HIGH-LEVEL TEMPERATURE-INDUCED SYNTHESIS OF AN ANTIBODY VH-DOMAIN IN ESCHERICHIA-COLI USING THE PELB SECRETION SIGNAL

被引:63
作者
POWER, BE
IVANCIC, N
HARLEY, VR
WEBSTER, RG
KORTT, AA
IRVING, RA
HUDSON, PJ
机构
[1] CSIRO, DIV BIOMOLEC ENGN, 343 ROYAL PARADE, PARKVILLE, VIC 3052, AUSTRALIA
[2] ST JUDE CHILDRENS RES HOSP, DEPT VIROL, MEMPHIS, TN 38105 USA
关键词
RECOMBINANT DNA; PCR; TANDEM PHAGE-LAMBDA PROMOTERS; NEURAMINIDASE; VARIABLE DOMAINS; FLAG PEPTIDE TAIL;
D O I
10.1016/0378-1119(92)90674-E
中图分类号
Q3 [遗传学];
学科分类号
071007 ; 090102 ;
摘要
We have constructed a temperature-inducible Escherichia coli expression vector (pPOW) for enhanced secretion of antibody (Ab) domains and other foreign proteins. The vector contains the lambda-p(R)p(L) promoters in tandem, and the cI857 gene encoding the temperature-sensitive repressor which provide tight control over protein production. The PelB secretion signal directs the synthesized foreign protein through the cytoplasmic membrane. A mouse Ab fragment (the variable heavy (VH) domain of NC41) was synthesized efficiently by this vector and accumulated with the cell membranes (not as inclusion bodies) at levels of 30 mg/l. This represents the highest yields reported to date for Ab fragments with a native N terminus. An octapeptide (FLAG) tail was fused to the C terminus of the VH domain to aid in purification, and remained intact throughout the protein purification process. The optimum conditions for protein production were controlled by the type of culture medium used, the age of the bacterial population at the time of induction, and the period of synthesis of the protein product. The purified Ab VH fragment showed binding affinity (K(a) < 10(4)/M) to its target antigen (neuraminidase).
引用
收藏
页码:95 / 99
页数:5
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