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1. Introduction
The complete genomes of many organisms including human, mouse, Arabidopsis, and rice have been sequenced. However, the functions of the proteins encoded by a large percentage of the genes in these organisms have not been determined. The immediate challenge of the post-genomic biology is to determine the biological functions of proteins coded for by those unknown genes. Many endogenous proteins occur in extremely low abundance (such as the anti-inflammatory protein tristetraprolin, TTP) (Cao et al., 2004) and are labile (such as omega-3 fatty-acid desaturase, FAD3) (O'Quin et al., 2010), which are major problems inherent to characterization of those proteins.
Recombinant proteins can be used as an alternative source to endogenous proteins. Production of active proteins in large quantities is necessary for the study of protein structure and function (Cao et al., 2003). Purified recombinant proteins are also important for the production of antibodies (Cao 2004; Cao et al., 2008; Cao et al., 2004) and pharmaceutical reagents. Unfortunately, a great number of proteins are difficult to express and purify. Those proteins include membrane proteins, lipid-associated proteins, and low-abundance proteins. The causes of the difficulties in protein expression and purification are various, among which are protein insolubility, protein degradation, and low-level protein expression (Cao 2010). Therefore, production of high-quality recombinant proteins requires optimization of protein expression and purification procedures in each case.
Over-production of DGATs has been the subject of a number of studies, but progress has been slow in the characterization of the enzymes because DGATs are integral membrane proteins (Shockey et al., 2006; Stone et al., 2006) and difficult to express and purify (Cheng et al., 2001; Weselake et al., 2006). Information regarding the expression of DGAT genes in E. coli is limited. The expression of DGAT1 and DGAT2 as full-length proteins in E. coli had not been reported. We recently developed a reliable procedure for the expression and purification of tung DGATs in E. coli (Cao et al., 2010; Cao et al., 2011).
2.1. DGAT genes have been identified in a wide range of organisms
Database search identified at least 115 DGAT sequences from 69 organisms including plants (such as Arabidopsis, barley, caster bean, cauliflower, corn, rape, rice, sorghum, soybean, tobacco, tung tree), animals (such as bird, chimpanzee, cow, dog, fish, fly, frog, monkey, mosquito, mouse, pig, rabbit, rat, sheep, worm), fungi (such as yeast), and human. The names of organisms, the subfamilies of DGATs (DGAT1 and DGAT2) and the GenBank accession numbers are listed in Table 1. Although more than two isoforms of DGATs are found in some species, most of them could be classified into the DGAT1 or DGAT2 subfamily according to their sequence similarities and phylogenetic analysis (data not shown). However, DGAT3 (Saha et al., 2006) and DGAT4 (Rani et al., 2010) were reported recently which have very different sequences with those of DGAT1 and DGAT2. DGAT1 and DGAT2 subfamilies have many conserved residues among the diverse species. However, addition of DGAT3 and DGAT4 from Arabidopsis (GenBank accession number: AAN31909.1), caster bean (GenBank accession number: XP_002519339.1), peanut (GenBank accession number: AY875644.1), and yeast (GenBank accession number: DG315417.1) to the multiple sequence alignment completely destroyed all the conserved residues (data not shown), which is contrary to the general belief that the active sites of the enzymes should have certain degree of conservation during the evolution because all are supposed to catalyze the same/similar biochemical reaction.
No.
Organism
DGAT
GenBank accession number
No.
Organism
DGAT
GenBank accession number
1
Aedes aegypti (A)
1
XP_001658299
59
Medicago truncatula (P)
2
ACJ84867.1
2
Ajellomyces capsulatus (F)
1
EGC41804.1
60
Nicotiana tabacum (P, tobacco)
1
AAF19345.1
3
Anolis carolinensis (A)
2
XP_003225477.1
61
Nematostella vectensis (A, worm)
2a
XP_001630435.1
4
Ashbya gossypii (F)
2
NP_983542.1
62
Nematostella vectensis (A, worm)
2b
XP_001633322.1
5
Arthroderma otae (F)
1
EEQ31683.1
63
Nematostella vectensis (A, worm)
2c
XP_001635548.1
6
Arabidopsis thaliana (P)
1
NP_179535.1
64
Ovis aries (A, sheep)
1
NP_001103634.1
7
Arabidopsis thaliana (P)
2
NP_566952
65
Ovis aries (A, sheep)
2
XP_001518899.1
8
Bubalus bubalis (A, buffalo)
1
AAZ22403.1
66
Oryctolagus cuniculus (A, rabbit)
1
XP_002724427.1
9
Brassica juncea (P)
1a
AAY40784.1
67
Olea europaea (P, tree)
1
AAS01606.1
10
Brassica juncea (P)
1b
AAY40785.1
68
Olea europaea (P, tree)
2
ADG22608.1
11
Brassica napus (P)
1a
AAD45536.1
69
Oryza sativa (P, rice)
1
NP_001054869.2
12
Brassica napus (P)
1b
AAD40881.1
70
Oryza sativa (P, rice)
2a
NP_001047917
13
Brassica napus (P)
2
ACO90187
71
Oryza sativa (P, rice)
2b
NP_001057530
14
Brassica napus (P)
2
ACO90188
72
Ostreococcus tauri (algae)
2
XP_003083539.1
15
Bos taurus (A, cow)
1
NP_777118.2
73
Pongo abelii (A)
2
XP_002822304.1
16
Bos taurus (A, cow)
2a
DAA21853.1
74
Paracoccidioides brasiliensis (F)
1
EEH17170.1
17
Bos taurus (A, cow)
2b
XP_875499.3
75
Perilla frutescens (P)
1
AAG23696.1
18
Bos taurus (A, cow)
2c
XP_002683800.1
76
Polysphondylium pallidum (F)
1
EFA85004.1
19
Caenorhabditis elegans (A, worm)
2a
NP_505413.1
77
Polysphondylium pallidum (F)
2
EFA83646.1
20
Caenorhabditis elegans (A, worm)
2b
NP_872180.1
78
Physcomitrella patens (P, moss)
1
XP_001770929.1
21
Canis familiaris (A, dog)
1b
XP_849176.1
79
Physcomitrella patens (P, moss)
1
XP_001758758.1
22
Canis familiaris (A, dog)
1c
XP_858062.1
80
Physcomitrella patens (P, moss)
2b
XP_001777726.1
23
Capra hircus (A, sheep)
1
ABD59375.1
81
Picea sitchensis (P, tree)
2
ABK26256.1
24
Ciona intestinalis (A)
2
XP_002120879.1
82
Pan troglodytes (A, chimpanzee)
1
XP_520014.2
25
Chlamydomonas reinhardtii (algae)
2a
XP_001694904.1
83
Pan troglodytes (A, chimpanzee)
2
XP_527842.2
26
Chlamydomonas reinhardtii (algae)
2b
XP_001693189.1
84
Phaeodactylum tricornutum (F)
1
XP_002177753.1
27
Chlorella variabilis (algae)
1
EFN50697.1
85
Populus trichocarpa (P, tree)
1a
XP_002308278.1
28
Chlorella variabilis (algae)
2
EFN51306.1
86
Populus trichocarpa (P, tree)
1b
XP_002330510.1
29
Dictyostelium discoideum (mold)
1
XP_645633.2
87
Populus trichocarpa (P, tree)
2
XP_002317635.1
30
Dictyostelium discoideum (mold)
2
XP_635762.1
88
Ricinus communis (P, castor bean)
1
XP_002514132.1
31
Drosophila melanogaster (A, fly)
1a
NP_609813.1
89
Ricinus communis (P, castor bean)
1
XP_002528531.1
32
Drosophila melanogaster (A, fly)
1d
NP_995724.1
90
Rattus norvegicus (A, rat)
1
NP_445889.1
33
Danio rerio (A, zebrafish)
1a
NP_956024.1
91
Rattus norvegicus (A, rat)
2
NP_001012345.1
34
Danio rerio ( A, zebrafish)
1b
NP_001002458.1
92
Sorghum bicolor (P, sorghum)
1a
XP_002437165.1
35
Danio rerio (A, zebrafish)
2
NP_001025367.1
93
Sorghum bicolor (P, sorghum)
1b
XP_002439419.1
36
Euonymus alatus (P)
1
AAV31083.1
94
Sorghum bicolor (P, sorghum)
2
XP_002452652.1
37
Euonymus alatus (P)
2
ADF57328.1
95
Saccharomyces cerevisiae (F, yeast)
2
NP_014888.1
38
Elaeis oleifera (P)
2
ACO35365.1
96
Saccoglossus kowalevskii (A, worm)
1
XP_002736160.1
39
Echium pitardii (P)
1
ACO55635.1
97
Selaginella moellendorffii (P)
1
XP_002964165.1
40
Glycine max (P, soybean)
1a
AAS78662.1
98
Selaginella moellendorffii (P)
2
XP_002972054.1
41
Glycine max (P, soybean)
1b
BAE93461.1
99
Spirodela polyrhiza (P)
2
AAQ89590.1
42
Glycine max (P, soybean)
2
ACU20344.1
100
Schizosaccharomyces pombe (F, yeast)
2
XP_001713160.1
43
Helianthus annuus (P)
2
ABU50328.1
101
Sus scrofa (A, pig)
1
NP_999216.1
44
Homo sapiens (human)
1
NP_036211.2
102
Tribolium castaneum (A)
1
XP_975142.1
45
Homo sapiens (human)
2a
AAQ88896.1
103
Tribolium castaneum (A)
2
XP_975146.1
46
Homo sapiens (human)
2b
NP_835470.1
104
Toxoplasma gondii (A)
1
AAP94209.1
47
Hordeum vulgare (P, barley)
2
BAJ85730.1
105
Taeniopygia guttata (A, bird)
2
XP_002187643.1
48
Ictalurus punctatus (A, catfish)
2b
NP_001188005.1
106
Tropaeolum majus (P)
1
AAM03340.2
49
Jatropha curcas (P)
1
ABB84383.1
107
Vernicia fordii (P, tung tree)
1
DQ356680.1
50
Lotus japonicas (P)
1
AAW51456.1
108
Vernicia fordii (P, tung tree)
2
DQ356682
51
Metarhizium acridum (F)
1a
EFY86774.1
109
Vernonia galamensis (P)
1
ABV21945.1
52
Metarhizium anisopliae (F)
1b
EFY97444.1
110
Vernonia galamensis (P)
2
ACV40232.1
53
Monodelphis domestica (A)
1
XP_001371565.1
111
Vitis vinifera (P, grape)
1
XP_002279345.1
54
Monodelphis domestica (A)
2
XP_001365685.1
112
Vitis vinifera (P, grape)
2
XP_002263626
55
Mus musculus (A, mouse)
1
NP_034176.1
113
Xenopus tropicalis (A, frog)
2
NP_989372.1
56
Mus musculus (A, mouse)
2
NP_080660.1
114
Zea mays (P, corn)
1b
EU039830
57
Macaca mulatta (A, monkey)
1
XP_001090134.1
115
Zea mays (P, corn)
2
NP_001150174.1
58
Medicago truncatula (P)
1
ABN09107.1
Table 1.
DGAT1 and DGAT2 sequence information (DGAT3 and DGAT4 are not included in the Table because of their divergent sequences). A: animal, F: fungus, P: plant.
2.2. Literature survey of DGAT expression
A literature survey was performed to find out how many publications related to DGATs have been collected by the two most popular databases, PubMed and Scopus. The data in Table 2 indicate that approximately 1000 papers had been collected by the two databases during the past 28 years when using DGAT and diacylglycerol acyltransferase as search terms in title/abstracts/keywords. Approximately four times of publications were obtained when using the full name of the enzyme “diacylglycerol acyltransferase” as a search term instead of using the abbreviation “DGAT” in the database search. More than half of the publications were from animals and approximately one quarter of the publications were from plants. Less than half of those publications dealt with expression of DGATs at the RNA and protein levels. Some of the publications reported of using more than one organism in the same paper, resulting in the total number of publications less than the number of publications from plants, animals, and human adding together (Table 2). Similarly, the total expression papers are less than the combination because more than one expression methods were used in the same paper. Approximately 5% of the publications were related to heterologous expression. However, only a few papers were from E. coli expression system.
Database
PubMed
PubMed
Scopus
Scopus
Search terms in title/abstracts/keywords
DGAT
diacylglycerol acyltransferase
DGAT
diacylglycerol acyltransferase
Total publications
216
817
255
1102
Plant
57
118
60
137
Human
74
203
72
316
Animal
138
588
164
760
Total expression papers
90
225
122
322
Plant expression
31
50
34
62
Human expression
31
85
42
131
Animal expression
53
144
78
220
E. coli expression
4
8
1
6
Yeast expression
17
32
17
33
Insect expression
5
12
7
15
Table 2.
Literature survey of publications related to DGAT expression in PubMed and Scopus databases (1982-2010).
2.4. Bioengineering recombinant DGAT for expression in bacteria
We recently described a procedure for over-expression of recombinant full-length DGAT1 and DGAT2 in a bacterial expression system (Cao et al., 2010; Cao et al., 2011). DGAT1 is much larger than DGAT2, although they are similar in other properties and amino acid composition (on % of frequency basis) (Table 3). The two DGAT isoforms have only limited sequence identity and similarity (Figure 1). We were able to express both proteins in E. coli as full-length recombinant proteins. In our study, we engineered a maltose binding protein (MBP) tag at the amino terminus and 6 histidine residues (His-tag) at the carboxyl terminus of full-length tung DGATs (Table 4).
Primer
Sequence (5’ to 3’)
Comments
DGAT1 forward
AATATTGGTACCCTGTTTCAGGGTCCGACAATCCTTGAAACGCCG
KpnI site underlined Codons for PreScission protease site Colored
Primers for PCR-amplification of the full-length DGAT1 and DGAT2 insert sequences.
We engineered plasmids pMBP-DGAT1-His and pMBP-DGAT2-His for expressing the full-length tung tree type 1 and type 2 diacylglycerol acyltransferases (DGAT1 and DGAT2, GenBank Accession No. DQ356680 and DQ356682, respectively (Shockey et al., 2006) as fusion proteins in E. coli. The recombinant proteins contained MBP at the amino terminus and His-tag at the carboxyl terminus. The cloning vector pMBP-hTTP (Figure 2) was
Figure 1.
Alignment of tung tree DGAT1 and DGAT2 amino acid sequences.
reported previously (Cao et al., 2003). Plasmids pMBP-DGAT1-His (Figure 3) and pMBP-DGAT2-His (Figure 4) were constructed by replacing the hTTP fragment in plasmid pMBP-hTTP (Figure 2) with the PCR-amplified DGAT1 and DGAT2 fragments at the KpnI and SpeI sites (Table 4). Existing DGAT plasmid DNAs were used as the templates for PCR-amplification of the DGAT DNA open reading frames (Shockey et al., 2006). DGAT forward primers contained DNA sequence for a KpnI/Asp718I restriction enzyme recognition site followed by a PreScission protease cleavage site (5′-CTGTTTCAGGGTCCG-3′) (Cao et al., 2003) which codes for 5 amino acid residues (LFQGP) between MBP and DGAT protein sequences (Table 4). DGAT reverse primers contained sequence for a His-tag (5′-ATGATGATGATGATGATG-3′) coding for 6 histidine residues at the carboxyl terminus of DGATs (Table 4).
The successful expression of full-length recombinant DGATs was probably due to the fusion to MBP, which was shown to increase the solubility of target proteins such as human and mouse TTP (Cao et al., 2003; Cao et al., 2008; Kapust & Waugh 1999). Although we engineered double affinity tags for facilitating purification of recombinant DGAT from E. coli, recombinant DGATs were only partially purified from the extract by either type of affinity beads [amylose resin and nickel-nitrilotriacetic agarose (Ni-NTA) beads] or both kinds of beads in tandem. Our data, together with the various published reports cited in the previous section, underline the tremendous challenges that exist for the purification of recombinant full-length DGAT proteins.
Figure 2.
Plasmid map of E. coli expression vector pMBP-hTTP.
Figure 3.
Plasmid map of E. coli expression vector pMBP-DGAT1-His.
Diacylglycerol acyltransferases (DGATs) catalyze the last and rate-limiting step of triacylglycerol (TAG) biosynthesis in eukaryotic organisms. At least 115 DGAT sequences are identified from 69 organisms in the GenBank databases. Only a few papers have been published in the last 28 years on the expression of the recombinant DGAT proteins in a bacterial expression system. None of the full-length DGAT1 or DGAT2 had been expressed in E. coli expression system. The difficulties in DGAT expression and purification are due to the nature of these proteins being integral membrane proteins with more than 40% of the total amino acid residues being hydrophobic. Therefore, progress in characterization of the enzymes has been slow. We recently developed a procedure for full-length DGAT expression in E. coli. Expression plasmids were engineered to express tung DGATs fused to maltose binding protein and poly-histidine. The development of the technique should help to purify full-length DGATs for further studies such as raising high-titer antibodies and studying the structure-function relationship. Understanding the roles of DGATs in plant oil
Figure 4.
Plasmid map of E. coli expression vector pMBP-DGAT2-His.
biosynthesis will help to create new oilseed crops with value-added properties. The elucidation of the precise roles of DGATs in animal and human fat synthesis and deposition may provide clues for nutritional and therapeutic intervention in obesity and related diseases.
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Written By
Heping Cao
Submitted: 31 October 2010Published: 01 August 2011