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Recovering corn germ enriched in recombinant protein by wet-fractionation
Bioresource Technology 101 (2010) 239–244
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Recovering corn germ enriched in recombinant protein by wet-fractionation Ilankovan Paraman a, Steven R. Fox a, Matthew T. Aspelund b, Charles E. Glatz b, Lawrence A. Johnson a,* a b
Center for Crops Utilization Research, Department of Food Science and Human Nutrition, Iowa State University, Ames, IA 50011-1061, USA Department of Chemical and Biological Engineering, Iowa State University, Ames, IA 50011-2230, USA
a r t i c l e
i n f o
Article history: Received 23 March 2009 Received in revised form 3 August 2009 Accepted 4 August 2009 Available online 1 September 2009 Keywords: Transgenic corn fractionation Quick-germ processing Wet milling Recombinant collagen-related proteins Corn
a b s t r a c t Corn wet-fractionation processes (quick-germ fractionation and traditional wet milling) were evaluated as means of recovering fractions rich in recombinant collagen-related proteins that were targeted for expression in the germ (embryo) of transgenic corn. Transgenic corn lines accumulating a recombinant full-length human collagen type-I-alpha-1 (full-length rCIa1) or a 44-kDa rCIa1 fragment targeted for seed expression with an embryo-specific promoter were used. Factors to consider in efficient recovery processes are the distribution of the peptides among botanical parts and process recovery efficiency. Both recombinant proteins were distributed 62–64% in germ comprising about 8.6% of the dry grain mass; 34– 38% in the endosperm comprising 84% of the dry grain mass; 1.7% in the pericarp comprising about 5% of the dry mass; and 1% in the tip-cap comprising 1.5–2% of the dry mass. The quick-germ method employed a short steeping period either in water or SO2–lactic acid solution followed by wet-milling degermination to recover a germ-rich fraction. Of the total recombinant protein expressed in germ, the quick-germ process recovered 40–43% of the total recombinant protein within 6–8% of the corn mass. The traditional corn wet-milling process produced higher purity germ but with lower recovery (24– 26%) of the recombinant protein. The two quick-germ methods, using water alone or SO2–lactic acid steeping, did not substantially differ in rCIa1 recovery, and the quick-germ processes recovered germ with less leaching and proteolytic losses of the recombinant proteins than did traditional wet milling. Thus, grain fractionation enriched the recombinant proteins 6-fold higher than that of unfractionated kernels. Such enrichment may improve downstream processing efficiency and enable utilizing the protein-lean co-products to produce biofuels and biorenewable chemicals by fermenting the remaining starch-rich fractions. Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction Transgenic plants as bioreactors to produce pharmaceuticals, vaccines, industrial proteins and enzymes have gained commercial interest due to many practical, economic and safety advantages over the traditional protein production systems such as microbial or cell culture. The production, purification, and commercialization of plant-made recombinant proteins have been reviewed numerous times (Ma et al., 2003; Menkhaus et al., 2004; Howard and Hood, 2005; Fox, 2006; Ramessar et al., 2008). Among the variety of crops considered as potential hosts for recombinant proteins, corn seed is preferred as a bioreactor because of its ability to produce stable recombinant proteins, absence of active endogenous proteases, and being rich in molecular chaperones and disulfide isomerases that facilitate proper protein folding (Ramessar et al., 2008); and protein in the seeds can remain stable for years without
* Corresponding author. Address: 1041 Food Sciences Building, Iowa State University, Ames, IA 50011, USA. Tel.: +1 515 294 4365; fax: +1 515 294 2689. E-mail address: [email protected] (L.A. Johnson). 0960-8524/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2009.08.023
refrigeration, providing practical and economic advantages in storing and processing. Extraction and purification of recombinant protein from plants, however, are difficult processes having low recovery yields and inconsistent end-product qualities. Furthermore, downstream processing consumes 40% or more of the total cost when producing pharmaceutical proteins from corn (Zhong et al., 2006). The normal practice has been to extract the target protein by grinding the whole grain and extracting with buffer; however, large volumes of extraction buffer (e.g. liquid-to-solids ratio of 10) are needed, low concentrations of target protein in the extract are produced, and co-extracted corn proteins complicate purification. Therefore, downstream processing costs are reduced when the protein is concentrated in the material to be extracted and protein-lean starch-rich fractions can be used as feedstocks for the fermentation industry to produce fuel ethanol and industrial chemicals (Johnson and May, 2003). Zhang et al. (2009a,b,c) recovered 60% of the total recombinant collagen-related protein (germ targeted) in 25% of the kernel mass by using dry-milling techniques, and found that such enrichment could reduce at least one step of chromatography in subsequent purification.
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Expression of recombinant proteins can be targeted to different grain tissues such as the germ (embryo) and endosperm. Wet milling is more effective (yield and purity considerations) in recovering germ from corn than dry-milling but care must be taken in selecting processing conditions that are compatible with the proteins of interest because some enzymes and pharmaceutical proteins lose their biological activities when exposed to typical grain-steeping conditions of traditional corn wet milling (Johnson and May, 2003). For instance, exposure to SO2, lactic acid and high temperatures (50–52 °C) for long periods (24–48 h) of traditional corn steeping may denature the target protein or promote endogenous and microbial protease activities (Dailey et al., 2000; Hull et al., 1996). Moreover, 10% of the recombinant collagen-related protein expressed in corn germ was lost by leaching when conventionally steeped for 40 h at 50 °C (Zhang et al., 2009a,b,c). To overcome these limitations, research efforts have focused on reducing steeping time and usage of detrimental chemicals when recovering recombinant proteins by wet-fractionation methods. Johnson and Fox (2002) modified the quick-germ process, a corn fractionation process designed to recover germ in dry-grind ethanol plants (Singh and Eckhoff, 1996), to recover recombinant b-glucuronidase expressed in corn germ. About 85% total grain enzyme activity was recovered in 10% of the corn mass (Johnson and Fox, 2002), much greater than using dry-fractionation methods where about 77% of the total b-glucuronidase was recovered in 20–25% of dry-milled corn mass (Kusnadi et al., 1998); 49% b-glucuronidase was recovered in 17% of corn mass (Yildirim et al., 2002); and 47.7% of total recombinant green fluorescence protein was recovered in 15% of corn mass (Shepherd et al., 2008a). Only a limited number of studies (Johnson and Fox, 2002; Zhang et al., 2009a,b,c) have focused on traditional wet milling as a means to recover recombinant proteins and the recombinant proteins have been found to be low in biological activities. The objectives of the present study were to determine the potential for wet-fractionation to recover recombinant collagen expressed in corn germ and to compare quick-germ methods (with water only and with steeping chemicals) and traditional wet milling for the recovery of collagen-related protein.
mated by ELISA. Unlike the hand-dissection procedure, for conservative sampling a small portion of tissue was taken from the center of each tissue without soaking the kernels; for hand-dissection, the entire kernel soaked in water was fractionated into tissue fractions, and, therefore, some minor amounts of contamination between tissues were unavoidable. 2.3. Hand-dissection About 100 g of kernels were steeped overnight at room temperature in 200 mL of deionized water. The tissues of the steeped kernels were hand-dissected to produce relatively pure fractions of germ, endosperm, pericarp, and tip-cap. The fractions were dried at 50 °C for 24 h and the mass yields were determined. The dried samples were finely ground in a coffee grinder and sieved through a 50-mesh sieve (0.30 mm opening) prior to determining moisture, oil, protein, and rCIa1 contents. 2.4. Corn fractionation Transgenic and normal dent corn lines were fractionated by using three methods: (a) the quick-germ process using 0.2% SO2 and 0.5% lactic acid steep solution (quick-germ-SO2–LA), by following the 100 g quick-germ procedure of Singh and Eckhoff (1996); (b) the quick-germ process using only water for steeping (quickgerm-water), similar to the previously described standard quickgerm-SO2–LA method except that the corn was steeped in 300 mL deionized water instead of SO2–LA steep solution; and (c) traditional wet milling (traditional-WM), a 100 g wet-milling procedure described by Vignaux et al. (2006). In the quick-germ processes, 100 g of corn was steeped in 300 mL of steep solution/ water at 59 °C in a water bath for 6 h; where as in traditionalWM process, 100 g of corn was steeped in 300 mL of steep solution at 50 °C in a water bath for 40 h. The steeped corn was further fractionated by following the procedures of Singh and Eckhoff (1996) and Vignaux et al. (2006) for the quick-germ and traditional wetmilling processes, respectively. 2.5. Compositional analysis
2. Methods 2.1. Materials Transgenic corn kernels containing recombinant full-length human collagen type-I-alpha-1 (full-length-rCIa1) and 44-kDa rCIa1fragment of human collagen (44-rCIa1) were provided by ProdiGene Inc. (College Station, TX, USA) and by FibroGen (South San Francisco, CA, USA). The full-length-rCIa1chain was expressed with its telopeptides and a C-terminus folding enhancing peptide. Both full-length-rCIa1 and 44-rCIa1 proteins were targeted for expression in the germ by the embryo-specific maize globulin-1 promoter. The grain was stored at 4 °C until used. The grain samples were hand-cleaned to minimize the effects of foreign material and broken kernels. 2.2. Conservative sampling To estimate the rCIa1 expression levels in the transgenic corn, tissues (germ, endosperm, and tip-cap) were conservatively sampled by using methods of Shepherd et al. (2008b). These samples consisted of a small amount of the pure tissue that was free of contamination from other tissues. The conservatively sampled tissues of transgenic and normal yellow dent (control) corn grains were ground by using a household coffee grinder to pass through a 50mesh sieve (0.30 mm opening) and the rCIa1 contents were esti-
The rCIa1 contents were determined by a competitive ELISA (Enzyme-Linked Immuno Sorbent Assay) as described in Zhang et al. (2009a) for 44-rCIa1 and Zhang et al. (2009b) for fulllength-rCIa1. Rabbit polyclonal antibody (FibroGen, South San Francisco, CA, USA) raised against a Pichia-derived 25-kDa CIa1 was used as a primary antibody. Horseradish peroxidase (HRP)conjugated goat anti-rabbit IgG (Zymed Laboratories, San Francisco, CA, USA) was used as a secondary antibody. Heat-denatured Pichia-derived CIa1 was used to provide the competitive binding sites for the primary antibody and to construct a standard curve. The standard curve was fitted to a four-parameter non-linear logistic equation according to the formula y = (A – B)/[1 - (x/C)D] + B, where A is the maximal absorbance, B is the minimum absorbance, C is the concentration producing 50% of the maximum absorbance, and D is the slope at the inflection point of the sigmoid curve. Protein contents were estimated by using the Dumas nitrogen combustion method with an Elementar Vario MAX CN analyzer (Elementar Analysesysteme GmbH, Hanau, Germany) and the conversion factor 6.25 nitrogen. Moisture contents were determined by using the 130 °C convection oven method 44–19 (AACC, 2000). Whole kernel moisture contents were determined by using the 103 °C convection oven method 44–18 (AACC, 2000). Crude free fat contents were determined by using AACC method 30–25 with the Goldfish apparatus (Labconco Corp., Kansas City, MO, USA) and hexane as solvent. Total starch contents were determined by using the amyloglucosidase/alpha-amylase enzymatic method
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79–13 (AACC, 2000) with an enzyme kit (Megazyme, Wicklow, Ireland). All the analyses were conducted in triplicate and expressed on moisture-free basis. 2.6. Grain physical properties One-hundred randomly selected whole kernels were used to determine 100-kernel weight. Kernel hardness was determined by measuring volume after grinding 20 g of corn in a Glenmills Stenvert Hardness Tester Microhammermill IV (Glenmills Model IV with a 2 mm screen, Clifton, NJ, USA) as described by Pomeranz et al. (1985). Moisture contents of the corn samples were adjusted to 15% by adding water to corn samples in a sealed bag and allowing to equilibrate for 4 h before grinding. The height of column of ground corn immediately after grinding was used as an indicator of kernel hardness with higher values indicating less hardness. 2.7. Statistical analysis Statistical analyses were carried out by using version 9.0.1 of SAS statistical software (SAS Institute Inc., 2003). A General Linear Model procedure was used to analyze the data. The least significant difference (LSD) was determined at the 5% level. 3. Results and discussion 3.1. Transgenic grain characterization Physical and compositional properties of the grain affect the separation of fractions during processing (kernel shape and grain hardness particularly affect grinding mill performance, shape, hardness and protein and crude fat contents may affect water absorption and action of steeping chemicals). The physical and compositional characteristics of the two transgenic corn lines were similar but differed from those of normal yellow dent corn (Table 1). The transgenic kernels were smaller and more irregular in shape than the normal dent corn kernels: 100-kernel weights were 29.1 g for full-length-rCIa1 grain, 28.6 g for 44-rCIa1 grain, and 33.4 g for normal dent grain. The germ crude protein contents of the two transgenic corns were higher (14.3–16.3%) than that of the normal yellow dent corn (12.4%) (data not shown). This was consistent with higher protein contents of whole kernels of the transgenic corn lines, full-length-rCIa1 (12.7%) and 44-rCIa1 (12.1%), compared to the protein content of the normal yellow dent grain (9.4%). Despite the differences in physical properties, crude
Table 1 Compositional and physical properties of transgenic and normal yellow dent corna. Kernel properties
rCIa1 (lg/g) Crude free fat (%) Protein (%) Starch (%) Moisture (%) Hardness (cm) Density (g/cm3) 100-Kernel weight (g)
Full-length-rCIa1 corn grain
44-rCIa1 corn grain
Hi-II cornb
Normal yellow dent corn
3.5 ± 0.4 4.0 ± 0.5 12.7 ± 0.8 61.5 ± 3.4 9.7 ± 0.4 8.6 ± 0.4 – 29.1 ± 1.4
22.8 ± 2.8 3.9 ± 0.5 12.1 ± 1.0 60.9 ± 3.3 10.4 ± 0.6 8.9 ± 0.5 – 28.6 ± 0.7
0.0 3.3 ± 0.6 13.4 ± 0.6 63.4 ± 2.9 9.3 ± 0.4 9.8 ± 0.2 1.30 22.4 ± 0.7
0.0 4.3 ± 0.6 9.4 ± 0.7 62.1 ± 2.6 12.3 ± 0.9 8.4 ± 0.4 1.24 33.4 ± 1.4
a Values are means ± standard deviations of three replicates. The 100-kernel weight, kernel hardness, and density are expressed at 15% moisture content; all other properties are expressed on dry weight basis. b Hi-II corn was the parent variety of the transgenic corn; recombinant protein genes were inserted into Hi-II corn and the Hi-II corn was crossed with a proprietary inbred.
free fat contents and kernel hardness values were similar for the transgenic corns and the dent corn (Table 1). Transgenic corn grains often have harder endosperm due to their high protein contents and the genetic backgrounds used (Shepherd et al., 2008a); however, the hardness values of the full-length- and 44-rCIa1 corn grains did not substantially differ from that of normal yellow dent corn. The parent variety of these transgenic corns, Hi-II corn, had unusual physical and compositional properties, such as smaller round kernels with higher protein content, compared to the transgenic grains and normal yellow dent corn (Table 1). Unexpectedly, the Stenvert hardness values did not correlate well with protein contents or kernel densities of the yellow dent and transgenic grains (normally higher protein content leads to harder endosperm and denser grain). The kernel properties might have segregated with generations causing the properties of transgenic grains to differ from the parent Hi-II kernels. The distribution of the target protein among botanical parts and the efficiency of separating and recovering fractions containing as pure as possible the botanical fraction determine the efficiency and practicality of employing wet-fractionation to enhance recovery of recombinant proteins. Unless the target protein is expressed in high amounts within a specific tissue that can be recovered in high yield and purity, wet-fractionation offers little advantage over extracting whole ground corn. Table 2 shows the mass yields and rCIa1 contents of the germ, endosperm, pericarp, and tip-cap tissues separated by hand-dissection. The germ masses of these transgenic corn lines (8.5–8.7%) were lower than the reported germ mass (10.2–11.9%) of non-transgenic commodity yellow dent corn (Watson, 2003). To determine the actual rCIa1 contents in the kernel tissues, small amounts of pure samples were conservatively taken from the tissues without steeping the kernels. Although the rCIa1was targeted to germ by using an embryo-specific maize globulin-1 promoter, both full-length- and 44-rCIa1 recombinant proteins were found to be expressed in all tissues in the following distribution: 63% in the germ, 34–38% in the endosperm, 1.6–1.8% in the pericarp, and 1% in the tip-cap of hand-dissected transgenic grains (Table 2). Conservatively sampled full-length-rCIa1 germ and endosperm contained 26.7 and 1.4 lg rCIa1/g tissue, respectively; similarly, 44-rCIa1 germ and endosperm contained 182.8 and 10.5 lg rCIa1/g tissue, respectively (Table 3). In both fulllength- and 44-rCIa1 corn lines, the hand-dissected germ contained slightly lower rCIa1 concentrations than those obtained by using the conservative sampling method (Tables 2 and 3), which
Table 2 Mass yields and rCIa1 contents of hand-dissected transgenic corn grainsa. rCIa1 concentration (lg/g tissue)
Total rCIa1 (lg/100 g kernel)
rCIa1 distribution (%)
Full-length-rCIa1 grain Germ 8.7 ± 0.7 Endosperm 84.2 ± 2.3 Pericarp 5.1 ± 0.2 Tip-cap 1.9 ± 0.3 Total 98.9 ± 1.7
24.9 ± 2.5 1.6 ± 0.2 1.2 ± 0.3 1.8 ± 0.5 –
216.4 ± 23.2 131.5 ± 14.1 6.3 ± 1.5 3.5 ± 0.4 357.7 ± 18.0
62.5 ± 6.3 38.0 ± 3.8 1.8 ± 0.4 1.0 ± 0.1 103.3 ± 2.3
44-rCIa1 grain Germ Endosperm Pericarp Tip-cap Total
169.2 ± 18.3 10.9 ± 0.8 7.1 ± 0.5 13.7 ± 4.9 –
1440.5 ± 107.9 903.4 ± 52.8 36.8 ± 4.7 21.1 ± 3.5 2401.8 ± 29.1
63.2 ± 4.8 33.9 ± 2.3 1.6 ± 0.2 0.9 ± 0.1 105.4 ± 4.0
Kernel fraction
Mass yield (%)
8.5 ± 0.4 84.0 ± 1.1 5.2 ± 0.3 1.6 ± 0.3 99.2 ± 0.7
b
a Values are means ± standard deviations of three replicates. Full-length-rCIa1 denotes 100 kDa human collagen type-I-alpha-1. 44-rCIa1 denotes 44-kDa fragment of human collagen type-I-alpha-1. b Calculated based on total rCIa1 expressed in whole kernels (346 lg/100 g grain for full-length-rCIa1 corn; 2279 lg/100 g grain for 44-rCIa1 corn).
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Table 3 Distributions of rCIa1 in transgenic corn grains determined by conservative samplinga. Total rCIa1b (lg/100 g kernel)
rCIa1 distributionc (%)
Full-length-rCIa1 grain Germ 26.7 ± 2.9 Endosperm 1.4 ± 0.1 Tip-cap 0.6 ± 0.2 Whole kernel 3.5 ± 0.4
229.2 ± 18.43 116.8 ± 14.1 1.2 ± 1.1 346.2 ± 38.2
66.4 ± 5.3 33.7 ± 4.1 0.3 ± 0.3 100
44-rCIa1 grain Germ Endosperm Tip-cap Whole kernel
1565.6 ± 165.8 882.2 ± 120.0 18.1 ± 4.7 2278.7 ± 279.0
68.7 ± 10.4 38.7 ± 8.1 0.8 ± 0.3 100
Transgenic kernel tissue
rCIa1 concentration (lg/g tissue)
182.8 ± 15.6 10.5 ± 1.8 11.1 ± 1.2 22.8 ± 2.8
a Values are means ± standard deviations of three replicates. Small amounts of pure samples were conservatively sampled from the tissues without socking the kernels. b Calculated by multiplying rCIa1 concentrations and mass yields determined by hand-dissection (Table 2). c Calculated based on total rCIa1 expressed in whole kernels (346 lg/100 g grain for full-length-rCIa1 corn; 2279 lg/100 g grain for 44-rCIa1 corn).
suggested the possibility of small amounts of protein migrating among tissues when the kernels were hydrated. The data presented in Table 3 (conservative sampling) express the actual amount of recombinant protein present in germ and endosperm tissues and these data were used to calculate the recombinant protein recovery. 3.2. Recovery of rCIa1-enriched germ The yields and compositional properties were compared for the germ fractions of the transgenic and normal yellow dent corns recovered by using the quick-germ-SO2–LA, quick-germ-water, and traditional-WM (Table 4). In all three corn lines, the traditional-WM process recovered germs in higher purity (45.4–48.5% crude free fat content) than using the quick-germ methods. Between the two quick-germ methods, quick-germ-SO2–LA produced germs with higher purities (42.5–46.0% germ crude free fat content) than did the quick-germ-water only process (34.2–38.7% germ crude free fat content). The germ fraction obtained by using the quick-germ-water process contained more non-germ tissues such as pericarp, tip-cap attached to germ (visual observation). SO2 and lactic acid are well known to improve corn grain fractionation (Johnson and May, 2003). Table 4 Germ yields and oil and protein contents of germ recovered by quick-germ and traditional wet-milling methodsa. Corn line/fractionation method
Germ yield (%)
Crude free fat (%)
Crude protein (%)
Full-length-rCIa1 grain Quick-germ-SO2–LA Quick-germ-water Traditional-WM
6.1b 7.9a 6.4b
45.2bc 37.4d 48.5a
16.4a 14.9bc 12.6d
44-rCIa1 grain Quick-germ-SO2–LA Quick-germ-water Traditional-WM
5.8c 7.6b 6.1c
46.2ab 39.1d 45.4b
15.4abc 14.7bc 12.9d
Normal dent corn Quick-germ-SO2–LA Quick-germ-water Traditional-WM
6.8d 8.1a 7.4c
42.5c 38.2d 47.6ab
15.8ab 14.5c 12.6d
a Means in the same column followed by different letters are significantly different (P < 0.05). Normal yellow dent corn was used as a control to compare fractionation methods.
The protein contents were higher in germ fractions recovered by the two quick-germ methods, quick-germ-SO2–LA (15.4– 16.4%) and quick-germ-water (14.1–14.9%) than that of traditional-WM (12.6–13.8%), indicating less loss of soluble protein from the germ than in traditional-WM. The long steeping period of traditional-WM allowed more protein leaching while the short steeping periods of the quick-germ processes enabled more protein to remain in the germ, which should increase recovery of the target recombinant protein in the germ. Data on proteins and other soluble solids found in steepwater discussed in a following section (Section 3.3) confirmed there was leaching loss of protein and other soluble substances into steepwater during the steeping step of wet milling, particularly during the long steeping periods of traditional-WM. Some important physicochemical properties (size, shape, protein content) of the transgenic grains differed from normal dent grain, but these differences did not adversely affect germ recovery in wet-fractionation processes. The germ yields were slightly lower for the transgenic corn lines than that of normal yellow dent corn. For instance, in quick-germ-SO2–LA, the germ recoveries were lower for full-length-rCIa1 (6.1%) and 44-rCIa1 (5.8%) than that of normal yellow dent corn (6.8%) (Table 4); however, these germ recoveries were consistent with the range of recoveries (6–8%) previously reported (Singh et al., 1997; Vignaux et al., 2006). The germ fractions recovered by the quick-germ methods contained higher concentrations of rCIa1 than the germ fractions produced by traditional-WM. In both full-length- and 44-rCIa1 corn germs, the rCIa1 contents decreased in the following order: hand-dissection > quick-germ > traditional-WM (Table 5). This decreasing trend indicated that a significant amount of rCIa1 was leached from the germ during steeping and degerming steps. Of the total full-length-rCIa1 expressed in germ, quick-germ-LA–SO2 recovered 39.9% of rCIa1 in 6.1% of the grain mass whereas traditional-WM recovered only 26.0% of rCIa1 in 6.4% of the grain mass. Quick-germ-water recovered 42.4% of rCIa1 in 7.9% of the grain mass. Similarly, of the total 44-rCIa1 expressed in germ, quickgerm-LA–SO2 recovered more rCIa1 (39.8% rCIa1 in 5.8% grain mass) than traditional-WM (23.7% rCIa1 in 6.1% grain mass). The two quick-germ methods using water or LA–SO2 steeping did not significantly differ in rCIa1 recovery, which indicated that lactic acid or SO2 did not adversely affect the protein structures.
Table 5 Mass yields and rCIa1 contents of germ fractions recovered by conservative sampling and various fractionation methodsa. rCIa1 concentration (lg/g tissue)
rCIa1 recovery in germb (%)
rCIa1 recovery expressed in germc (%)
Full-length-rCIa1 grain Conservative – Hand-dissection 8.7a Quick-germ-SO2–LA 6.1b Quick-germ-water 7.9a Traditional-WM 6.4b
26.7a 24.9a 22.8ab 18.6bc 13.2c
67.0a 62.5a 39.9b 42.4b 26.0c
100.0a 93.3a 59.5b 63.3b 38.9c
44-rCIa1 grain Conservative Hand-dissection Quick-germ-SO2–LA Quick-germ-water Traditional-WM
182.8a 169.2a 155.7b 129.1b 88.3c
68.2a 63.2a 39.8b 42.8b 23.7c
100.0a 92.7a 58.4b 62.7b 34.7c
Corn line/ fractionation method
Germ yield (%)
– 8.5a 5.8c 7.6b 6.1c
a Means within the same corn line and column followed by different letters are significantly different (P < 0.05). b Percentage rCIa1 recovered in germ divided by rCIa1 expressed in whole kernels (3.5 lg/g grain for full-length-rCIa1 corn; 22.8 lg/g grain for 44-rCIa1 corn). c Percentage rCIa1 recovered in germ divided by rCIa1 expressed in germ (26.7 lg/g germ tissue for full-length-rCIa1 corn grain; 182.8 lg/g germ tissue for 44-rCIa1 corn grain) as determined by conservative sampling (Table 3).
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I. Paraman et al. / Bioresource Technology 101 (2010) 239–244 Table 6 Steepwater crude protein, 44-rCIa1, full-length-rCIa1, and total dry matter contents of quick-germ-SO2–LA, quick-germ-water, and traditional-WMa. Corn line/fractionation method
rCIa1 recombinant protein (%)
Crude protein (%)
Total solids (%)
Full-length-rCIa1 grain Quick-germ-SO2–LA Quick-germ-water Traditional-WM
2.3c 2.7c 7.4b
5.0b 3.5c 14.5a
2.3b 1.8c 6.3a
44-rCIa1 grain Quick-germ-SO2–LA Quick-germ-water Traditional-WM
3.8bc 4.0bc 11.7a
4.7b 3.8c 13.6a
2.7b 1.8c 6.9a
a Means in the same column followed by different letters are significantly different (P < 0.05). Each component was expressed as a percentage of their original total respective compounds of the grain.
3.3. Loss of rCIa1 into steepwater The steepwater fractions of quick-germ and traditional-WM processing methods were analyzed for rCIa1, crude protein, and total dry matter contents (Table 6). In all three fractionation methods, rCIa1 and crude protein were observed in steepwater, which confirmed the diffusional loss of protein into steepwater; however, the losses were substantially lower in the quick-germ methods (2.3–3.8%) than in traditional-WM (7.4%) for full-length-rCIa1. A similar trend was observed for 44-rCIa1 as well; however, the loss of 44-rCIa1 in steepwater was higher than for full-length-rCIa1 using all three fractionation methods, likely due to the high solubility of 44-rCIa1 (Tables 6 and 7). Similarly, crude protein and total dry matter contents of quick-germ steepwater were much lower than those of traditional-WM. Between the two quick-germ methods, steepwater of quickgerm-SO2–LA contained slightly higher crude protein and rCIa1 contents than those of quick-germ-water, which indicated SO2– LA assist protein solubilization during steeping (Table 6). Dailey et al. (2000) noted that direct protein solubilization was the main cause for protein leaching but cell wall degradation might be required for leaching to occur. In addition to SO2, lactic acid also directly affects protein release as it helps soften the kernel by promoting water and SO2 absorption. Table 7 shows the overall mass balances and the amount of recombinant protein lost in the fractionation processes. Prolonged steeping in the presence of SO2 as in traditional-WM caused unde-
sirable effects on recombinant proteins. In addition to leaching, a small amount of rCIa1 was lost in these fractionation processes, unaccounted for by mass balance of rCIa1. The loss was greater in traditional-WM (11.7% for full-length-rCIa1 and 14.0% for 44rCIa1) than those of quick-germ-SO2–LA (3.7% for full-lengthrCIa1 and 4.2% for 44-rCIa1) and quick-germ-water (6.5% for full-length-rCIa1 and 1.8% for 44-rCIa1). The reason for this loss was unclear, but it may be due to proteolytic degradation of these proteins during steeping. Although conventional steeping conditions might have enhanced protease activity, Hull et al. (1996) found no protease activity in industrial corn steepwater. 3.4. rCIa1 in traditional-WM fractions In traditional-WM, the degermed fractions were further fractionated into fiber-, starch-, and gluten-rich fractions (data not shown). In both full-length- and 44-rCIa1 grain, the fiber fractions contained considerable amounts of rCIa1 (34.6% for full-lengthrCIa1 and 41.4% for 44-rCIa1); but the starch fraction contained the least amount of the total rCIa1 (0.3% for full-length-rCIa1 and 0.4% for 44-rCIa1). The high retention of rCIa1 in the fiber fraction of traditional-WM may be due to: (i) rCIa1 protein leachedout from germ during steeping and degerming and adsorbed onto fiber; (ii) germ not recovered in the germ-separation process ended up in the fiber-rich fractions; or (iii) incomplete subcellular localization of protein attached to endosperm cell walls ending up with fiber. Zhang et al. (2009a,b,c) indicated that recovering a combined germ-fiber fraction of traditional-WM could enrich to about 65% of the total rCIa1 in 17% of the kernel mass; the germ-fiber fraction contained 4.6-fold higher rCIa1 concentration than the unfractionated kernels (Zhang et al., 2009a,b,c). 3.5. Advantages of grain fractionation The fractionation of corn by using the quick-germ process enriched rCIa1 concentration 5.5–6.5-fold compared to unfractionated whole grain. Such enrichment can substantially improve recovery and purification of recombinant proteins. Compared to traditional-WM, the quick-germ methods offer advantages because of the short steeping period. The quick-germ process produced germ with high purity, equivalent to traditional wet milling, and recovered more rCIa1 in germ fraction than did traditional-WM. The quick-germ process is simpler and may be more cost-effective than the traditional-WM to recover recombinant proteins. Cost-
Table 7 Material balances for mass yields and rCIa1 of corn fractions recovered by quick-germ and traditional wet-milling methods. Methods
a b
Fractions
Full-length-rCIa1 grain
44-rCIa1 corn grain
Fraction mass yielda (%)
rCIa1 distributionb (%)
Fraction mass yielda (%)
rCIa1 distributionb (%)
Quick-germ-SO2–LA
Germ Non-germ Steepwater Total Unaccounted
6.1 ± 0.3 89.5 ± 1.0 2.3 ± 0.2 97.8 ± 0.8 2.2 ± 0.8
39.9 ± 6.4 54.2 ± 3.9 2.3 ± 1.9 96.3 ± 8.8 3.7 ± 8.8
5.8 ± 0.4 89.2 ± 1.6 2.7 ± 0.8 97.7 ± 0.6 2.3 ± 0.6
39.8 ± 2.1 52.1 ± 9.5 3.8 ± 2.5 95.8 ± 9.7 4.2 ± 9.7
Quick-germ-water
Germ Non-germ Steepwater Total Unaccounted
7.9 ± 0.5 88.2 ± 1.5 1.8 ± 0.1 97.9 ± 1.7 2.1 ± 1.7
42.4 ± 4.1 48.4 ± 10.9 2.7 ± 0.7 93.5 ± 6.2 6.5 ± 6.2
7.6 ± 0.5 87.1 ± 1.9 1.8 ± 0.8 96.4 ± 3.0 3.6 ± 3.0
42.8 ± 3.2 55.0 ± 6.2 4.0 ± 2.0 101.8 ± 8.5 -1.8 ± 8.5
Traditional-WM
Germ Non-germ Steepwater Total Unaccounted
6.4 ± 0.2 85.6 ± 1.0 6.3 ± 0.2 98.3 ± 1.1 1.7 ± 1.1
26.0 ± 3.4 54.9 ± 3.8 7.4 ± 3.7 88.3 ± 7.5 11.7 ± 7.5
6.1 ± 0.4 85.8 ± 2.6 6.9 ± 1.5 98.8 ± 2.8 1.2 ± 2.8
23.7 ± 3.2 50.6 ± 4.3 11.7 ± 2.3 86.0 ± 3.8 14.0 ± 3.8
Mass yield of fractions obtained from the total corn. Calculated based on total rCIa1 expressed in whole kernels (346 lg/100 g grain for full-length-rCIa1 corn; 2279 lg/100 g grain for 44-rCIa1 corn).
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effectiveness of any alternative process for producing purified recombinant proteins will depend on recovery, purity and ease of downstream purification. Germ separation at an early stage of processing is beneficial regardless of tissue (germ/endosperm) in which a recombinant protein is expressed. If the target protein is expressed in germ, as in the present study, recovering the germ enriches the protein in a small mass of grain dry matter with less endosperm protein to separate during purification. If the protein is expressed in endosperm, removing germ at an early stage of processing would eliminate oil and water-soluble germ proteins that interfere with downstream processing. Corn grain fractionation enhances the economics of recombinant protein recovery and enables utilizing the protein-lean co-products of transgenic grains in various nonfood applications such as production of fuel ethanol and biorenewable chemicals. 4. Conclusion Quick-germ-SO2–LA, quick-germ-water, and traditional-WM methods were evaluated to recover recombinant rCIa1 that accumulated in corn germ. Quick-germ methods recovered more rCIa1 (40–43%) than traditional-WM (24–26%). The two quick-germ methods showed no differences in total rCIa1 recovery. Use of a short steeping period and wet-mill degermination in quick-germ methods reduced leaching and possibly proteolytic loss of the proteins. The quick-germ processes achieved 43% recovery of rCIa1 in germ comprising 6–8% of the corn mass; the recovered germ contained 6-fold higher rCIa1 concentration than the unfractionated kernels and 1.7-fold higher concentration than traditional wet-milled germs. Although the physicochemical properties (size, shape, protein content) of the transgenic kernels differed from non-transgenic kernels, those differences did not adversely affect germ recovery in wet-fractionation processes. Acknowledgements The authors express their gratitude to the USDA CREES for its financial support and the FibroGen, South San Francisco, CA, USA, for providing transgenic corns and analytical methods for this research. References AACC International, 2000. Approved Methods of the American Association of Cereal Chemists Methods 30–25, 44–18, 44–19, 79–13, 10th ed. The Association, St. Paul, MN.
Dailey Jr., O.D., Dowd, M.K., Mayorga, J.C., 2000. Influence of lactic acid on the solubilization of protein during corn steeping. J. Agric. Food Chem. 48, 1352– 1357. Fox, J.L., 2006. Turning plants into protein factories. Nat. Biotechnol. 24, 1191–1193. Howard, J.A., Hood, E., 2005. Bioindustrial and biopharmaceutical products produced in plants. Adv. Agron. 85, 91–124. Hull, S.R., Yang, B.Y., Venzke, D., Kulhavy, K., Montgomery, R., 1996. Composition of corn steepwater during steeping. J. Agric. Food Chem. 44, 1857–1863. Johnson, L.A., Fox, S.R., 2002. Processing transgenic corn for industrial enzymes. In: Proceedings of Corn Utilization and Technology Conference 2002. Corn Refiners Association, Washington, DC, p. 180. Johnson, L.A., May, J.B., 2003. Wet milling: the basis for corn biorefineries. In: White, P.J., Johnson, L.A. (Eds.), Corn: Chemistry and Technology, second ed. American Association of Cereal Chemists, St. Paul, MN, pp. 449–494. Kusnadi, A.R., Evangelista, R.L., Hood, E.E., Howard, J.A., Nikolov, Z.L., 1998. Processing of transgenic corn seed and its effect on the recovery of recombinant beta-glucuronidase. Biotechnol. Bioeng. 60, 44–52. Ma, J.K., Drake, P.M.W., Christou, P., 2003. The production of recombinant pharmaceutical proteins in plants. Nat. Rev. Genet. 4, 794–805. Menkhaus, T.J., Bai, Y., Zhang, C.M., Nikolov, Z.L., Glatz, C.E., 2004. Considerations for the recovery of recombinant proteins from plants. Biotechnol. Prog. 20, 1001– 1014. Pomeranz, Y., Czuchajowska, Z., Martin, C.R., Lai, F.S., 1985. Determination of corn hardness by the Stenvert hardness tester. Cereal Chem. 62, 108–112. Ramessar, K., Sabalzaa, M., Capella, T., Christou, P., 2008. Maize plants: an ideal production platform for effective and safe molecular pharming. Plant Sci. 174, 409–419. Shepherd, C.T., Vignaux, N., Peterson, J.M., Scott, M.P., Johnson, L.A., 2008a. Drymilling and fractionation of transgenic maize seed tissues with green florescent protein as a tissue marker. Cereal Chem. 85, 196–201. Shepherd, C., Vignaux, N., Peterson, J., Johnson, L., Scott, M., 2008b. Green fluorescent protein as a tissue marker in transgenic maize seed. Cereal Chem. 85, 188–195. Singh, S.K., Johnson, L.A., Pollak, L.M., Fox, S.R., Bailey, T.B., 1997. Comparison of laboratory and pilot-plant corn wet-milling procedures. Cereal Chem. 74, 40– 48. Singh, V., Eckhoff, S.R., 1996. Effect of soak time, soak temperature, and lactic acid on germ recovery parameters. Cereal Chem. 73, 716–720. Vignaux, N., Fox, S.R., Johnson, L.A., 2006. A 10-g laboratory wet-milling procedure for maize and comparison with larger scale laboratory procedures. Cereal Chem. 83, 482–490. Watson, S.A., 2003. Description, development, structure and composition of the corn kernel. In: White, P.J., Johnson, L.A. (Eds.), Corn: Chemistry and Technology, second ed. American Association of Cereal Chemists, St. Paul, MN, p. 82. Yildirim, S., Fuentes, R.G., Evangelista, R., Nikolov, Z.L., 2002. Fractionation of transgenic corn for recovery of recombinant enzymes. J. Am. Oil Chem. Soc. 79, 809–814. Zhang, C., Baez, J., Glatz, C.E., 2009a. Purification and characterization of a 44 kDa recombinant collagen I alpha 1 fragment from corn seed. J. Agric Food Chem. 57, 880–887. Zhang, C., Baez, J., Glatz, C.E., 2009b. Purification and characterization of recombinant collagen type I alpha 1 from transgenic corn seed. Biotechnol. Prog. (in review). Zhang, C., Johnson, L.A., Glatz, C.E., 2009c. Fractionation of transgenic corn seed by dry and wet-milling to recover recombinant collagens. Biotechnol. Prog. (in review). Zhong, Q.X., Gu, Z.R., Glatz, C.E., 2006. Extraction of recombinant dog gastric lipase from transgenic corn seed. J. Agric. Food Chem. 54, 8086–8092.
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Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Recovering corn germ enriched in recombinant protein by wet-fractionation Ilankovan Paraman a, Steven R. Fox a, Matthew T. Aspelund b, Charles E. Glatz b, Lawrence A. Johnson a,* a b
Center for Crops Utilization Research, Department of Food Science and Human Nutrition, Iowa State University, Ames, IA 50011-1061, USA Department of Chemical and Biological Engineering, Iowa State University, Ames, IA 50011-2230, USA
a r t i c l e
i n f o
Article history: Received 23 March 2009 Received in revised form 3 August 2009 Accepted 4 August 2009 Available online 1 September 2009 Keywords: Transgenic corn fractionation Quick-germ processing Wet milling Recombinant collagen-related proteins Corn
a b s t r a c t Corn wet-fractionation processes (quick-germ fractionation and traditional wet milling) were evaluated as means of recovering fractions rich in recombinant collagen-related proteins that were targeted for expression in the germ (embryo) of transgenic corn. Transgenic corn lines accumulating a recombinant full-length human collagen type-I-alpha-1 (full-length rCIa1) or a 44-kDa rCIa1 fragment targeted for seed expression with an embryo-specific promoter were used. Factors to consider in efficient recovery processes are the distribution of the peptides among botanical parts and process recovery efficiency. Both recombinant proteins were distributed 62–64% in germ comprising about 8.6% of the dry grain mass; 34– 38% in the endosperm comprising 84% of the dry grain mass; 1.7% in the pericarp comprising about 5% of the dry mass; and 1% in the tip-cap comprising 1.5–2% of the dry mass. The quick-germ method employed a short steeping period either in water or SO2–lactic acid solution followed by wet-milling degermination to recover a germ-rich fraction. Of the total recombinant protein expressed in germ, the quick-germ process recovered 40–43% of the total recombinant protein within 6–8% of the corn mass. The traditional corn wet-milling process produced higher purity germ but with lower recovery (24– 26%) of the recombinant protein. The two quick-germ methods, using water alone or SO2–lactic acid steeping, did not substantially differ in rCIa1 recovery, and the quick-germ processes recovered germ with less leaching and proteolytic losses of the recombinant proteins than did traditional wet milling. Thus, grain fractionation enriched the recombinant proteins 6-fold higher than that of unfractionated kernels. Such enrichment may improve downstream processing efficiency and enable utilizing the protein-lean co-products to produce biofuels and biorenewable chemicals by fermenting the remaining starch-rich fractions. Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction Transgenic plants as bioreactors to produce pharmaceuticals, vaccines, industrial proteins and enzymes have gained commercial interest due to many practical, economic and safety advantages over the traditional protein production systems such as microbial or cell culture. The production, purification, and commercialization of plant-made recombinant proteins have been reviewed numerous times (Ma et al., 2003; Menkhaus et al., 2004; Howard and Hood, 2005; Fox, 2006; Ramessar et al., 2008). Among the variety of crops considered as potential hosts for recombinant proteins, corn seed is preferred as a bioreactor because of its ability to produce stable recombinant proteins, absence of active endogenous proteases, and being rich in molecular chaperones and disulfide isomerases that facilitate proper protein folding (Ramessar et al., 2008); and protein in the seeds can remain stable for years without
* Corresponding author. Address: 1041 Food Sciences Building, Iowa State University, Ames, IA 50011, USA. Tel.: +1 515 294 4365; fax: +1 515 294 2689. E-mail address: [email protected] (L.A. Johnson). 0960-8524/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2009.08.023
refrigeration, providing practical and economic advantages in storing and processing. Extraction and purification of recombinant protein from plants, however, are difficult processes having low recovery yields and inconsistent end-product qualities. Furthermore, downstream processing consumes 40% or more of the total cost when producing pharmaceutical proteins from corn (Zhong et al., 2006). The normal practice has been to extract the target protein by grinding the whole grain and extracting with buffer; however, large volumes of extraction buffer (e.g. liquid-to-solids ratio of 10) are needed, low concentrations of target protein in the extract are produced, and co-extracted corn proteins complicate purification. Therefore, downstream processing costs are reduced when the protein is concentrated in the material to be extracted and protein-lean starch-rich fractions can be used as feedstocks for the fermentation industry to produce fuel ethanol and industrial chemicals (Johnson and May, 2003). Zhang et al. (2009a,b,c) recovered 60% of the total recombinant collagen-related protein (germ targeted) in 25% of the kernel mass by using dry-milling techniques, and found that such enrichment could reduce at least one step of chromatography in subsequent purification.
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Expression of recombinant proteins can be targeted to different grain tissues such as the germ (embryo) and endosperm. Wet milling is more effective (yield and purity considerations) in recovering germ from corn than dry-milling but care must be taken in selecting processing conditions that are compatible with the proteins of interest because some enzymes and pharmaceutical proteins lose their biological activities when exposed to typical grain-steeping conditions of traditional corn wet milling (Johnson and May, 2003). For instance, exposure to SO2, lactic acid and high temperatures (50–52 °C) for long periods (24–48 h) of traditional corn steeping may denature the target protein or promote endogenous and microbial protease activities (Dailey et al., 2000; Hull et al., 1996). Moreover, 10% of the recombinant collagen-related protein expressed in corn germ was lost by leaching when conventionally steeped for 40 h at 50 °C (Zhang et al., 2009a,b,c). To overcome these limitations, research efforts have focused on reducing steeping time and usage of detrimental chemicals when recovering recombinant proteins by wet-fractionation methods. Johnson and Fox (2002) modified the quick-germ process, a corn fractionation process designed to recover germ in dry-grind ethanol plants (Singh and Eckhoff, 1996), to recover recombinant b-glucuronidase expressed in corn germ. About 85% total grain enzyme activity was recovered in 10% of the corn mass (Johnson and Fox, 2002), much greater than using dry-fractionation methods where about 77% of the total b-glucuronidase was recovered in 20–25% of dry-milled corn mass (Kusnadi et al., 1998); 49% b-glucuronidase was recovered in 17% of corn mass (Yildirim et al., 2002); and 47.7% of total recombinant green fluorescence protein was recovered in 15% of corn mass (Shepherd et al., 2008a). Only a limited number of studies (Johnson and Fox, 2002; Zhang et al., 2009a,b,c) have focused on traditional wet milling as a means to recover recombinant proteins and the recombinant proteins have been found to be low in biological activities. The objectives of the present study were to determine the potential for wet-fractionation to recover recombinant collagen expressed in corn germ and to compare quick-germ methods (with water only and with steeping chemicals) and traditional wet milling for the recovery of collagen-related protein.
mated by ELISA. Unlike the hand-dissection procedure, for conservative sampling a small portion of tissue was taken from the center of each tissue without soaking the kernels; for hand-dissection, the entire kernel soaked in water was fractionated into tissue fractions, and, therefore, some minor amounts of contamination between tissues were unavoidable. 2.3. Hand-dissection About 100 g of kernels were steeped overnight at room temperature in 200 mL of deionized water. The tissues of the steeped kernels were hand-dissected to produce relatively pure fractions of germ, endosperm, pericarp, and tip-cap. The fractions were dried at 50 °C for 24 h and the mass yields were determined. The dried samples were finely ground in a coffee grinder and sieved through a 50-mesh sieve (0.30 mm opening) prior to determining moisture, oil, protein, and rCIa1 contents. 2.4. Corn fractionation Transgenic and normal dent corn lines were fractionated by using three methods: (a) the quick-germ process using 0.2% SO2 and 0.5% lactic acid steep solution (quick-germ-SO2–LA), by following the 100 g quick-germ procedure of Singh and Eckhoff (1996); (b) the quick-germ process using only water for steeping (quickgerm-water), similar to the previously described standard quickgerm-SO2–LA method except that the corn was steeped in 300 mL deionized water instead of SO2–LA steep solution; and (c) traditional wet milling (traditional-WM), a 100 g wet-milling procedure described by Vignaux et al. (2006). In the quick-germ processes, 100 g of corn was steeped in 300 mL of steep solution/ water at 59 °C in a water bath for 6 h; where as in traditionalWM process, 100 g of corn was steeped in 300 mL of steep solution at 50 °C in a water bath for 40 h. The steeped corn was further fractionated by following the procedures of Singh and Eckhoff (1996) and Vignaux et al. (2006) for the quick-germ and traditional wetmilling processes, respectively. 2.5. Compositional analysis
2. Methods 2.1. Materials Transgenic corn kernels containing recombinant full-length human collagen type-I-alpha-1 (full-length-rCIa1) and 44-kDa rCIa1fragment of human collagen (44-rCIa1) were provided by ProdiGene Inc. (College Station, TX, USA) and by FibroGen (South San Francisco, CA, USA). The full-length-rCIa1chain was expressed with its telopeptides and a C-terminus folding enhancing peptide. Both full-length-rCIa1 and 44-rCIa1 proteins were targeted for expression in the germ by the embryo-specific maize globulin-1 promoter. The grain was stored at 4 °C until used. The grain samples were hand-cleaned to minimize the effects of foreign material and broken kernels. 2.2. Conservative sampling To estimate the rCIa1 expression levels in the transgenic corn, tissues (germ, endosperm, and tip-cap) were conservatively sampled by using methods of Shepherd et al. (2008b). These samples consisted of a small amount of the pure tissue that was free of contamination from other tissues. The conservatively sampled tissues of transgenic and normal yellow dent (control) corn grains were ground by using a household coffee grinder to pass through a 50mesh sieve (0.30 mm opening) and the rCIa1 contents were esti-
The rCIa1 contents were determined by a competitive ELISA (Enzyme-Linked Immuno Sorbent Assay) as described in Zhang et al. (2009a) for 44-rCIa1 and Zhang et al. (2009b) for fulllength-rCIa1. Rabbit polyclonal antibody (FibroGen, South San Francisco, CA, USA) raised against a Pichia-derived 25-kDa CIa1 was used as a primary antibody. Horseradish peroxidase (HRP)conjugated goat anti-rabbit IgG (Zymed Laboratories, San Francisco, CA, USA) was used as a secondary antibody. Heat-denatured Pichia-derived CIa1 was used to provide the competitive binding sites for the primary antibody and to construct a standard curve. The standard curve was fitted to a four-parameter non-linear logistic equation according to the formula y = (A – B)/[1 - (x/C)D] + B, where A is the maximal absorbance, B is the minimum absorbance, C is the concentration producing 50% of the maximum absorbance, and D is the slope at the inflection point of the sigmoid curve. Protein contents were estimated by using the Dumas nitrogen combustion method with an Elementar Vario MAX CN analyzer (Elementar Analysesysteme GmbH, Hanau, Germany) and the conversion factor 6.25 nitrogen. Moisture contents were determined by using the 130 °C convection oven method 44–19 (AACC, 2000). Whole kernel moisture contents were determined by using the 103 °C convection oven method 44–18 (AACC, 2000). Crude free fat contents were determined by using AACC method 30–25 with the Goldfish apparatus (Labconco Corp., Kansas City, MO, USA) and hexane as solvent. Total starch contents were determined by using the amyloglucosidase/alpha-amylase enzymatic method
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79–13 (AACC, 2000) with an enzyme kit (Megazyme, Wicklow, Ireland). All the analyses were conducted in triplicate and expressed on moisture-free basis. 2.6. Grain physical properties One-hundred randomly selected whole kernels were used to determine 100-kernel weight. Kernel hardness was determined by measuring volume after grinding 20 g of corn in a Glenmills Stenvert Hardness Tester Microhammermill IV (Glenmills Model IV with a 2 mm screen, Clifton, NJ, USA) as described by Pomeranz et al. (1985). Moisture contents of the corn samples were adjusted to 15% by adding water to corn samples in a sealed bag and allowing to equilibrate for 4 h before grinding. The height of column of ground corn immediately after grinding was used as an indicator of kernel hardness with higher values indicating less hardness. 2.7. Statistical analysis Statistical analyses were carried out by using version 9.0.1 of SAS statistical software (SAS Institute Inc., 2003). A General Linear Model procedure was used to analyze the data. The least significant difference (LSD) was determined at the 5% level. 3. Results and discussion 3.1. Transgenic grain characterization Physical and compositional properties of the grain affect the separation of fractions during processing (kernel shape and grain hardness particularly affect grinding mill performance, shape, hardness and protein and crude fat contents may affect water absorption and action of steeping chemicals). The physical and compositional characteristics of the two transgenic corn lines were similar but differed from those of normal yellow dent corn (Table 1). The transgenic kernels were smaller and more irregular in shape than the normal dent corn kernels: 100-kernel weights were 29.1 g for full-length-rCIa1 grain, 28.6 g for 44-rCIa1 grain, and 33.4 g for normal dent grain. The germ crude protein contents of the two transgenic corns were higher (14.3–16.3%) than that of the normal yellow dent corn (12.4%) (data not shown). This was consistent with higher protein contents of whole kernels of the transgenic corn lines, full-length-rCIa1 (12.7%) and 44-rCIa1 (12.1%), compared to the protein content of the normal yellow dent grain (9.4%). Despite the differences in physical properties, crude
Table 1 Compositional and physical properties of transgenic and normal yellow dent corna. Kernel properties
rCIa1 (lg/g) Crude free fat (%) Protein (%) Starch (%) Moisture (%) Hardness (cm) Density (g/cm3) 100-Kernel weight (g)
Full-length-rCIa1 corn grain
44-rCIa1 corn grain
Hi-II cornb
Normal yellow dent corn
3.5 ± 0.4 4.0 ± 0.5 12.7 ± 0.8 61.5 ± 3.4 9.7 ± 0.4 8.6 ± 0.4 – 29.1 ± 1.4
22.8 ± 2.8 3.9 ± 0.5 12.1 ± 1.0 60.9 ± 3.3 10.4 ± 0.6 8.9 ± 0.5 – 28.6 ± 0.7
0.0 3.3 ± 0.6 13.4 ± 0.6 63.4 ± 2.9 9.3 ± 0.4 9.8 ± 0.2 1.30 22.4 ± 0.7
0.0 4.3 ± 0.6 9.4 ± 0.7 62.1 ± 2.6 12.3 ± 0.9 8.4 ± 0.4 1.24 33.4 ± 1.4
a Values are means ± standard deviations of three replicates. The 100-kernel weight, kernel hardness, and density are expressed at 15% moisture content; all other properties are expressed on dry weight basis. b Hi-II corn was the parent variety of the transgenic corn; recombinant protein genes were inserted into Hi-II corn and the Hi-II corn was crossed with a proprietary inbred.
free fat contents and kernel hardness values were similar for the transgenic corns and the dent corn (Table 1). Transgenic corn grains often have harder endosperm due to their high protein contents and the genetic backgrounds used (Shepherd et al., 2008a); however, the hardness values of the full-length- and 44-rCIa1 corn grains did not substantially differ from that of normal yellow dent corn. The parent variety of these transgenic corns, Hi-II corn, had unusual physical and compositional properties, such as smaller round kernels with higher protein content, compared to the transgenic grains and normal yellow dent corn (Table 1). Unexpectedly, the Stenvert hardness values did not correlate well with protein contents or kernel densities of the yellow dent and transgenic grains (normally higher protein content leads to harder endosperm and denser grain). The kernel properties might have segregated with generations causing the properties of transgenic grains to differ from the parent Hi-II kernels. The distribution of the target protein among botanical parts and the efficiency of separating and recovering fractions containing as pure as possible the botanical fraction determine the efficiency and practicality of employing wet-fractionation to enhance recovery of recombinant proteins. Unless the target protein is expressed in high amounts within a specific tissue that can be recovered in high yield and purity, wet-fractionation offers little advantage over extracting whole ground corn. Table 2 shows the mass yields and rCIa1 contents of the germ, endosperm, pericarp, and tip-cap tissues separated by hand-dissection. The germ masses of these transgenic corn lines (8.5–8.7%) were lower than the reported germ mass (10.2–11.9%) of non-transgenic commodity yellow dent corn (Watson, 2003). To determine the actual rCIa1 contents in the kernel tissues, small amounts of pure samples were conservatively taken from the tissues without steeping the kernels. Although the rCIa1was targeted to germ by using an embryo-specific maize globulin-1 promoter, both full-length- and 44-rCIa1 recombinant proteins were found to be expressed in all tissues in the following distribution: 63% in the germ, 34–38% in the endosperm, 1.6–1.8% in the pericarp, and 1% in the tip-cap of hand-dissected transgenic grains (Table 2). Conservatively sampled full-length-rCIa1 germ and endosperm contained 26.7 and 1.4 lg rCIa1/g tissue, respectively; similarly, 44-rCIa1 germ and endosperm contained 182.8 and 10.5 lg rCIa1/g tissue, respectively (Table 3). In both fulllength- and 44-rCIa1 corn lines, the hand-dissected germ contained slightly lower rCIa1 concentrations than those obtained by using the conservative sampling method (Tables 2 and 3), which
Table 2 Mass yields and rCIa1 contents of hand-dissected transgenic corn grainsa. rCIa1 concentration (lg/g tissue)
Total rCIa1 (lg/100 g kernel)
rCIa1 distribution (%)
Full-length-rCIa1 grain Germ 8.7 ± 0.7 Endosperm 84.2 ± 2.3 Pericarp 5.1 ± 0.2 Tip-cap 1.9 ± 0.3 Total 98.9 ± 1.7
24.9 ± 2.5 1.6 ± 0.2 1.2 ± 0.3 1.8 ± 0.5 –
216.4 ± 23.2 131.5 ± 14.1 6.3 ± 1.5 3.5 ± 0.4 357.7 ± 18.0
62.5 ± 6.3 38.0 ± 3.8 1.8 ± 0.4 1.0 ± 0.1 103.3 ± 2.3
44-rCIa1 grain Germ Endosperm Pericarp Tip-cap Total
169.2 ± 18.3 10.9 ± 0.8 7.1 ± 0.5 13.7 ± 4.9 –
1440.5 ± 107.9 903.4 ± 52.8 36.8 ± 4.7 21.1 ± 3.5 2401.8 ± 29.1
63.2 ± 4.8 33.9 ± 2.3 1.6 ± 0.2 0.9 ± 0.1 105.4 ± 4.0
Kernel fraction
Mass yield (%)
8.5 ± 0.4 84.0 ± 1.1 5.2 ± 0.3 1.6 ± 0.3 99.2 ± 0.7
b
a Values are means ± standard deviations of three replicates. Full-length-rCIa1 denotes 100 kDa human collagen type-I-alpha-1. 44-rCIa1 denotes 44-kDa fragment of human collagen type-I-alpha-1. b Calculated based on total rCIa1 expressed in whole kernels (346 lg/100 g grain for full-length-rCIa1 corn; 2279 lg/100 g grain for 44-rCIa1 corn).
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Table 3 Distributions of rCIa1 in transgenic corn grains determined by conservative samplinga. Total rCIa1b (lg/100 g kernel)
rCIa1 distributionc (%)
Full-length-rCIa1 grain Germ 26.7 ± 2.9 Endosperm 1.4 ± 0.1 Tip-cap 0.6 ± 0.2 Whole kernel 3.5 ± 0.4
229.2 ± 18.43 116.8 ± 14.1 1.2 ± 1.1 346.2 ± 38.2
66.4 ± 5.3 33.7 ± 4.1 0.3 ± 0.3 100
44-rCIa1 grain Germ Endosperm Tip-cap Whole kernel
1565.6 ± 165.8 882.2 ± 120.0 18.1 ± 4.7 2278.7 ± 279.0
68.7 ± 10.4 38.7 ± 8.1 0.8 ± 0.3 100
Transgenic kernel tissue
rCIa1 concentration (lg/g tissue)
182.8 ± 15.6 10.5 ± 1.8 11.1 ± 1.2 22.8 ± 2.8
a Values are means ± standard deviations of three replicates. Small amounts of pure samples were conservatively sampled from the tissues without socking the kernels. b Calculated by multiplying rCIa1 concentrations and mass yields determined by hand-dissection (Table 2). c Calculated based on total rCIa1 expressed in whole kernels (346 lg/100 g grain for full-length-rCIa1 corn; 2279 lg/100 g grain for 44-rCIa1 corn).
suggested the possibility of small amounts of protein migrating among tissues when the kernels were hydrated. The data presented in Table 3 (conservative sampling) express the actual amount of recombinant protein present in germ and endosperm tissues and these data were used to calculate the recombinant protein recovery. 3.2. Recovery of rCIa1-enriched germ The yields and compositional properties were compared for the germ fractions of the transgenic and normal yellow dent corns recovered by using the quick-germ-SO2–LA, quick-germ-water, and traditional-WM (Table 4). In all three corn lines, the traditional-WM process recovered germs in higher purity (45.4–48.5% crude free fat content) than using the quick-germ methods. Between the two quick-germ methods, quick-germ-SO2–LA produced germs with higher purities (42.5–46.0% germ crude free fat content) than did the quick-germ-water only process (34.2–38.7% germ crude free fat content). The germ fraction obtained by using the quick-germ-water process contained more non-germ tissues such as pericarp, tip-cap attached to germ (visual observation). SO2 and lactic acid are well known to improve corn grain fractionation (Johnson and May, 2003). Table 4 Germ yields and oil and protein contents of germ recovered by quick-germ and traditional wet-milling methodsa. Corn line/fractionation method
Germ yield (%)
Crude free fat (%)
Crude protein (%)
Full-length-rCIa1 grain Quick-germ-SO2–LA Quick-germ-water Traditional-WM
6.1b 7.9a 6.4b
45.2bc 37.4d 48.5a
16.4a 14.9bc 12.6d
44-rCIa1 grain Quick-germ-SO2–LA Quick-germ-water Traditional-WM
5.8c 7.6b 6.1c
46.2ab 39.1d 45.4b
15.4abc 14.7bc 12.9d
Normal dent corn Quick-germ-SO2–LA Quick-germ-water Traditional-WM
6.8d 8.1a 7.4c
42.5c 38.2d 47.6ab
15.8ab 14.5c 12.6d
a Means in the same column followed by different letters are significantly different (P < 0.05). Normal yellow dent corn was used as a control to compare fractionation methods.
The protein contents were higher in germ fractions recovered by the two quick-germ methods, quick-germ-SO2–LA (15.4– 16.4%) and quick-germ-water (14.1–14.9%) than that of traditional-WM (12.6–13.8%), indicating less loss of soluble protein from the germ than in traditional-WM. The long steeping period of traditional-WM allowed more protein leaching while the short steeping periods of the quick-germ processes enabled more protein to remain in the germ, which should increase recovery of the target recombinant protein in the germ. Data on proteins and other soluble solids found in steepwater discussed in a following section (Section 3.3) confirmed there was leaching loss of protein and other soluble substances into steepwater during the steeping step of wet milling, particularly during the long steeping periods of traditional-WM. Some important physicochemical properties (size, shape, protein content) of the transgenic grains differed from normal dent grain, but these differences did not adversely affect germ recovery in wet-fractionation processes. The germ yields were slightly lower for the transgenic corn lines than that of normal yellow dent corn. For instance, in quick-germ-SO2–LA, the germ recoveries were lower for full-length-rCIa1 (6.1%) and 44-rCIa1 (5.8%) than that of normal yellow dent corn (6.8%) (Table 4); however, these germ recoveries were consistent with the range of recoveries (6–8%) previously reported (Singh et al., 1997; Vignaux et al., 2006). The germ fractions recovered by the quick-germ methods contained higher concentrations of rCIa1 than the germ fractions produced by traditional-WM. In both full-length- and 44-rCIa1 corn germs, the rCIa1 contents decreased in the following order: hand-dissection > quick-germ > traditional-WM (Table 5). This decreasing trend indicated that a significant amount of rCIa1 was leached from the germ during steeping and degerming steps. Of the total full-length-rCIa1 expressed in germ, quick-germ-LA–SO2 recovered 39.9% of rCIa1 in 6.1% of the grain mass whereas traditional-WM recovered only 26.0% of rCIa1 in 6.4% of the grain mass. Quick-germ-water recovered 42.4% of rCIa1 in 7.9% of the grain mass. Similarly, of the total 44-rCIa1 expressed in germ, quickgerm-LA–SO2 recovered more rCIa1 (39.8% rCIa1 in 5.8% grain mass) than traditional-WM (23.7% rCIa1 in 6.1% grain mass). The two quick-germ methods using water or LA–SO2 steeping did not significantly differ in rCIa1 recovery, which indicated that lactic acid or SO2 did not adversely affect the protein structures.
Table 5 Mass yields and rCIa1 contents of germ fractions recovered by conservative sampling and various fractionation methodsa. rCIa1 concentration (lg/g tissue)
rCIa1 recovery in germb (%)
rCIa1 recovery expressed in germc (%)
Full-length-rCIa1 grain Conservative – Hand-dissection 8.7a Quick-germ-SO2–LA 6.1b Quick-germ-water 7.9a Traditional-WM 6.4b
26.7a 24.9a 22.8ab 18.6bc 13.2c
67.0a 62.5a 39.9b 42.4b 26.0c
100.0a 93.3a 59.5b 63.3b 38.9c
44-rCIa1 grain Conservative Hand-dissection Quick-germ-SO2–LA Quick-germ-water Traditional-WM
182.8a 169.2a 155.7b 129.1b 88.3c
68.2a 63.2a 39.8b 42.8b 23.7c
100.0a 92.7a 58.4b 62.7b 34.7c
Corn line/ fractionation method
Germ yield (%)
– 8.5a 5.8c 7.6b 6.1c
a Means within the same corn line and column followed by different letters are significantly different (P < 0.05). b Percentage rCIa1 recovered in germ divided by rCIa1 expressed in whole kernels (3.5 lg/g grain for full-length-rCIa1 corn; 22.8 lg/g grain for 44-rCIa1 corn). c Percentage rCIa1 recovered in germ divided by rCIa1 expressed in germ (26.7 lg/g germ tissue for full-length-rCIa1 corn grain; 182.8 lg/g germ tissue for 44-rCIa1 corn grain) as determined by conservative sampling (Table 3).
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I. Paraman et al. / Bioresource Technology 101 (2010) 239–244 Table 6 Steepwater crude protein, 44-rCIa1, full-length-rCIa1, and total dry matter contents of quick-germ-SO2–LA, quick-germ-water, and traditional-WMa. Corn line/fractionation method
rCIa1 recombinant protein (%)
Crude protein (%)
Total solids (%)
Full-length-rCIa1 grain Quick-germ-SO2–LA Quick-germ-water Traditional-WM
2.3c 2.7c 7.4b
5.0b 3.5c 14.5a
2.3b 1.8c 6.3a
44-rCIa1 grain Quick-germ-SO2–LA Quick-germ-water Traditional-WM
3.8bc 4.0bc 11.7a
4.7b 3.8c 13.6a
2.7b 1.8c 6.9a
a Means in the same column followed by different letters are significantly different (P < 0.05). Each component was expressed as a percentage of their original total respective compounds of the grain.
3.3. Loss of rCIa1 into steepwater The steepwater fractions of quick-germ and traditional-WM processing methods were analyzed for rCIa1, crude protein, and total dry matter contents (Table 6). In all three fractionation methods, rCIa1 and crude protein were observed in steepwater, which confirmed the diffusional loss of protein into steepwater; however, the losses were substantially lower in the quick-germ methods (2.3–3.8%) than in traditional-WM (7.4%) for full-length-rCIa1. A similar trend was observed for 44-rCIa1 as well; however, the loss of 44-rCIa1 in steepwater was higher than for full-length-rCIa1 using all three fractionation methods, likely due to the high solubility of 44-rCIa1 (Tables 6 and 7). Similarly, crude protein and total dry matter contents of quick-germ steepwater were much lower than those of traditional-WM. Between the two quick-germ methods, steepwater of quickgerm-SO2–LA contained slightly higher crude protein and rCIa1 contents than those of quick-germ-water, which indicated SO2– LA assist protein solubilization during steeping (Table 6). Dailey et al. (2000) noted that direct protein solubilization was the main cause for protein leaching but cell wall degradation might be required for leaching to occur. In addition to SO2, lactic acid also directly affects protein release as it helps soften the kernel by promoting water and SO2 absorption. Table 7 shows the overall mass balances and the amount of recombinant protein lost in the fractionation processes. Prolonged steeping in the presence of SO2 as in traditional-WM caused unde-
sirable effects on recombinant proteins. In addition to leaching, a small amount of rCIa1 was lost in these fractionation processes, unaccounted for by mass balance of rCIa1. The loss was greater in traditional-WM (11.7% for full-length-rCIa1 and 14.0% for 44rCIa1) than those of quick-germ-SO2–LA (3.7% for full-lengthrCIa1 and 4.2% for 44-rCIa1) and quick-germ-water (6.5% for full-length-rCIa1 and 1.8% for 44-rCIa1). The reason for this loss was unclear, but it may be due to proteolytic degradation of these proteins during steeping. Although conventional steeping conditions might have enhanced protease activity, Hull et al. (1996) found no protease activity in industrial corn steepwater. 3.4. rCIa1 in traditional-WM fractions In traditional-WM, the degermed fractions were further fractionated into fiber-, starch-, and gluten-rich fractions (data not shown). In both full-length- and 44-rCIa1 grain, the fiber fractions contained considerable amounts of rCIa1 (34.6% for full-lengthrCIa1 and 41.4% for 44-rCIa1); but the starch fraction contained the least amount of the total rCIa1 (0.3% for full-length-rCIa1 and 0.4% for 44-rCIa1). The high retention of rCIa1 in the fiber fraction of traditional-WM may be due to: (i) rCIa1 protein leachedout from germ during steeping and degerming and adsorbed onto fiber; (ii) germ not recovered in the germ-separation process ended up in the fiber-rich fractions; or (iii) incomplete subcellular localization of protein attached to endosperm cell walls ending up with fiber. Zhang et al. (2009a,b,c) indicated that recovering a combined germ-fiber fraction of traditional-WM could enrich to about 65% of the total rCIa1 in 17% of the kernel mass; the germ-fiber fraction contained 4.6-fold higher rCIa1 concentration than the unfractionated kernels (Zhang et al., 2009a,b,c). 3.5. Advantages of grain fractionation The fractionation of corn by using the quick-germ process enriched rCIa1 concentration 5.5–6.5-fold compared to unfractionated whole grain. Such enrichment can substantially improve recovery and purification of recombinant proteins. Compared to traditional-WM, the quick-germ methods offer advantages because of the short steeping period. The quick-germ process produced germ with high purity, equivalent to traditional wet milling, and recovered more rCIa1 in germ fraction than did traditional-WM. The quick-germ process is simpler and may be more cost-effective than the traditional-WM to recover recombinant proteins. Cost-
Table 7 Material balances for mass yields and rCIa1 of corn fractions recovered by quick-germ and traditional wet-milling methods. Methods
a b
Fractions
Full-length-rCIa1 grain
44-rCIa1 corn grain
Fraction mass yielda (%)
rCIa1 distributionb (%)
Fraction mass yielda (%)
rCIa1 distributionb (%)
Quick-germ-SO2–LA
Germ Non-germ Steepwater Total Unaccounted
6.1 ± 0.3 89.5 ± 1.0 2.3 ± 0.2 97.8 ± 0.8 2.2 ± 0.8
39.9 ± 6.4 54.2 ± 3.9 2.3 ± 1.9 96.3 ± 8.8 3.7 ± 8.8
5.8 ± 0.4 89.2 ± 1.6 2.7 ± 0.8 97.7 ± 0.6 2.3 ± 0.6
39.8 ± 2.1 52.1 ± 9.5 3.8 ± 2.5 95.8 ± 9.7 4.2 ± 9.7
Quick-germ-water
Germ Non-germ Steepwater Total Unaccounted
7.9 ± 0.5 88.2 ± 1.5 1.8 ± 0.1 97.9 ± 1.7 2.1 ± 1.7
42.4 ± 4.1 48.4 ± 10.9 2.7 ± 0.7 93.5 ± 6.2 6.5 ± 6.2
7.6 ± 0.5 87.1 ± 1.9 1.8 ± 0.8 96.4 ± 3.0 3.6 ± 3.0
42.8 ± 3.2 55.0 ± 6.2 4.0 ± 2.0 101.8 ± 8.5 -1.8 ± 8.5
Traditional-WM
Germ Non-germ Steepwater Total Unaccounted
6.4 ± 0.2 85.6 ± 1.0 6.3 ± 0.2 98.3 ± 1.1 1.7 ± 1.1
26.0 ± 3.4 54.9 ± 3.8 7.4 ± 3.7 88.3 ± 7.5 11.7 ± 7.5
6.1 ± 0.4 85.8 ± 2.6 6.9 ± 1.5 98.8 ± 2.8 1.2 ± 2.8
23.7 ± 3.2 50.6 ± 4.3 11.7 ± 2.3 86.0 ± 3.8 14.0 ± 3.8
Mass yield of fractions obtained from the total corn. Calculated based on total rCIa1 expressed in whole kernels (346 lg/100 g grain for full-length-rCIa1 corn; 2279 lg/100 g grain for 44-rCIa1 corn).
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effectiveness of any alternative process for producing purified recombinant proteins will depend on recovery, purity and ease of downstream purification. Germ separation at an early stage of processing is beneficial regardless of tissue (germ/endosperm) in which a recombinant protein is expressed. If the target protein is expressed in germ, as in the present study, recovering the germ enriches the protein in a small mass of grain dry matter with less endosperm protein to separate during purification. If the protein is expressed in endosperm, removing germ at an early stage of processing would eliminate oil and water-soluble germ proteins that interfere with downstream processing. Corn grain fractionation enhances the economics of recombinant protein recovery and enables utilizing the protein-lean co-products of transgenic grains in various nonfood applications such as production of fuel ethanol and biorenewable chemicals. 4. Conclusion Quick-germ-SO2–LA, quick-germ-water, and traditional-WM methods were evaluated to recover recombinant rCIa1 that accumulated in corn germ. Quick-germ methods recovered more rCIa1 (40–43%) than traditional-WM (24–26%). The two quick-germ methods showed no differences in total rCIa1 recovery. Use of a short steeping period and wet-mill degermination in quick-germ methods reduced leaching and possibly proteolytic loss of the proteins. The quick-germ processes achieved 43% recovery of rCIa1 in germ comprising 6–8% of the corn mass; the recovered germ contained 6-fold higher rCIa1 concentration than the unfractionated kernels and 1.7-fold higher concentration than traditional wet-milled germs. Although the physicochemical properties (size, shape, protein content) of the transgenic kernels differed from non-transgenic kernels, those differences did not adversely affect germ recovery in wet-fractionation processes. Acknowledgements The authors express their gratitude to the USDA CREES for its financial support and the FibroGen, South San Francisco, CA, USA, for providing transgenic corns and analytical methods for this research. References AACC International, 2000. Approved Methods of the American Association of Cereal Chemists Methods 30–25, 44–18, 44–19, 79–13, 10th ed. The Association, St. Paul, MN.
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