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Methods for the production of multimeric proteins, and related compositions

A technology of protein complexes and multimers, which is applied in the field of multimeric protein complexes and can solve problems such as difficulties in the preparation of multimeric proteins

Inactive Publication Date: 2004-03-31
SEMBIOSYS GENETICS INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, given their complexity, multimeric proteins often pose significant difficulties for the preparation

Method used

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  • Methods for the production of multimeric proteins, and related compositions
  • Methods for the production of multimeric proteins, and related compositions
  • Methods for the production of multimeric proteins, and related compositions

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0311] Isolation of thioredoxin and NADPH thioredoxin reductase genes

[0312] The Arabidopsis silique cDNA library CD4-12 was obtained from the Arabidopsis Biological Resource Center (ABRC, http: / / aims.cps.msu.edu) Arabidopsis collection and used as a template to isolate the mouse ear Thioredoxin h (Trxh) and thioredoxin reductase genes from mustard. In order to isolate the Trxh gene, the following primers were synthesized: GVR833: 5'TA 3' (SEQ ID NO: 1)

[0313] Bold shows the sequence identical to the 5' end of the Trxh gene published by Rivera-Madrid et al. (1993) Plant Physiol 102:327-328. Underlined are NcoI restriction sites to facilitate cloning. GVR834: 5'GA 3' (SEQ ID NO: 2)

[0314] Bold shows the sequence complementary to the 3' end of the Trxh gene published by Rivera-Madrid et al. (1993) Plant Physiol 102:327-328. Underlined is the HindII restriction site to facilitate cloning.

[0315] Polymerase chain reaction (PCR) was performed using GVR833 and GVR8...

Embodiment 2

[0320] Construction of plant expression vectors

[0321] Expression vectors were constructed to allow overexpression of thioredoxin and NADPH thioredoxin reductase in seeds in a seed-specific manner. The vector is constructed to allow overexpression at its natural subcellular location and accumulation on oil bodies.

[0322] Construction of Plant Transformation Vector pSBS2520

[0323]The Arabidopsis thaliana thioredoxin h gene described in Example 1 was placed under the control of the phaseolin promoter and phaseolin terminator derived from Phaseolus vulgaris (Slighton et al. (1983) Proc. Natl Acad Sc USA 80:1897-1901; Sengupta-Gopalan et al. (1985) PNAS USA 82:3320-3324)). Gene splicing by overlap extension technique (Horton et al. (1989) 15:61-68) fused the phaseolin promoter and Trxh gene. Standard molecular biology laboratory techniques (see, e.g., Sambrook et al. (1990) Molecular Cloning, 2nd edition, Cold Spring Harbor Press) were used to provide the phaseolin promot...

Embodiment 3

[0340] Polyacrylamide Gel Electrophoresis and Western Blotting of Transgenic Seed Extracts

[0341] Arabidopsis Thioredoxin, Thioredoxin Reductase and Oleosin

[0342] source of antibodies

[0343] The Arabidopsis thaliana thioredoxin and thioredoxin reductase genes were cloned in-frame into the bacterial expression vector pRSETB (Invitrogen) to allow expression of the Arabidopsis thaliana thioredoxin and thioredoxin reductase proteins. Express. These proteins were purified using standard procedures (see, eg, Invitrogen Protocols) and used to raise antibodies in rabbits by standard biochemical techniques (see, eg, Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989)). The Arabidopsis oleosin gene was cloned in-frame in the bacterial expression vector pRSETB (Invitrogen) to allow overexpression of the Arabidopsis oleosin. The protein is purified using standard procedures (see, eg, Invitrogen Protocols) and used to prepare mouse monoclonal antibodies by stan...

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Abstract

The present invention discloses improved methods for preparing multimeric protein complexes, such as redoxins and immunoglobulins, associated with oil bodies. The redoxin is enzymatically active when prepared in combination with oil bodies. The invention also provides related nucleic acids, proteins, cells, plants and compositions.

Description

[0001] related application [0002] Priority is claimed under 35 U.S.C. §119(e) to U.S. Provisional Application 60 / 302,885, filed July 5, 2001 by van Rooijen et al., entitled "Methods for Making Redoxins." This application is also a continuation in part of U.S. Invention Application No. 10 / 006,038, filed December 4, 2001, entitled "Methods for the Preparation of Redoxins" by van Rooijen et al; Continuation-in-Part of US Invention Application Serial No. 09 / 742,900 for "Methods of Preparation and Delivery of Thioredoxin." This application is also a continuation-in-part of US application serial number 09 / 742,900. The subject matter of these Provisional and Invention Applications are hereby incorporated by reference in their entirety. technical field [0003] The present invention relates to multimeric protein complexes, redoxins, and recombinant polypeptides; and improved methods of making them. Background o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01H5/00A23L1/30A23L1/305A61K38/00A61K38/43A61P1/04A61P3/10A61P9/00A61P11/00A61P17/00A61P17/02A61P17/06A61P25/00A61P27/02A61P29/00A61P31/04A61P35/00A61P37/08A61P43/00C07K14/315C07K14/415C07K16/00C07K19/00C11B1/04C11D7/44C12N5/00C12N5/10C12N9/02C12N15/09C12N15/82C12P7/64C12P21/02
CPCC12N15/8258C12N15/8257A61P1/04A61P11/00A61P17/00A61P17/02A61P17/06A61P25/00A61P27/02A61P27/12A61P29/00A61P31/04A61P35/00A61P37/08A61P43/00A61P9/00A61P3/10C07K19/00
Inventor G·范罗伊珍H·德克尔斯P·B·海费茨S·P·布里格斯B·K·道尔米亚G·戴尔瓦尔S·萨普拉琴斯基M·莫洛尼
Owner SEMBIOSYS GENETICS INC
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