Raftery M J, Harrison C A, Geczy C L
Cytokine Research Unit, School of Pathology, University of New South Wales, Kensington, Australia.
Rapid Commun Mass Spectrom. 1997;11(4):405-9. doi: 10.1002/(SICI)1097-0231(19970228)11:4<405::AID-RCM823>3.0.CO;2-A.
Two recombinant proteins derived by thrombin cleavage of a fusion protein between glutathione-S-transferase and CP10 (Chemotactic protein 10 kDa) were separated by C4 reversed-phase high-performance liquid chromatography (RP-HPLC). Both proteins were recognised by a polyclonal antibody to native CP10 following sodium dodecyl sulphate/polyacryamide gel electrophoresis (SDS/PAGE) and Western blotting. The major form (approximately 90%) had a mass of 10308 Da, by electrospray mass spectrometry (ESI-MS), which compared well with the theoretical mass of rCP10 (10307.6 Da) whereas the minor component (approximately 10%) had a mass of 11333 Da, 1025 mass units greater than expected. One sequence was obtained by N-terminal sequencing, suggesting that the N-terminus was not modified. The mass of peptides isolated after Asp-N digestion and C18 RP-HPLC were determined by ESI-MS and each assigned a probable sequence based on the expected peptide man of rCP10. The mutant protein produced one additional peak at 10.0 min with mass 1639 Da and the sequence DSHKEQQRGIPGNSS by Edman degradation. The first 5 amino acids corresponded to the last 5 C-terminal amino acids of rCP10. Analysis of the cDNA sequence of the expression vector used to produce rCP10 indicated that the 10 additional C-terminal amino acids were translated after the insertion of glutamine at the normal TAG stop codon. Another stop codon (TGA) located 27 base pairs downstream halts translation. The calculated mass of the mutant protein is 11332.7 Da, in good agreement with the experimental mass. Readthrough occurs in strains of E. coli (eg JPA101) with the amber mutation supE, and this allowed substitution of glutamine at TAG codons in approximately 5-10% of transcripts.
通过凝血酶切割谷胱甘肽-S-转移酶与CP10（10 kDa趋化蛋白）之间的融合蛋白得到的两种重组蛋白，采用C4反相高效液相色谱（RP-HPLC）进行分离。经十二烷基硫酸钠/聚丙烯酰胺凝胶电泳（SDS/PAGE）和蛋白质印迹法检测，两种蛋白均能被抗天然CP10的多克隆抗体识别。通过电喷雾质谱（ESI-MS）分析，主要形式（约90%）的分子量为10308 Da，与重组CP10（rCP10）的理论分子量（10307.6 Da）相符，而次要成分（约10%）的分子量为11333 Da，比预期大1025个质量单位。通过N端测序获得了一个序列，表明N端未被修饰。经天冬酰胺酶N消化和C18 RP-HPLC分离后得到的肽段，通过ESI-MS测定其分子量，并根据rCP10预期的肽图谱为每个肽段确定一个可能的序列。通过埃德曼降解法分析，突变蛋白在10.0分钟处产生了一个额外的峰，分子量为1639 Da，序列为DSHKEQQRGIPGNSS。前5个氨基酸与rCP10的最后5个C端氨基酸相对应。对用于生产rCP10的表达载体的cDNA序列分析表明，在正常的TAG终止密码子处插入谷氨酰胺后，额外翻译出了10个C端氨基酸。位于下游27个碱基对处的另一个终止密码子（TGA）使翻译终止。突变蛋白的计算分子量为11332.7 Da，与实验测得的分子量高度一致。在带有琥珀突变supE的大肠杆菌菌株（如JPA101）中会发生通读现象，这使得大约5-10%的转录本中的TAG密码子处的谷氨酰胺得以取代。
