Datasets:
vgainullin
/
xciting_data

Dataset card Data Studio Files
xet
 Community
1
paragraph_index
int64
sec
string
p_has_citation
int64
cites
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pmid
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int64
2
DISCUSSION
1
12
[ "b12", "b12", "b43", "b31" ]
16,914,440
pmid-9050851|pmid-12727889|pmid-15610738|pmid-15610738|pmid-12242451|pmid-9843952
Thus, pol II EC assembled on scaffold having an 8 bp RNA:DNA hybrid appeared to be in the posttranslocated state (12), whereas the 8 bp Tth ECs described here are in the pretranslocated state.
[ "12", "12", "43", "31" ]
192
400
1
false
Thus, pol II EC assembled on scaffold having an 8 bp RNA:DNA hybrid appeared to be in the posttranslocated state, whereas the 8 bp Tth ECs described here are in the pretranslocated state.
[ "12" ]
Thus, pol II EC assembled on scaffold having an 8 bp RNA:DNA hybrid appeared to be in the posttranslocated state, whereas the 8 bp Tth ECs described here are in the pretranslocated state.
true
true
true
true
true
62
2
DISCUSSION
1
12
[ "b12", "b12", "b43", "b31" ]
16,914,440
pmid-9050851|pmid-12727889|pmid-15610738|pmid-15610738|pmid-12242451|pmid-9843952
While this discrepancy might be attributed simply to differences in the nature of the two proteins, it may also reflect differences in scaffold design (especially in view of the high conservation of the RNA:DNA hybrid binding site in eukaryotic and prokaryotic RNAPs).
[ "12", "12", "43", "31" ]
268
401
0
false
While this discrepancy might be attributed simply to differences in the nature of the two proteins, it may also reflect differences in scaffold design (especially in view of the high conservation of the RNA:DNA hybrid binding site in eukaryotic and prokaryotic RNAPs).
[]
While this discrepancy might be attributed simply to differences in the nature of the two proteins, it may also reflect differences in scaffold design (especially in view of the high conservation of the RNA:DNA hybrid binding site in eukaryotic and prokaryotic RNAPs).
true
true
true
true
true
62
2
DISCUSSION
1
12
[ "b12", "b12", "b43", "b31" ]
16,914,440
pmid-9050851|pmid-12727889|pmid-15610738|pmid-15610738|pmid-12242451|pmid-9843952
In contrast to scaffolds employed for Tth ECs, the pol II EC was assembled on a ‘bubble’ scaffold template that contained 11 bp of artificially melted DNA in the region of the RNA:DNA hybrid and 14 bp of upstream DNA duplex (12).
[ "12", "12", "43", "31" ]
229
402
1
false
In contrast to scaffolds employed for Tth ECs, the pol II EC was assembled on a ‘bubble’ scaffold template that contained 11 bp of artificially melted DNA in the region of the RNA:DNA hybrid and 14 bp of upstream DNA duplex.
[ "12" ]
In contrast to scaffolds employed for Tth ECs, the pol II EC was assembled on a ‘bubble’ scaffold template that contained 11 bp of artificially melted DNA in the region of the RNA:DNA hybrid and 14 bp of upstream DNA duplex.
true
true
true
true
true
62
2
DISCUSSION
1
43
[ "b12", "b12", "b43", "b31" ]
16,914,440
pmid-9050851|pmid-12727889|pmid-15610738|pmid-15610738|pmid-12242451|pmid-9843952
As in the previous crystallization studies with T7 RNAP EC (43), a significant portion of the upstream DNA duplex was missing from the electron density in the pol II EC, suggesting limited protein–nucleic acid interactions in that region.
[ "12", "12", "43", "31" ]
238
403
1
false
As in the previous crystallization studies with T7 RNAP EC, a significant portion of the upstream DNA duplex was missing from the electron density in the pol II EC, suggesting limited protein–nucleic acid interactions in that region.
[ "43" ]
As in the previous crystallization studies with T7 RNAP EC, a significant portion of the upstream DNA duplex was missing from the electron density in the pol II EC, suggesting limited protein–nucleic acid interactions in that region.
true
true
true
true
true
62
2
DISCUSSION
1
31
[ "b12", "b12", "b43", "b31" ]
16,914,440
pmid-9050851|pmid-12727889|pmid-15610738|pmid-15610738|pmid-12242451|pmid-9843952
Since the lack of upstream DNA duplex does not compromise the stability of bacterial ECs (31), we used such ‘minimal’ scaffolds for both biochemical and structural experiments.
[ "12", "12", "43", "31" ]
176
404
1
false
Since the lack of upstream DNA duplex does not compromise the stability of bacterial ECs, we used such ‘minimal’ scaffolds for both biochemical and structural experiments.
[ "31" ]
Since the lack of upstream DNA duplex does not compromise the stability of bacterial ECs, we used such ‘minimal’ scaffolds for both biochemical and structural experiments.
true
true
true
true
true
62
3
DISCUSSION
1
12
[ "b12", "b12", "b44", "b45" ]
16,914,440
pmid-15610738|pmid-15610738|pmid-8509420|pmid-8530472
The results of exonuclease footprinting experiments suggest that the tested complexes are mostly in pre (EC14) and posttranslocated (EC15) states.
[ "12", "12", "44", "45" ]
146
405
0
false
The results of exonuclease footprinting experiments suggest that the tested complexes are mostly in pre (EC14) and posttranslocated (EC15) states.
[]
The results of exonuclease footprinting experiments suggest that the tested complexes are mostly in pre and posttranslocated states.
true
true
true
true
true
63
3
DISCUSSION
1
12
[ "b12", "b12", "b44", "b45" ]
16,914,440
pmid-15610738|pmid-15610738|pmid-8509420|pmid-8530472
Intriguingly, we observed GreA stimulated endonuclease activity in a highly stable, posttranslocated EC15 (Figure 4A and B).
[ "12", "12", "44", "45" ]
124
406
0
false
Intriguingly, we observed GreA stimulated endonuclease activity in a highly stable, posttranslocated EC15 (Figure 4A and B).
[]
Intriguingly, we observed GreA stimulated endonuclease activity in a highly stable, posttranslocated EC15 (Figure 4A and B).
true
true
true
true
true
63
3
DISCUSSION
1
12
[ "b12", "b12", "b44", "b45" ]
16,914,440
pmid-15610738|pmid-15610738|pmid-8509420|pmid-8530472
Our data indicate that GreA-induced RNAP nuclease activity cleavage produced exclusively di-nucleotide products (Figure 4A).
[ "12", "12", "44", "45" ]
124
407
0
false
Our data indicate that GreA-induced RNAP nuclease activity cleavage produced exclusively di-nucleotide products (Figure 4A).
[]
Our data indicate that GreA-induced RNAP nuclease activity cleavage produced exclusively di-nucleotide products (Figure 4A).
true
true
true
true
true
63
3
DISCUSSION
1
12
[ "b12", "b12", "b44", "b45" ]
16,914,440
pmid-15610738|pmid-15610738|pmid-8509420|pmid-8530472
It is unclear, however, why during progressive cleavage the EC would backtrack by exactly 2 nt.
[ "12", "12", "44", "45" ]
95
408
0
false
It is unclear, however, why during progressive cleavage the EC would backtrack by exactly 2 nt.
[]
It is unclear, however, why during progressive cleavage the EC would backtrack by exactly 2 nt.
true
true
true
true
true
63
3
DISCUSSION
1
12
[ "b12", "b12", "b44", "b45" ]
16,914,440
pmid-15610738|pmid-15610738|pmid-8509420|pmid-8530472
It is tempting to speculate that GreA can shift the equilibrium towards backtracked conformation, e.g.
[ "12", "12", "44", "45" ]
102
409
0
false
It is tempting to speculate that GreA can shift the equilibrium towards backtracked conformation, e.g.
[]
It is tempting to speculate that GreA can shift the equilibrium towards backtracked conformation, e.g.
true
true
true
true
true
63
3
DISCUSSION
1
12
[ "b12", "b12", "b44", "b45" ]
16,914,440
pmid-15610738|pmid-15610738|pmid-8509420|pmid-8530472
by inducing large conformational changes in the RNAP.
[ "12", "12", "44", "45" ]
53
410
0
false
by inducing large conformational changes in the RNAP.
[]
by inducing large conformational changes in the RNAP.
false
true
true
true
false
63
3
DISCUSSION
1
12
[ "b12", "b12", "b44", "b45" ]
16,914,440
pmid-15610738|pmid-15610738|pmid-8509420|pmid-8530472
Such changes were observed in the case of a functional homolog of GreA, TFIIS, as revealed by the crystal structure of this transcription factor in complex with pol II EC (12).
[ "12", "12", "44", "45" ]
176
411
1
false
Such changes were observed in the case of a functional homolog of GreA, TFIIS, as revealed by the crystal structure of this transcription factor in complex with pol II EC.
[ "12" ]
Such changes were observed in the case of a functional homolog of GreA, TFIIS, as revealed by the crystal structure of this transcription factor in complex with pol II EC.
true
true
true
true
true
63
3
DISCUSSION
1
12
[ "b12", "b12", "b44", "b45" ]
16,914,440
pmid-15610738|pmid-15610738|pmid-8509420|pmid-8530472
In that case, binding of TFIIS resulted in conformational changes in RNAP that led to realignment of the RNA in the active center and stabilization of an alternative conformation of the EC (12).
[ "12", "12", "44", "45" ]
194
412
1
false
In that case, binding of TFIIS resulted in conformational changes in RNAP that led to realignment of the RNA in the active center and stabilization of an alternative conformation of the EC.
[ "12" ]
In that case, binding of TFIIS resulted in conformational changes in RNAP that led to realignment of the RNA in the active center and stabilization of an alternative conformation of the EC.
true
true
true
true
true
63
3
DISCUSSION
1
12
[ "b12", "b12", "b44", "b45" ]
16,914,440
pmid-15610738|pmid-15610738|pmid-8509420|pmid-8530472
Interestingly, dinucleotides are also the predominant products of TFIIS-stimulated cleavage (44,45).
[ "12", "12", "44", "45" ]
100
413
0
false
Interestingly, dinucleotides are also the predominant products of TFIIS-stimulated cleavage.
[ "44,45" ]
Interestingly, dinucleotides are also the predominant products of TFIIS-stimulated cleavage.
true
true
true
true
true
63
3
DISCUSSION
1
12
[ "b12", "b12", "b44", "b45" ]
16,914,440
pmid-15610738|pmid-15610738|pmid-8509420|pmid-8530472
An alternative, more parsimonious explanation of sensitivity of EC15 to GreA is that it may serve as a more powerful (and thus more revealing) probe, compared to PPi, in monitoring oscillations of the EC between pre and posttranslocated conformations.
[ "12", "12", "44", "45" ]
251
414
0
false
An alternative, more parsimonious explanation of sensitivity of EC15 to GreA is that it may serve as a more powerful (and thus more revealing) probe, compared to PPi, in monitoring oscillations of the EC between pre and posttranslocated conformations.
[]
An alternative, more parsimonious explanation of sensitivity of EC15 to GreA is that it may serve as a more powerful (and thus more revealing) probe, compared to PPi, in monitoring oscillations of the EC between pre and posttranslocated conformations.
true
true
true
true
true
63
4
DISCUSSION
1
33
[ "b33", "b41" ]
16,914,440
pmid-16049026|pmid-10860741
Interestingly, in contrast to E.coli RNAP (33), we did not find significant intrinsic cleavage activity in most of the Tth RNAP scaffold ECs and promoter complexes (though we tested only a limited number of such complexes).
[ "33", "41" ]
223
415
1
false
Interestingly, in contrast to E.coli RNAP, we did not find significant intrinsic cleavage activity in most of the Tth RNAP scaffold ECs and promoter complexes (though we tested only a limited number of such complexes).
[ "33" ]
Interestingly, in contrast to E.coli RNAP, we did not find significant intrinsic cleavage activity in most of the Tth RNAP scaffold ECs and promoter complexes (though we tested only a limited number of such complexes).
true
true
true
true
true
64
4
DISCUSSION
1
33
[ "b33", "b41" ]
16,914,440
pmid-16049026|pmid-10860741
However, when the length of the RNA:DNA hybrid in the EC was increased to 10–11 bp we observed efficient endonucleolytic activity (Figure 3).
[ "33", "41" ]
141
416
0
false
However, when the length of the RNA:DNA hybrid in the EC was increased to 10–11 bp we observed efficient endonucleolytic activity (Figure 3).
[]
However, when the length of the RNA:DNA hybrid in the EC was increased to 10–11 bp we observed efficient endonucleolytic activity.
true
true
true
true
true
64
4
DISCUSSION
1
41
[ "b33", "b41" ]
16,914,440
pmid-16049026|pmid-10860741
It is likely that complexes assembled on scaffolds lacking the NT strand in the region of the transcription bubble, (such as EC16 and EC17, Figure 3) cannot efficiently displace the 5′ end of the transcript (41).
[ "33", "41" ]
212
417
1
false
It is likely that complexes assembled on scaffolds lacking the NT strand in the region of the transcription bubble, (such as EC16 and EC17, Figure 3) cannot efficiently displace the 5′ end of the transcript.
[ "41" ]
It is likely that complexes assembled on scaffolds lacking the NT strand in the region of the transcription bubble, cannot efficiently displace the 5′ end of the transcript.
true
true
true
true
true
64
4
DISCUSSION
1
33
[ "b33", "b41" ]
16,914,440
pmid-16049026|pmid-10860741
Thus the formation of the overextended RNA:DNA hybrid results in a complex that is prone to endonucleolytic cleavage (Figure 3C).
[ "33", "41" ]
129
418
0
false
Thus the formation of the overextended RNA:DNA hybrid results in a complex that is prone to endonucleolytic cleavage (Figure 3C).
[]
Thus the formation of the overextended RNA:DNA hybrid results in a complex that is prone to endonucleolytic cleavage.
true
true
true
true
true
64
5
DISCUSSION
1
21
[ "b21", "b28", "b11", "b9", "b11", "b13", "b43", "b46", "b25", "b47" ]
16,914,440
pmid-9094712|pmid-9702191|pmid-15016374|pmid-12422209|pmid-15016374|pmid-15016373|pmid-12242451|pmid-10600732|pmid-12351656|pmid-11095736
All together, the results of biochemical assays indicate that ECs assembled on nucleic acid scaffolds with different structures (topology) possess quite distinct conformations.
[ "21", "28", "11", "9", "11", "13", "43", "46", "25", "47" ]
176
419
0
false
All together, the results of biochemical assays indicate that ECs assembled on nucleic acid scaffolds with different structures (topology) possess quite distinct conformations.
[]
All together, the results of biochemical assays indicate that ECs assembled on nucleic acid scaffolds with different structures (topology) possess quite distinct conformations.
true
true
true
true
true
65
5
DISCUSSION
1
21
[ "b21", "b28", "b11", "b9", "b11", "b13", "b43", "b46", "b25", "b47" ]
16,914,440
pmid-9094712|pmid-9702191|pmid-15016374|pmid-12422209|pmid-15016374|pmid-15016373|pmid-12242451|pmid-10600732|pmid-12351656|pmid-11095736
In the absence of NTPs, one conformation is greatly stabilized and preferred over another.
[ "21", "28", "11", "9", "11", "13", "43", "46", "25", "47" ]
90
420
0
false
In the absence of NTPs, one conformation is greatly stabilized and preferred over another.
[]
In the absence of NTPs, one conformation is greatly stabilized and preferred over another.
true
true
true
true
true
65
5
DISCUSSION
1
21
[ "b21", "b28", "b11", "b9", "b11", "b13", "b43", "b46", "b25", "b47" ]
16,914,440
pmid-9094712|pmid-9702191|pmid-15016374|pmid-12422209|pmid-15016374|pmid-15016373|pmid-12242451|pmid-10600732|pmid-12351656|pmid-11095736
Our data suggest that the length of the RNA:DNA hybrid plays a primary role not only in stabilization of EC (21,28) but also shifting the equilibrium between pre and posttranslocated conformations.
[ "21", "28", "11", "9", "11", "13", "43", "46", "25", "47" ]
197
421
0
false
Our data suggest that the length of the RNA:DNA hybrid plays a primary role not only in stabilization of EC but also shifting the equilibrium between pre and posttranslocated conformations.
[ "21,28" ]
Our data suggest that the length of the RNA:DNA hybrid plays a primary role not only in stabilization of EC but also shifting the equilibrium between pre and posttranslocated conformations.
true
true
true
true
true
65
5
DISCUSSION
1
21
[ "b21", "b28", "b11", "b9", "b11", "b13", "b43", "b46", "b25", "b47" ]
16,914,440
pmid-9094712|pmid-9702191|pmid-15016374|pmid-12422209|pmid-15016374|pmid-15016373|pmid-12242451|pmid-10600732|pmid-12351656|pmid-11095736
Interestingly, the pretranslocated state of the EC (as observed in EC14) appears to be stable and preferred even in the absence of NTP, since very distinct patterns of footprinting and cross-links were observed (Figure 2).
[ "21", "28", "11", "9", "11", "13", "43", "46", "25", "47" ]
222
422
0
false
Interestingly, the pretranslocated state of the EC (as observed in EC14) appears to be stable and preferred even in the absence of NTP, since very distinct patterns of footprinting and cross-links were observed (Figure 2).
[]
Interestingly, the pretranslocated state of the EC appears to be stable and preferred even in the absence of NTP, since very distinct patterns of footprinting and cross-links were observed.
true
true
true
true
true
65
5
DISCUSSION
1
11
[ "b21", "b28", "b11", "b9", "b11", "b13", "b43", "b46", "b25", "b47" ]
16,914,440
pmid-9094712|pmid-9702191|pmid-15016374|pmid-12422209|pmid-15016374|pmid-15016373|pmid-12242451|pmid-10600732|pmid-12351656|pmid-11095736
This observation argues against the power stroke mechanism of transcription elongation, which suggests that PPi release is required for the transition from a pre to a posttranslocated state in multi-subunit RNAPs (11).
[ "21", "28", "11", "9", "11", "13", "43", "46", "25", "47" ]
218
423
1
false
This observation argues against the power stroke mechanism of transcription elongation, which suggests that PPi release is required for the transition from a pre to a posttranslocated state in multi-subunit RNAPs.
[ "11" ]
This observation argues against the power stroke mechanism of transcription elongation, which suggests that PPi release is required for the transition from a pre to a posttranslocated state in multi-subunit RNAPs.
true
true
true
true
true
65
5
DISCUSSION
1
21
[ "b21", "b28", "b11", "b9", "b11", "b13", "b43", "b46", "b25", "b47" ]
16,914,440
pmid-9094712|pmid-9702191|pmid-15016374|pmid-12422209|pmid-15016374|pmid-15016373|pmid-12242451|pmid-10600732|pmid-12351656|pmid-11095736
However, in contrast to bacterial ECs (which can assume different translocation conformations), halted T7 RNAP ECs appear to exist preferentially in a posttranslocated state (9,11,13,43,46).
[ "21", "28", "11", "9", "11", "13", "43", "46", "25", "47" ]
190
424
0
false
However, in contrast to bacterial ECs (which can assume different translocation conformations), halted T7 RNAP ECs appear to exist preferentially in a posttranslocated state.
[ "9,11,13,43,46" ]
However, in contrast to bacterial ECs (which can assume different translocation conformations), halted T7 RNAP ECs appear to exist preferentially in a posttranslocated state.
true
true
true
true
true
65
5
DISCUSSION
1
21
[ "b21", "b28", "b11", "b9", "b11", "b13", "b43", "b46", "b25", "b47" ]
16,914,440
pmid-9094712|pmid-9702191|pmid-15016374|pmid-12422209|pmid-15016374|pmid-15016373|pmid-12242451|pmid-10600732|pmid-12351656|pmid-11095736
Moreover, ECs of T7 RNAP assembled on scaffolds similar or identical to those used in the current work are highly resistant to pyrophosphorolysis (25,47).
[ "21", "28", "11", "9", "11", "13", "43", "46", "25", "47" ]
154
425
0
false
Moreover, ECs of T7 RNAP assembled on scaffolds similar or identical to those used in the current work are highly resistant to pyrophosphorolysis.
[ "25,47" ]
Moreover, ECs of T7 RNAP assembled on scaffolds similar or identical to those used in the current work are highly resistant to pyrophosphorolysis.
true
true
true
true
true
65
5
DISCUSSION
1
21
[ "b21", "b28", "b11", "b9", "b11", "b13", "b43", "b46", "b25", "b47" ]
16,914,440
pmid-9094712|pmid-9702191|pmid-15016374|pmid-12422209|pmid-15016374|pmid-15016373|pmid-12242451|pmid-10600732|pmid-12351656|pmid-11095736
Hence, it is possible that the single subunit T7-like RNAPs and bacterial RNAPs utilize different mechanisms of translocation.
[ "21", "28", "11", "9", "11", "13", "43", "46", "25", "47" ]
126
426
0
false
Hence, it is possible that the single subunit T7-like RNAPs and bacterial RNAPs utilize different mechanisms of translocation.
[]
Hence, it is possible that the single subunit T7-like RNAPs and bacterial RNAPs utilize different mechanisms of translocation.
true
true
true
true
true
65
6
DISCUSSION
0
null
null
16,914,440
null
The results of biochemical experiments suggesting that Tth RNAP ECs possess distinct conformations are in a good agreement with finding that pretranslocated EC14 and posttranslocated EC15 crystallized under different conditions.
null
228
427
0
false
null
null
The results of biochemical experiments suggesting that Tth RNAP ECs possess distinct conformations are in a good agreement with finding that pretranslocated EC14 and posttranslocated EC15 crystallized under different conditions.
true
true
true
true
true
66
6
DISCUSSION
0
null
null
16,914,440
null
Since scaffold components of EC are buried inside of the polymerase, they are unlikely to affect contacts in the crystal lattice.
null
129
428
0
false
null
null
Since scaffold components of EC are buried inside of the polymerase, they are unlikely to affect contacts in the crystal lattice.
true
true
true
true
true
66
6
DISCUSSION
0
null
null
16,914,440
null
Instead, yet to be identified conformational changes in the RNAP itself are likely to result in packing of the molecules into distinct crystal forms.
null
149
429
0
false
null
null
Instead, yet to be identified conformational changes in the RNAP itself are likely to result in packing of the molecules into distinct crystal forms.
true
true
true
true
true
66
6
DISCUSSION
0
null
null
16,914,440
null
Thus, it will be possible to use scaffold ECs to monitor different intermediates of transcription complexes of bacterial polymerases during the nucleotide addition cycle by structural analysis.
null
193
430
0
false
null
null
Thus, it will be possible to use scaffold ECs to monitor different intermediates of transcription complexes of bacterial polymerases during the nucleotide addition cycle by structural analysis.
true
true
true
true
true
66
6
DISCUSSION
0
null
null
16,914,440
null
Model building of the posttranslocated EC15 and refinement of the structure are under way.
null
90
431
0
false
null
null
Model building of the posttranslocated EC15 and refinement of the structure are under way.
true
true
true
true
true
66
0
INTRODUCTION
0
null
null
17,317,681
pmid-16289702|pmid-11075367|pmid-15750721|pmid-16211504
The Oxford Protein Production Facility (OPPF) is a structural proteomics facility funded to produce high quality structural data for proteins from a diverse range of host organisms, including viruses, bacterial human pathogens and human proteins associated with the aetiology of human diseases such as cancers.
null
310
432
0
false
null
null
The Oxford Protein Production Facility (OPPF) is a structural proteomics facility funded to produce high quality structural data for proteins from a diverse range of host organisms, including viruses, bacterial human pathogens and human proteins associated with the aetiology of human diseases such as cancers.
true
true
true
true
true
67
0
INTRODUCTION
0
null
null
17,317,681
pmid-16289702|pmid-11075367|pmid-15750721|pmid-16211504
As such, the OPPF target list contains many proteins that may be viewed as problematic to express and crystallize.
null
114
433
0
false
null
null
As such, the OPPF target list contains many proteins that may be viewed as problematic to express and crystallize.
true
true
true
true
true
67
0
INTRODUCTION
0
null
null
17,317,681
pmid-16289702|pmid-11075367|pmid-15750721|pmid-16211504
In order to investigate these proteins either in large numbers or to investigate, in parallel, many multiple domains of smaller numbers of these proteins, a highly efficient cloning and expression screening strategy was required.
null
229
434
0
false
null
null
In order to investigate these proteins either in large numbers or to investigate, in parallel, many multiple domains of smaller numbers of these proteins, a highly efficient cloning and expression screening strategy was required.
true
true
true
true
true
67
1
INTRODUCTION
0
null
null
17,317,681
pmid-15177282|pmid-15284479|pmid-17001101|pmid-11788735|pmid-15803471|pmid-16211514
The prime characteristics of this process must be: The ability to clone genes encoding proteins, or domains thereof, in a rapid and reliable parallel or high-throughput (HTP) fashion.
null
183
435
0
false
null
null
The prime characteristics of this process must be: The ability to clone genes encoding proteins, or domains thereof, in a rapid and reliable parallel or high-throughput (HTP) fashion.
true
true
true
true
true
68
1
INTRODUCTION
0
null
null
17,317,681
pmid-15177282|pmid-15284479|pmid-17001101|pmid-11788735|pmid-15803471|pmid-16211514
The ability to accurately determine the final constructs without the addition of extraneous/vector or restriction-site-derived amino acids to the expressed protein.
null
164
436
0
false
null
null
The ability to accurately determine the final constructs without the addition of extraneous/vector or restriction-site-derived amino acids to the expressed protein.
true
true
true
true
true
68
1
INTRODUCTION
0
null
null
17,317,681
pmid-15177282|pmid-15284479|pmid-17001101|pmid-11788735|pmid-15803471|pmid-16211514
The process must be versatile in terms of insert sequence independence.
null
71
437
0
false
null
null
The process must be versatile in terms of insert sequence independence.
true
true
true
true
true
68
1
INTRODUCTION
0
null
null
17,317,681
pmid-15177282|pmid-15284479|pmid-17001101|pmid-11788735|pmid-15803471|pmid-16211514
The process would preferably be single step, to give rapid and cost-effective vector construction.
null
98
438
0
false
null
null
The process would preferably be single step, to give rapid and cost-effective vector construction.
true
true
true
true
true
68
1
INTRODUCTION
0
null
null
17,317,681
pmid-15177282|pmid-15284479|pmid-17001101|pmid-11788735|pmid-15803471|pmid-16211514
The constructs should be suitable for expressing proteins from multiple hosts, i.e.
null
83
439
0
false
null
null
The constructs should be suitable for expressing proteins from multiple hosts, i.e.
true
true
true
true
true
68
1
INTRODUCTION
0
null
null
17,317,681
pmid-15177282|pmid-15284479|pmid-17001101|pmid-11788735|pmid-15803471|pmid-16211514
a single vector capable of expression in E. coli, mammalian cell lines (e.g.
null
76
440
0
false
null
null
a single vector capable of expression in E. coli, mammalian cell lines (e.g.
false
true
true
true
false
68
1
INTRODUCTION
0
null
null
17,317,681
pmid-15177282|pmid-15284479|pmid-17001101|pmid-11788735|pmid-15803471|pmid-16211514
HEK293T cells) and insect cell lines (e.g.
null
42
441
0
false
null
null
HEK293T cells) and insect cell lines (e.g.
true
true
true
true
true
68
1
INTRODUCTION
0
null
null
17,317,681
pmid-15177282|pmid-15284479|pmid-17001101|pmid-11788735|pmid-15803471|pmid-16211514
Sf9 cells).The expressed proteins must be capable of purification in HTP mode, i.e.
null
83
442
0
false
null
null
Sf9 cells).The expressed proteins must be capable of purification in HTP mode, i.e.
true
true
true
true
true
68
1
INTRODUCTION
0
null
null
17,317,681
pmid-15177282|pmid-15284479|pmid-17001101|pmid-11788735|pmid-15803471|pmid-16211514
they must all be fused to a common affinity purification ‘tag’ which may be removed, if desired, by enzymatic digest prior to crystallization.
null
142
443
0
false
null
null
they must all be fused to a common affinity purification ‘tag’ which may be removed, if desired, by enzymatic digest prior to crystallization.
false
true
true
true
false
68
1
INTRODUCTION
0
null
null
17,317,681
pmid-15177282|pmid-15284479|pmid-17001101|pmid-11788735|pmid-15803471|pmid-16211514
The process should be amenable to automation.
null
45
444
0
false
null
null
The process should be amenable to automation.
true
true
true
true
true
68
2
INTRODUCTION
0
null
null
17,317,681
null
The ability to clone genes encoding proteins, or domains thereof, in a rapid and reliable parallel or high-throughput (HTP) fashion.
null
132
445
0
false
null
null
The ability to clone genes encoding proteins, or domains thereof, in a rapid and reliable parallel or high-throughput (HTP) fashion.
true
true
true
true
true
69
3
INTRODUCTION
0
null
null
17,317,681
null
The ability to accurately determine the final constructs without the addition of extraneous/vector or restriction-site-derived amino acids to the expressed protein.
null
164
446
0
false
null
null
The ability to accurately determine the final constructs without the addition of extraneous/vector or restriction-site-derived amino acids to the expressed protein.
true
true
true
true
true
70
4
INTRODUCTION
0
null
null
17,317,681
null
The process must be versatile in terms of insert sequence independence.
null
71
447
0
false
null
null
The process must be versatile in terms of insert sequence independence.
true
true
true
true
true
71
5
INTRODUCTION
0
null
null
17,317,681
null
The process would preferably be single step, to give rapid and cost-effective vector construction.
null
98
448
0
false
null
null
The process would preferably be single step, to give rapid and cost-effective vector construction.
true
true
true
true
true
72
6
INTRODUCTION
0
null
null
17,317,681
null
The constructs should be suitable for expressing proteins from multiple hosts, i.e.
null
83
449
0
false
null
null
The constructs should be suitable for expressing proteins from multiple hosts, i.e.
true
true
true
true
true
73
6
INTRODUCTION
0
null
null
17,317,681
null
a single vector capable of expression in E. coli, mammalian cell lines (e.g.
null
76
450
0
false
null
null
a single vector capable of expression in E. coli, mammalian cell lines (e.g.
false
true
true
true
false
73
6
INTRODUCTION
0
null
null
17,317,681
null
HEK293T cells) and insect cell lines (e.g.
null
42
451
0
false
null
null
HEK293T cells) and insect cell lines (e.g.
true
true
true
true
true
73
6
INTRODUCTION
0
null
null
17,317,681
null
Sf9 cells).
null
11
452
0
false
null
null
Sf9 cells).
true
true
true
true
true
73
7
INTRODUCTION
0
null
null
17,317,681
null
The expressed proteins must be capable of purification in HTP mode, i.e.
null
72
453
0
false
null
null
The expressed proteins must be capable of purification in HTP mode, i.e.
true
true
true
true
true
74
7
INTRODUCTION
0
null
null
17,317,681
null
they must all be fused to a common affinity purification ‘tag’ which may be removed, if desired, by enzymatic digest prior to crystallization.
null
142
454
0
false
null
null
they must all be fused to a common affinity purification ‘tag’ which may be removed, if desired, by enzymatic digest prior to crystallization.
false
true
true
true
false
74
8
INTRODUCTION
0
null
null
17,317,681
null
The process should be amenable to automation.
null
45
455
0
false
null
null
The process should be amenable to automation.
true
true
true
true
true
75
9
INTRODUCTION
1
1
[ "B1", "B2", "B3", "B4" ]
17,317,681
pmid-16211504|NA|pmid-2235490|pmid-1362067
Two options are available for constructing the expression vectors required for protein production, namely ligation-dependent and ligation-independent cloning.
[ "1", "2", "3", "4" ]
158
456
0
false
Two options are available for constructing the expression vectors required for protein production, namely ligation-dependent and ligation-independent cloning.
[]
Two options are available for constructing the expression vectors required for protein production, namely ligation-dependent and ligation-independent cloning.
true
true
true
true
true
76
9
INTRODUCTION
1
1
[ "B1", "B2", "B3", "B4" ]
17,317,681
pmid-16211504|NA|pmid-2235490|pmid-1362067
The former uses restriction enzyme digestion in combination with DNA ligation to produce the vectors, whereas the latter utilizes either some form of recombination or the production and annealing of single-stranded overhangs, to avoid the need to restriction digest the input DNA insert.
[ "1", "2", "3", "4" ]
287
457
0
false
The former uses restriction enzyme digestion in combination with DNA ligation to produce the vectors, whereas the latter utilizes either some form of recombination or the production and annealing of single-stranded overhangs, to avoid the need to restriction digest the input DNA insert.
[]
The former uses restriction enzyme digestion in combination with DNA ligation to produce the vectors, whereas the latter utilizes either some form of recombination or the production and annealing of single-stranded overhangs, to avoid the need to restriction digest the input DNA insert.
true
true
true
true
true
76
9
INTRODUCTION
1
1
[ "B1", "B2", "B3", "B4" ]
17,317,681
pmid-16211504|NA|pmid-2235490|pmid-1362067
Typically, in both cases the starting DNA is a PCR product corresponding to a complete open reading frame (ORF), or domains thereof, produced from either a genomic or cDNA template.
[ "1", "2", "3", "4" ]
181
458
0
false
Typically, in both cases the starting DNA is a PCR product corresponding to a complete open reading frame (ORF), or domains thereof, produced from either a genomic or cDNA template.
[]
Typically, in both cases the starting DNA is a PCR product corresponding to a complete open reading frame (ORF), or domains thereof, produced from either a genomic or cDNA template.
true
true
true
true
true
76
9
INTRODUCTION
1
1
[ "B1", "B2", "B3", "B4" ]
17,317,681
pmid-16211504|NA|pmid-2235490|pmid-1362067
The PCR primers incorporate either restriction enzyme recognition sites or the sequences required for ligation-independent cloning (LIC) reactivity.
[ "1", "2", "3", "4" ]
148
459
0
false
The PCR primers incorporate either restriction enzyme recognition sites or the sequences required for ligation-independent cloning (LIC) reactivity.
[]
The PCR primers incorporate either restriction enzyme recognition sites or the sequences required for ligation-independent cloning (LIC) reactivity.
true
true
true
true
true
76
9
INTRODUCTION
1
1
[ "B1", "B2", "B3", "B4" ]
17,317,681
pmid-16211504|NA|pmid-2235490|pmid-1362067
By using rare cutting restriction enzyme sites, ligation-based cloning has been used effectively for semi-automated high-throughput cloning (1).
[ "1", "2", "3", "4" ]
144
460
1
false
By using rare cutting restriction enzyme sites, ligation-based cloning has been used effectively for semi-automated high-throughput cloning.
[ "1" ]
By using rare cutting restriction enzyme sites, ligation-based cloning has been used effectively for semi-automated high-throughput cloning.
true
true
true
true
true
76
9
INTRODUCTION
1
2
[ "B1", "B2", "B3", "B4" ]
17,317,681
pmid-16211504|NA|pmid-2235490|pmid-1362067
However, most projects have adopted ligation-independent cloning for the obvious reason that it is independent of the input sequence (2).
[ "1", "2", "3", "4" ]
137
461
1
false
However, most projects have adopted ligation-independent cloning for the obvious reason that it is independent of the input sequence.
[ "2" ]
However, most projects have adopted ligation-independent cloning for the obvious reason that it is independent of the input sequence.
true
true
true
true
true
76
9
INTRODUCTION
1
1
[ "B1", "B2", "B3", "B4" ]
17,317,681
pmid-16211504|NA|pmid-2235490|pmid-1362067
A number of ligation-independent cloning methods are commercially available but these require either multiple rounds of cloning or substantial preparation of inserts and vector prior to cloning.
[ "1", "2", "3", "4" ]
194
462
0
false
A number of ligation-independent cloning methods are commercially available but these require either multiple rounds of cloning or substantial preparation of inserts and vector prior to cloning.
[]
A number of ligation-independent cloning methods are commercially available but these require either multiple rounds of cloning or substantial preparation of inserts and vector prior to cloning.
true
true
true
true
true
76
9
INTRODUCTION
1
1
[ "B1", "B2", "B3", "B4" ]
17,317,681
pmid-16211504|NA|pmid-2235490|pmid-1362067
In addition, these ligation-independent methods, without exception, introduce extra codons into the sequence.
[ "1", "2", "3", "4" ]
109
463
0
false
In addition, these ligation-independent methods, without exception, introduce extra codons into the sequence.
[]
In addition, these ligation-independent methods, without exception, introduce extra codons into the sequence.
true
true
true
true
true
76
9
INTRODUCTION
1
1
[ "B1", "B2", "B3", "B4" ]
17,317,681
pmid-16211504|NA|pmid-2235490|pmid-1362067
These are either predefined, for example the att recombination sites in the 2-step Gateway™ system (InVitrogen, Paisley, UK), or may be composed from only three of the four bases, where the fourth base acts as a ‘lock’ during single-strand production by the 3′ to 5′ processing activity of T4 polymerase, for example ligation-independent cloning [LIC—see (3,4) and also commercially available as the Radiance™ system Novagen, Nottingham, UK].
[ "1", "2", "3", "4" ]
442
464
0
false
These are either predefined, for example the att recombination sites in the 2-step Gateway™ system (InVitrogen, Paisley, UK), or may be composed from only three of the four bases, where the fourth base acts as a ‘lock’ during single-strand production by the 3′ to 5′ processing activity of T4 polymerase, for example ligation-independent cloning.
[ "LIC—see (3,4) and also commercially available as the Radiance™ system Novagen, Nottingham, UK" ]
These are either predefined, for example the att recombination sites in the 2-step Gateway™ system (InVitrogen, Paisley, UK), or may be composed from only three of the four bases, where the fourth base acts as a ‘lock’ during single-strand production by the 3′ to 5′ processing activity of T4 polymerase, for example ligation-independent cloning.
true
true
true
true
true
76
10
INTRODUCTION
0
null
null
17,317,681
null
The heterologous expression of large, multi-domain mammalian or viral proteins in E. coli can be problematic, often producing either very low yields of the target protein or miss-folded protein, targeted to inclusion bodies.
null
224
465
0
false
null
null
The heterologous expression of large, multi-domain mammalian or viral proteins in E. coli can be problematic, often producing either very low yields of the target protein or miss-folded protein, targeted to inclusion bodies.
true
true
true
true
true
77
10
INTRODUCTION
0
null
null
17,317,681
null
These problems highlight the necessity for expression screening in more than one host type (e.g.
null
96
466
0
false
null
null
These problems highlight the necessity for expression screening in more than one host type (e.g.
true
true
true
true
true
77
10
INTRODUCTION
0
null
null
17,317,681
null
mammalian or insect cells in addition to E. coli).
null
50
467
0
false
null
null
mammalian or insect cells in addition to E. coli).
false
true
true
true
false
77
10
INTRODUCTION
0
null
null
17,317,681
null
The current HTP cloning and expression platforms only accommodate this by the use of multiple host-specific vectors.
null
116
468
0
false
null
null
The current HTP cloning and expression platforms only accommodate this by the use of multiple host-specific vectors.
true
true
true
true
true
77
11
INTRODUCTION
0
null
null
17,317,681
null
The limitations of existing systems led us to the development of a ligation-independent cloning method that satisfies our pipeline requirements and cloning specifications by utilizing the unique properties of the commercially available In-Fusion™ enzyme (Clontech–Takara Bio Europe, St. Germain en Laye, France).
null
312
469
0
false
null
null
The limitations of existing systems led us to the development of a ligation-independent cloning method that satisfies our pipeline requirements and cloning specifications by utilizing the unique properties of the commercially available In-Fusion™ enzyme (Clontech–Takara Bio Europe, St. Germain en Laye, France).
true
true
true
true
true
78
11
INTRODUCTION
0
null
null
17,317,681
null
We have produced a versatile suite of vectors for the expression of proteins or protein domains without, or with minimal, extraneous recombination site, or vector-derived amino acids added to the expressed product.
null
214
470
0
false
null
null
We have produced a versatile suite of vectors for the expression of proteins or protein domains without, or with minimal, extraneous recombination site, or vector-derived amino acids added to the expressed product.
true
true
true
true
true
78
11
INTRODUCTION
0
null
null
17,317,681
null
The vectors described here utilize multiple promoter systems such that a single construct may be screened for expression in E. coli, mammalian or insect hosts, thereby avoiding the need to make multiple, host-specific, vectors for each target.
null
243
471
0
false
null
null
The vectors described here utilize multiple promoter systems such that a single construct may be screened for expression in E. coli, mammalian or insect hosts, thereby avoiding the need to make multiple, host-specific, vectors for each target.
true
true
true
true
true
78
11
INTRODUCTION
0
null
null
17,317,681
null
In combination with automated liquid handling, we show that the method enables rapid one-step cloning and rapid expression screening of recombinant proteins in E. coli, mammalian cells and insect cells (from baculovirus).
null
221
472
0
false
null
null
In combination with automated liquid handling, we show that the method enables rapid one-step cloning and rapid expression screening of recombinant proteins in E. coli, mammalian cells and insect cells (from baculovirus).
true
true
true
true
true
78
0
DISCUSSION
1
18
[ "B18", "B19", "B20", "B1" ]
17,317,681
pmid-16289702|pmid-11075367|pmid-15750721|pmid-16211504
The In-Fusion™ cloning method was originally developed to produce a common entry vector for a multi-vector cloning system based on the cre-lox recombination (Clontech).
[ "18", "19", "20", "1" ]
168
473
0
false
The In-Fusion™ cloning method was originally developed to produce a common entry vector for a multi-vector cloning system based on the cre-lox recombination (Clontech).
[]
The In-Fusion™ cloning method was originally developed to produce a common entry vector for a multi-vector cloning system based on the cre-lox recombination (Clontech).
true
true
true
true
true
79
0
DISCUSSION
1
18
[ "B18", "B19", "B20", "B1" ]
17,317,681
pmid-16289702|pmid-11075367|pmid-15750721|pmid-16211504
As shown here and by others (18) (and Andrew Farmer: Clontech, personal communication), the enzyme can be used in combination with any 15 bp homology region to enable ligation-independent cloning of PCR products.
[ "18", "19", "20", "1" ]
212
474
1
false
As shown here and by others (and Andrew Farmer: Clontech, personal communication), the enzyme can be used in combination with any 15 bp homology region to enable ligation-independent cloning of PCR products.
[ "18" ]
As shown here and by others (and Andrew Farmer: Clontech, personal communication), the enzyme can be used in combination with any 15 bp homology region to enable ligation-independent cloning of PCR products.
true
true
true
true
true
79
0
DISCUSSION
1
18
[ "B18", "B19", "B20", "B1" ]
17,317,681
pmid-16289702|pmid-11075367|pmid-15750721|pmid-16211504
We have exploited this property, in combination with single and multiple promoter vectors, to produce a simple and versatile system for expression of recombinant proteins.
[ "18", "19", "20", "1" ]
171
475
0
false
We have exploited this property, in combination with single and multiple promoter vectors, to produce a simple and versatile system for expression of recombinant proteins.
[]
We have exploited this property, in combination with single and multiple promoter vectors, to produce a simple and versatile system for expression of recombinant proteins.
true
true
true
true
true
79
0
DISCUSSION
1
18
[ "B18", "B19", "B20", "B1" ]
17,317,681
pmid-16289702|pmid-11075367|pmid-15750721|pmid-16211504
We have incorporated the method into a semi-automated pipeline for both vector construction and expression screening using standard laboratory liquid handling instruments.
[ "18", "19", "20", "1" ]
171
476
0
false
We have incorporated the method into a semi-automated pipeline for both vector construction and expression screening using standard laboratory liquid handling instruments.
[]
We have incorporated the method into a semi-automated pipeline for both vector construction and expression screening using standard laboratory liquid handling instruments.
true
true
true
true
true
79
0
DISCUSSION
1
18
[ "B18", "B19", "B20", "B1" ]
17,317,681
pmid-16289702|pmid-11075367|pmid-15750721|pmid-16211504
The pilot experiments in 96-well format showed cloning efficiencies of 89% after two clones per target tested, with 94% achievable with four clones tested, which we would recommend to maximize cloning efficiency.
[ "18", "19", "20", "1" ]
212
477
0
false
The pilot experiments in 96-well format showed cloning efficiencies of 89% after two clones per target tested, with 94% achievable with four clones tested, which we would recommend to maximize cloning efficiency.
[]
The pilot experiments in 96-well format showed cloning efficiencies of 89% after two clones per target tested, with 94% achievable with four clones tested, which we would recommend to maximize cloning efficiency.
true
true
true
true
true
79
0
DISCUSSION
1
18
[ "B18", "B19", "B20", "B1" ]
17,317,681
pmid-16289702|pmid-11075367|pmid-15750721|pmid-16211504
The reason(s) for some PCR products not cloning is not clear although the quality and quantity of the input DNA appears to be important.
[ "18", "19", "20", "1" ]
136
478
0
false
The reason(s) for some PCR products not cloning is not clear although the quality and quantity of the input DNA appears to be important.
[]
The reason(s) for some PCR products not cloning is not clear although the quality and quantity of the input DNA appears to be important.
true
true
true
true
true
79
0
DISCUSSION
1
18
[ "B18", "B19", "B20", "B1" ]
17,317,681
pmid-16289702|pmid-11075367|pmid-15750721|pmid-16211504
Over the last 12 months in the OPPF, we have used In-Fusion™ to construct a total of 661 vectors from 703 PCR products, an overall cloning efficiency of 94%.
[ "18", "19", "20", "1" ]
157
479
0
false
Over the last 12 months in the OPPF, we have used In-Fusion™ to construct a total of 661 vectors from 703 PCR products, an overall cloning efficiency of 94%.
[]
Over the last 12 months in the OPPF, we have used In-Fusion™ to construct a total of 661 vectors from 703 PCR products, an overall cloning efficiency of 94%.
true
true
true
true
true
79
0
DISCUSSION
1
18
[ "B18", "B19", "B20", "B1" ]
17,317,681
pmid-16289702|pmid-11075367|pmid-15750721|pmid-16211504
These results compare favourably with data reported by other HTP structural genomics consortia using either the Gateway™ recombinatorial system (e.g.
[ "18", "19", "20", "1" ]
149
480
0
false
These results compare favourably with data reported by other HTP structural genomics consortia using either the Gateway™ recombinatorial system (e.g.
[]
These results compare favourably with data reported by other HTP structural genomics consortia using either the Gateway™ recombinatorial system (e.g.
true
true
true
true
true
79
0
DISCUSSION
1
18
[ "B18", "B19", "B20", "B1" ]
17,317,681
pmid-16289702|pmid-11075367|pmid-15750721|pmid-16211504
79% PCR product to expression clone efficiency (19,20)) or ‘classical’ restriction enzyme and ligation-dependent methods (e.g.
[ "18", "19", "20", "1" ]
126
481
0
false
79% PCR product to expression clone efficiency ) or ‘classical’ restriction enzyme and ligation-dependent methods (e.g.
[ "19,20" ]
79% PCR product to expression clone efficiency ) or ‘classical’ restriction enzyme and ligation-dependent methods (e.g.
false
false
true
true
false
79
1
DISCUSSION
1
21
[ "B21", "B22", "B7", "B12", "B23", "B24" ]
17,317,681
pmid-15177282|pmid-15284479|pmid-17001101|pmid-11788735|pmid-15803471|pmid-16211514
Multi-promoter vectors have been used to express recombinant proteins in more than one host in parallel, typically baculovirus-infected insect cells and E. coli e.g.
[ "21", "22", "7", "12", "23", "24" ]
165
482
0
false
Multi-promoter vectors have been used to express recombinant proteins in more than one host in parallel, typically baculovirus-infected insect cells and E. coli e.g.
[]
Multi-promoter vectors have been used to express recombinant proteins in more than one host in parallel, typically baculovirus-infected insect cells and E. coli e.g.
true
true
true
true
true
80
1
DISCUSSION
1
21
[ "B21", "B22", "B7", "B12", "B23", "B24" ]
17,317,681
pmid-15177282|pmid-15284479|pmid-17001101|pmid-11788735|pmid-15803471|pmid-16211514
Similarly, the pOPINE, pOPINF, pOPINJ or pOPINM vectors based on pTriEx2 (Novagen) described in this study can be used to survey expression in E. coli, insect cells, and mammalian cells from a single vector, which can be easily constructed in HTP mode with the associated savings in time and resources.
[ "21", "22", "7", "12", "23", "24" ]
302
483
0
false
Similarly, the pOPINE, pOPINF, pOPINJ or pOPINM vectors based on pTriEx2 (Novagen) described in this study can be used to survey expression in E. coli, insect cells, and mammalian cells from a single vector, which can be easily constructed in HTP mode with the associated savings in time and resources.
[]
Similarly, the pOPINE, pOPINF, pOPINJ or pOPINM vectors based on pTriEx2 (Novagen) described in this study can be used to survey expression in E. coli, insect cells, and mammalian cells from a single vector, which can be easily constructed in HTP mode with the associated savings in time and resources.
true
true
true
true
true
80
1
DISCUSSION
1
21
[ "B21", "B22", "B7", "B12", "B23", "B24" ]
17,317,681
pmid-15177282|pmid-15284479|pmid-17001101|pmid-11788735|pmid-15803471|pmid-16211514
Transient transfection of mammalian cells, notably COS7 and HEK293, is widely used to investigate the expression of eukaryotic proteins and can be readily scaled up for production purposes (7,12,23,24).
[ "21", "22", "7", "12", "23", "24" ]
202
484
0
false
Transient transfection of mammalian cells, notably COS7 and HEK293, is widely used to investigate the expression of eukaryotic proteins and can be readily scaled up for production purposes.
[ "7,12,23,24" ]
Transient transfection of mammalian cells, notably COS7 and HEK293, is widely used to investigate the expression of eukaryotic proteins and can be readily scaled up for production purposes.
true
true
true
true
true
80
1
DISCUSSION
1
21
[ "B21", "B22", "B7", "B12", "B23", "B24" ]
17,317,681
pmid-15177282|pmid-15284479|pmid-17001101|pmid-11788735|pmid-15803471|pmid-16211514
We describe the derivation and use of a secretion vector based on the pOPINF/pTriEx2 vector backbone for the HTP expression of extra-cellular proteins and domains.
[ "21", "22", "7", "12", "23", "24" ]
163
485
0
false
We describe the derivation and use of a secretion vector based on the pOPINF/pTriEx2 vector backbone for the HTP expression of extra-cellular proteins and domains.
[]
We describe the derivation and use of a secretion vector based on the pOPINF/pTriEx2 vector backbone for the HTP expression of extra-cellular proteins and domains.
true
true
true
true
true
80
1
DISCUSSION
1
21
[ "B21", "B22", "B7", "B12", "B23", "B24" ]
17,317,681
pmid-15177282|pmid-15284479|pmid-17001101|pmid-11788735|pmid-15803471|pmid-16211514
In addition, the expression from single vector constructs in all three hosts (E. coli, HEK293T and Sf9 cells) has been demonstrated for five target proteins.
[ "21", "22", "7", "12", "23", "24" ]
157
486
0
false
In addition, the expression from single vector constructs in all three hosts (E. coli, HEK293T and Sf9 cells) has been demonstrated for five target proteins.
[]
In addition, the expression from single vector constructs in all three hosts has been demonstrated for five target proteins.
true
true
true
true
true
80
1
DISCUSSION
1
21
[ "B21", "B22", "B7", "B12", "B23", "B24" ]
17,317,681
pmid-15177282|pmid-15284479|pmid-17001101|pmid-11788735|pmid-15803471|pmid-16211514
This ability to screen in multiple hosts would enable rapid scale-up in the most appropriate host (determined by small-scale screening) without the necessity for additional rounds of sub-cloning.
[ "21", "22", "7", "12", "23", "24" ]
195
487
0
false
This ability to screen in multiple hosts would enable rapid scale-up in the most appropriate host (determined by small-scale screening) without the necessity for additional rounds of sub-cloning.
[]
This ability to screen in multiple hosts would enable rapid scale-up in the most appropriate host (determined by small-scale screening) without the necessity for additional rounds of sub-cloning.
true
true
true
true
true
80
1
DISCUSSION
1
21
[ "B21", "B22", "B7", "B12", "B23", "B24" ]
17,317,681
pmid-15177282|pmid-15284479|pmid-17001101|pmid-11788735|pmid-15803471|pmid-16211514
The transient transfection protocol (HEK293 cells) and co-transfection protocols (Sf9 cells), which make use of a commercial transfection reagents, have both been automated to enable consistent HTP operation for functional/structural proteomic applications.
[ "21", "22", "7", "12", "23", "24" ]
257
488
0
false
The transient transfection protocol (HEK293 cells) and co-transfection protocols (Sf9 cells), which make use of a commercial transfection reagents, have both been automated to enable consistent HTP operation for functional/structural proteomic applications.
[]
The transient transfection protocol and co-transfection protocols (Sf9 cells), which make use of a commercial transfection reagents, have both been automated to enable consistent HTP operation for functional/structural proteomic applications.
true
true
true
true
true
80
1
DISCUSSION
1
21
[ "B21", "B22", "B7", "B12", "B23", "B24" ]
17,317,681
pmid-15177282|pmid-15284479|pmid-17001101|pmid-11788735|pmid-15803471|pmid-16211514
As far as we are aware, this is the first report of such implementations.
[ "21", "22", "7", "12", "23", "24" ]
73
489
0
false
As far as we are aware, this is the first report of such implementations.
[]
As far as we are aware, this is the first report of such implementations.
true
true
true
true
true
80
2
DISCUSSION
0
null
null
17,317,681
null
In summary, we have addressed the primary characteristics we set for an improved strategy for HTP cloning as follows: The ability to clone genes-encoding proteins, or domains thereof, in a rapid and reliable parallel or high-throughput (HTP) fashion.
null
250
490
0
false
null
null
In summary, we have addressed the primary characteristics we set for an improved strategy for HTP cloning as follows: The ability to clone genes-encoding proteins, or domains thereof, in a rapid and reliable parallel or high-throughput (HTP) fashion.
true
true
true
true
true
81
2
DISCUSSION
0
null
null
17,317,681
null
Cloning efficiencies of >90% have been achieved in a parallel 96-well plate format using a generic vector in combination with the In-Fusion™ enzyme.
null
148
491
0
false
null
null
Cloning efficiencies of >90% have been achieved in a parallel 96-well plate format using a generic vector in combination with the In-Fusion™ enzyme.
true
true
true
true
true
81
2
DISCUSSION
0
null
null
17,317,681
null
The ability to accurately determine the final constructs without the addition of extraneous/vector or restriction-site-derived amino acids to the expressed protein.
null
164
492
0
false
null
null
The ability to accurately determine the final constructs without the addition of extraneous/vector or restriction-site-derived amino acids to the expressed protein.
true
true
true
true
true
81
2
DISCUSSION
0
null
null
17,317,681
null
A suite of vectors has been described which make use of In-Fusion™ cloning to precisely engineer the expressed sequence, for example, introducing an N-terminal His6 tag and 3C protease cleavage site to enable removal of the tag during purification.
null
248
493
0
false
null
null
A suite of vectors has been described which make use of In-Fusion™ cloning to precisely engineer the expressed sequence, for example, introducing an N-terminal His6 tag and 3C protease cleavage site to enable removal of the tag during purification.
true
true
true
true
true
81
2
DISCUSSION
0
null
null
17,317,681
null
The utility of this format has been exemplified (Case study I).The process must be versatile in terms of insert sequence independence.
null
134
494
0
false
null
null
The utility of this format has been exemplified (Case study I).The process must be versatile in terms of insert sequence independence.
true
true
true
true
true
81
2
DISCUSSION
0
null
null
17,317,681
null
Three case studies have been described in which eighty-two expression vectors have been produced from a variety of target genes using the vector system (Case studies I, II, III).The process would preferably be single-step, to give rapid and cost-effective vector construction.
null
276
495
0
false
null
null
Three case studies have been described in which eighty-two expression vectors have been produced from a variety of target genes using the vector system (Case studies I, II, III).The process would preferably be single-step, to give rapid and cost-effective vector construction.
true
true
true
true
true
81
2
DISCUSSION
0
null
null
17,317,681
null
Vector construction involves a single-step reaction that enables the simple and rapid construction of vectors in parallel using a 96-well plate format.
null
151
496
0
false
null
null
Vector construction involves a single-step reaction that enables the simple and rapid construction of vectors in parallel using a 96-well plate format.
true
true
true
true
true
81
2
DISCUSSION
0
null
null
17,317,681
null
Further, certain PCR products are compatible with multiple fusion vector formats (e.g.
null
86
497
0
false
null
null
Further, certain PCR products are compatible with multiple fusion vector formats (e.g.
true
true
true
true
true
81
2
DISCUSSION
0
null
null
17,317,681
null
N-His, N-His-GST and N-His-MBP—see examples in Case study III) thereby enabling cost-effective use of PCR primers.
null
114
498
0
false
null
null
N-His, N-His-GST and N-His-MBP—see examples in Case study III) thereby enabling cost-effective use of PCR primers.
true
true
true
true
true
81
2
DISCUSSION
0
null
null
17,317,681
null
The constructs should be capable of expressing proteins from multiple hosts, i.e.
null
81
499
0
false
null
null
The constructs should be capable of expressing proteins from multiple hosts, i.e.
true
true
true
true
true
81