealed no detectable glycosylation.;The 100 kDa CIalpha1 was purified to close to 100% purity and 16% yield by using membrane filtration followed by four chromatographic steps (two cation-exchange, one hydrophobic interaction, and one gel filtration chromatography). The 1.2% hydroxyproline in the corn-derived 100 kDa CIalpha1, corresponding to about 10% of the hydroxyproline content in native human collagens, confirmed both some ability of corn to hydroxylate proline, but also the necessity of co-expression of a heterologous P4H if higher content is desired. The low hydroxyproline content was well correlated with its relatively low melting temperate (26°C vs 40°C in native human collagen). A fraction of the expressed CIalpha1 formed triple helices resistant to pepsin hydrolysis. A 29 amino acid foldon at the C-terminus was not cleaved from the CIalpha1 chains but could be removed by pepsin treatment. The amino acid composition and immunoreactivity of the purified CIalpha1 was as expected and similar to that of an analogous CIalpha1 produced by the yeast Pichia.;The evidence that the CIalpha1s accumulated in corn grain with the intended composition and post-translational modifications established transgenic corn as a viable way to produce this particular full-length 100 kDa CIalpha1 and its 44kD CIalpha1 fragment, and potentially other "designer" gelatins with specific molecular weights and properties tailored to suit various material property specifications.;Two milling processes have been evaluated for their potential to reduce the downstream cost by enriching the CIalpha1s in fractionated tissues prior to protein extraction and purification. Both dry-milling and wet-milling processes were able to concentrate and capture ca. 60% of the total CIalpha1s in ca. 20-25% of the total kernel mass. The 44 kDa and 100 kDa CIalpha1s were enriched 2.5 and 5.8 times respectively in the dry-milled germ-rich fractions compared to whole corn kernel. For the wet milling, the 44 kDa CIalpha1 was captured in fine fiber-, coarse fiber-, and gluten-rich fractions with 7.7 times enrichment, and the 100 kDa CIalpha1 was recovered in fine fiber-, coarse fiber-, and germ-rich fractions with 4.6 times enrichment. In summary, both milling processes can increase the CIalpha1 purity in selected fractions. While wet and dry milling achieved similar performance in enriching the 100 kDa CIalpha1, wet milling is superior to dry milling in enriching the 44 kDa CIalpha1, as the former achieved higher purity in the extract and higher purification factor (PF). CIalpha1s were associated with both coarse fiber and fine fiber when wet-milled, but the mechanism behind that discovery was not clear. While the main advantage offered by fractionation is the reduction in volume and impurity load to the separation train, an initial on-farm dry-milling step would eliminate the need to transport viable transgenic seeds, thus reducing risk of accidental release.</p>