| Project/Area Number |
12680618
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Structural biochemistry
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| Research Institution | Teikyo University |
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Principal Investigator |
SHIMURA Kiyohito Teikyo Univ., Faculty of Pharmceut. Scis., Associate Professor, 薬学部, 助教授 (30130008)
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| Project Period (FY) |
2000 – 2001
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| Project Status |
Completed (Fiscal Year 2001)
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| Budget Amount *help |
¥2,200,000 (Direct Cost: ¥2,200,000)
Fiscal Year 2001: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 2000: ¥1,100,000 (Direct Cost: ¥1,100,000)
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| Keywords | recombinat antibody / charge / isolectric point / deamidation / fluorescence labeling / capillary isoelectric focusing / site-directed mutagenesis |
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| Research Abstract |
1 Charge heterogeneity of recombinant Fab' should partially represent charge heterogeneity of immunoglobulin molecule. Deamidation of an asparagine residue adjacent to glycine and serine at its carboxyl side has been reported to be accelerated. Five asparagine residues of recombinant Fab' (mouse γl, K) and one glutamine residue were mutated to serine residue to produce six different mutants.
2 The six recombinant Fab' and the original wild type Fab' were produced from E. coli and the Fab's were labeled at a cysteine residue in the hinge region with tetramethyl rhodamine dye. The labeled Fab's were purified by isoelectric focusing in an agarose slab gel to 99 % homogeneity.
3 The labeled homogeneous Fab's were incubated at pH 7.5 for 15 h and 30 h, and the heterogeneity of the products in terms of pi was analyzed by the use of a house made capillary isoelectric focusing instrument with a scanning laser-induced fluorescence detector. Incubation produced acidic pi isoforms with the decrease of the original peak. When the velocity of the reduction of the original peaks were compared to that of the wild type, two mutants among the six showed marked reduction in the decrease. The results showed Ansl35 of γl chain and Asnl57 of κ chain contribute 65 % and 30 % of the overall charge heterogeneity of Fab' molecule, respectively.
4 A double mutant of γlN135S and κN157S was constructed and its velocity to be heterogeneous was determined to be as small as 8 % in comparison with the wild type.
5 The discovery of the quantitative contribution of the two asparagine reside in the charge heterogeneity of antibody is important in protein chemistry and would be useful in the production of stable affinity probes and antibody medicines.
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