國立高雄大學圖資館 |

Language: English

Back

Cell-free protein synthesis of prote...

Knapp, Kurtis Gale.

Cell-free protein synthesis of proteins that require disulfide bonds.

Record Type:	Electronic resources : Monograph/item
Title/Author:	Cell-free protein synthesis of proteins that require disulfide bonds.
Author:	Knapp, Kurtis Gale.
Description:	78 p.
Notes:	Adviser: James R. Swartz.
Notes:	Source: Dissertation Abstracts International, Volume: 67-05, Section: B, page: 2708.
Contained By:	Dissertation Abstracts International67-05B.
Subject:	Engineering, Chemical.
Online resource:	http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=3219308
ISBN:	9780542707087

Cell-free protein synthesis of proteins that require disulfide bonds.
Knapp, Kurtis Gale.

Cell-free protein synthesis of proteins that require disulfide bonds. - 78 p.

Adviser: James R. Swartz.

Thesis (Ph.D.)--Stanford University, 2006.

Active urokinase and GM-CSF were produced in reactions with KGK10 extract, either with affinity removal of TrxB or with 50 muM IAM pretreatment. This lower concentration of IAM now allows glucose to be used as an energy source for the production of valuable disulfide bond containing proteins such as human pharmaceuticals.

ISBN: 9780542707087Subjects--Topical Terms:

226989
Engineering, Chemical.

Cell-free protein synthesis of proteins that require disulfide bonds.
LDR:03190nmm _2200289 _450 001 180573
005 20080111103742.5
008 090528s2006 eng d
020 $a 9780542707087
035 $a 00311598
040 $a UMI $c UMI
100 0 $a Knapp, Kurtis Gale. $3 264149
245 1 0 $a Cell-free protein synthesis of proteins that require disulfide bonds.
300 $a 78 p.
500 $a Adviser: James R. Swartz.
500 $a Source: Dissertation Abstracts International, Volume: 67-05, Section: B, page: 2708.
502 $a Thesis (Ph.D.)--Stanford University, 2006.
520 # $a Active urokinase and GM-CSF were produced in reactions with KGK10 extract, either with affinity removal of TrxB or with 50 muM IAM pretreatment. This lower concentration of IAM now allows glucose to be used as an energy source for the production of valuable disulfide bond containing proteins such as human pharmaceuticals.
520 # $a In our Cell-free Protein Synthesis (CFPS) reactions, an E. coli cell extract is incubated with amino acids, an energy source, T7 RNA polymerase, and a DNA template encoding the product protein. In order for CFPS to become the technology of choice for the production of recombinant proteins, the system must be able to produce complex proteins including those that require disulfide bonds.
520 # $a The cytoplasm of E. coli, and therefore CFPS reactions as well, are kept reduced by two pathways mediated by thioredoxin reductase (trxB) and glutathione reductase (gor). This function appears to be essential, and the deletion of both trxB and gor results in the mutational transformation of a peroxiredoxin into a disulfide reductase. However, for CFPS, the reductases can be chemically inactivated by blocking cysteines in their active sites with iodoacetamide (IAM). Unfortunately, IAM also destroys a key enzyme in glucose metabolism.
520 # $a The primary objective of this work was to create a cell-free protein synthesis extract that does not require IAM pretreatment to produce proteins that require disulfide bonds. The glutathione reductase mediated system was disabled by deleting the gor gene from the KC6 strain. The thioredoxin reductase mediated system was modified by adding a hemagglutinin purification tag sequence to the trxB gene in the chromosome, creating strain KGK10. It was expected that affinity removal of the tagged TrxB would result in a cell extract devoid of all reduction systems. This would therefore eliminate the reduction of glutathione, but this was not the case. The concentration of IAM required to stabilize oxidized glutathione in KGK10 extract was reduced twenty-fold, but not eliminated. Experiments with analogs of NAD(P)H demonstrated that the unknown reductase utilizes cysteines in its active site, is inhibited by NADH, and utilizes NADPH as an electron carrier.
590 $a School code: 0212.
650 # 0 $a Engineering, Chemical. $3 226989
690 $a 0542
710 0 # $a Stanford University. $3 212607
773 0 # $g 67-05B. $t Dissertation Abstracts International
790 $a 0212
790 1 0 $a Swartz, James R., $e advisor
791 $a Ph.D.
792 $a 2006
856 4 0 $u http://libsw.nuk.edu.tw:81/login?url=http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=3219308 $z http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=3219308