| 1. |
- Hober, Sophia, et al.
(författare)
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Folding of insulin-like growth factor I is thermodynamically controlled by insulin-like growth factor binding protein.
- 1994
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 33:22
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Tidskriftsartikel (refereegranskat)abstract
- Insulin-like growth factor I (IGF-I) is thermodynamically unable to quantitatively form its native disulfides under reversible redox conditions in vitro [Hober et al. (1992) Biochemistry 31, 1749-1756]. These results prompted the question of how IGF-I may overcome this energetic problem in its folding in vivo. Here, we report that an IGF-I precursor, IGF-I-Ea, shows disulfide-exchange folding properties similar to those of mature IGF-I and, thus, is concluded not to overcome the identified folding problem of mature IGF-I. However, correct disulfide bonds are formed very efficiently when insulin-like growth factor binding protein 1 is added in equimolar amounts to IGF-I to the refolding mixture. On the basis of these results, we propose that one important function of at least one of the six homologous insulin-like growth factor binding proteins is to assist in the formation and maintenance of the native disulfides of IGF-I. To our knowledge, this is the first example where the folding of a mammalian protein or peptide in circulation has been demonstrated to be thermodynamically controlled by its binding protein. Speculatively, this could provide a mechanism to regulate the half-life of IGF-I in vivo by altering the interaction with insulin-like growth factor binding proteins.
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| 2. |
- Holmberg, A., et al.
(författare)
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Automatic Preparation of DNA Templates for Sequencing on the ABI Catalyst Robotic Workstation
- 1994
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Ingår i: Automated DNA Sequencing and Analysis. - : Elsevier Ltd. ; , s. 139-145
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Bokkapitel (övrigt vetenskapligt/konstnärligt)abstract
- The need for automation of the various steps in DNA sequencing increases as the number of applications and genomic sequencing applications grow. It is therefore necessary to design robotic workstations for template preparation and sequencing reactions in order to allow high throughput of samples with reproducible results of high quality. Also, it is not economically feasible to use manual labor in such large-scale projects.Several protocols based on paramagnetic microbeads for the recovery and purification of DNA templates to be used for sequencing have recently been described. These protocols involve both purification of polymerase chain reaction (PCR) products (Hultman et al., 1989) and M13 templates (Fry et al., 1992) and allow the isolation of specific single-stranded DNA templates. Protocols using both T7 DNA polymerase (Tabor & Richardson, 1989) and Taq cycle sequencing (Carothers et al., 1989) have been investigated. The fact that pure single-stranded templates are obtained allows accurate sequencing of heterozygotes using T7 DNA polymerase (Gibbs et al., 1989; Syvänen et al. 1991), mixed viral populations such as human immunodeficiency virus (HIV) (Wahlberg et al., 1992a) and direct mitochondrial sequencing of hair shafts and semen (Hopgood et al., 1992). The advantages of using magnetic beads as solid support include the possibility to perform manipulations such as strand melting and hybridization in a small volume with rapid kinetics (Uhlén, 1989). It is also important to point out that the magnetic beads can be transferred with a simple pipette tool, thus allowing immobilized DNA to be aliquoted and mixed within different wells or tubes within a robotic workstation. Thus, easy liquid handling is combined with rapid phase separation using a magnetic field. Recently, several automated protocols for solid phase sequencing have been described based on the Beckman Biomek-1000 laboratory workstation (Hultman et al., 1991; Wahlberg et al., 1992b).Here, we describe an experimental model of a robotic workstation (ABI Catalyst) to allow for magnetic preparation of PCR products and/or M13 templates to be used directly for sequencing either with T7 DNA polymerase or Taq DNA polymerase. A novel user interface to allow for modifications of the protocols is also described. Thus, automatic protocols with flexible parameter settings are obtained, allowing an easy setup of integrated protocols for template preparations and sequencing reactions.
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