A three-step PCR protocol for construction of chimeric proteins

被引:19
作者
Grandori, R [1 ]
Struck, K [1 ]
Giovanielli, K [1 ]
Carey, J [1 ]
机构
[1] Princeton Univ, Dept Chem, Princeton, NJ 08544 USA
来源
PROTEIN ENGINEERING | 1997年 / 10卷 / 09期
关键词
chimeric proteins; PCR; protein structure; protein subdomains; WrbA;
D O I
10.1093/protein/10.9.1099
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
A general method is described for creating chimeric proteins by transposition of subdomain-sized gene fragments. The method uses three sequential PCR steps to circumvent several limitations inherent in transposition of small insertions and has been optimized to avoid purification of intermediate products. The sequence-independence of the method permits flexibility in the choice of host molecule and insertion site.
引用
收藏
页码:1099 / 1100
页数:2
相关论文
共 11 条
[1]   6 NEW CANDIDATE MEMBERS OF THE ALPHA/BETA TWISTED OPEN-SHEET FAMILY DETECTED BY SEQUENCE SIMILARITY TO FLAVODOXIN [J].
GRANDORI, R ;
CAREY, J .
PROTEIN SCIENCE, 1994, 3 (12) :2185-2193
[2]   A GENERAL-METHOD OF INVITRO PREPARATION AND SPECIFIC MUTAGENESIS OF DNA FRAGMENTS - STUDY OF PROTEIN AND DNA INTERACTIONS [J].
HIGUCHI, R ;
KRUMMEL, B ;
SAIKI, RK .
NUCLEIC ACIDS RESEARCH, 1988, 16 (15) :7351-7367
[3]   CLEAVAGE OF STRUCTURAL PROTEINS DURING ASSEMBLY OF HEAD OF BACTERIOPHAGE-T4 [J].
LAEMMLI, UK .
NATURE, 1970, 227 (5259) :680-+
[4]   STRUCTURAL PATTERNS IN GLOBULAR PROTEINS [J].
LEVITT, M ;
CHOTHIA, C .
NATURE, 1976, 261 (5561) :552-558
[5]  
MCCLELLAND M, 1992, METHOD ENZYMOL, V216, P279
[6]  
Sambrook J., 2002, MOL CLONING LAB MANU
[7]   DNA SEQUENCING WITH CHAIN-TERMINATING INHIBITORS [J].
SANGER, F ;
NICKLEN, S ;
COULSON, AR .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1977, 74 (12) :5463-5467
[8]   3-DIMENSIONAL STRUCTURE OF CHEY, THE RESPONSE REGULATOR OF BACTERIAL CHEMOTAXIS [J].
STOCK, AM ;
MOTTONEN, JM ;
STOCK, JB ;
SCHUTT, CE .
NATURE, 1989, 337 (6209) :745-749
[9]   PROTEIN-PHOSPHORYLATION AND REGULATION OF ADAPTIVE RESPONSES IN BACTERIA [J].
STOCK, JB ;
NINFA, AJ ;
STOCK, AM .
MICROBIOLOGICAL REVIEWS, 1989, 53 (04) :450-490
[10]  
WU LC, 1994, PROTEIN SCI, V3, P369