Molecular Biology Problem Solver 48. Sách được nhiều nhà khoa học có uy tín, nhiều kinh nghiệm trong nghiên cứu thực nghiệm trình bày những vấn đề thường hay phát sinh trong phòng thí nghiệm. Do vậy mà sách không trình bày các protocol hay quy trình như các sách khác, thay vào đó các tác giả sẽ trình bày các vấn đề nhằm giúp giúp đọc giả: Tự nâng cao khả năng chẩn đoán nguyên nhân khi gặp các vấn để về kỹ thuật, quy trình, hóa chất, thuốc thử trong quá trình thực nghiệm trong phòng thí. | effect on translation when they occur close to the initiator codon Chen and Inouye 1990 . While codon usage is not the only or most important factor be aware that it may influence translation efficiency. Secondary Structure Secondary structures that occur near the start codon may block translation initiation Gold et al. 1981 Buell et al. 1985 or serve as translation pause sites resulting in premature termination and truncated protein. These can be found using DNA or RNA analysis software. Structures with clear stem structures greater than eight bases long may be disrupted by site-specific mutation or by making all or a portion of the coding sequence synthetically. Depending on the size of the gene and the importance of obtaining high-expression levels it may be worth synthesizing the gene. This has been generally done by synthesizing overlapping oligonucleotides that when annealed can be extended using PCR and ligated to form the full-length coding sequence. There are several examples where this approach has been used to optimize codon usage for E. coli Koshiba et al. 1999 Beck von Bodman et al. 1986 . In addition if one takes on the work and expense of synthesizing a gene secondary structures in the predicted RNA that might stall translation can be removed and sites for restriction endonucleases can be introduced. Size of a Gene or Protein As a rule very large 100kDa and very small 5kDa proteins are more difficult to express in E. coli. Small polypeptides with little secondary structure tend to be rapidly degraded in E. coli. Degradation can be minimized by expressing such short oligopeptides as concatemers with proteolytic or chemical cleavage sites in between the monomeric units Hostomsky Smrt and Paces 1985 . Short peptides are also successfully expressed as fusion proteins. Fusion with GST MalB or other larger well-folded partners will tend to stabilize a short peptide making expression possible and purification relatively simple. One publication has shown MBP
