Ikeda R A, Bowman B H, Yang F, Lokey L K
School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta 30332-0400.
Gene. 1992 Aug 15;117(2):265-9. doi: 10.1016/0378-1119(92)90737-a.
Transferrin (Tf) crystals diffract to only medium resolution. The mediocre quality of the crystals may be due to two factors: (1) the genetic variations naturally present in the primary sequence of Tf, and (2) the glycosylation of the protein. To control genetic variations and glycosylation of samples of Tf, it would be desirable to express the Tf gene from a recombinant clone. Additionally, expression of Tf from a clone would allow for manipulation of the structure of Tf. The cDNA encoding Tf has been cloned into the pL-based expression vector, pRE1, and the T7-based expression vectors, pRSETA and pET11A. The Tf expression plasmids, pTF-SSn and pTF-ESn, based on the T7 expression vectors, efficiently produce a 76-kDa protein that is approximately the same size as deglycosylated Tf, cross reacts with anti-Tf antibodies, and matches the deduced N-terminal amino acid sequence. Expression of Tf in Escherichia coli will allow the production of genetically pure, unglycosylated protein.
转铁蛋白（Tf）晶体仅能衍射到中等分辨率。晶体质量一般可能归因于两个因素：（1）Tf一级序列中天然存在的基因变异，以及（2）蛋白质的糖基化。为了控制Tf样品的基因变异和糖基化，从重组克隆中表达Tf基因是很有必要的。此外，从克隆中表达Tf将有助于对Tf的结构进行操作。编码Tf的cDNA已被克隆到基于pL的表达载体pRE1以及基于T7的表达载体pRSETA和pET11A中。基于T7表达载体的Tf表达质粒pTF-SSn和pTF-ESn能高效产生一种76 kDa的蛋白质，其大小与去糖基化的Tf大致相同，能与抗Tf抗体发生交叉反应，并且与推导的N端氨基酸序列相符。在大肠杆菌中表达Tf将能够产生基因纯合的非糖基化蛋白质。
