Preliminary Study On DNA Family Shuffling Of Tissue-type Plasminogen Activator Genes

tPA(tissue-type plasminogen activator) is the most effective therapeutic agent in pharmacological thrombolysis. However, rapid clearance of tPA from plasma, with a half-life of a few minutes in human, is a drawback to the therapy. We suppose a higher activity of tPA may solve this problem partly.DNA shuffling(molecular breeding) is known as a powerful technology of directed evolution. It's a process for recursive recombination and mutation, performed by random fragmentation of a pool of related genes, followed by reassembly of the fragments by primerless PCR. From this diversity of recombination, some evolution genes (shuffled DNA) which express the super-activity protein can be expected. It was reported that, in some proteins, the activity of the shuffled gene product was hundreds of thousands of times higher than its wild type. Encouraged by these achievements of DNA shuffling, we attempted to screen for the higher activity tPA via tPA DNA family shuffling.An effective approach of screening is a key step to DNA shuffling. The strategy of DNA shuffling is usually based on the prokaryotic expression. In all reported articles, shuffled DNA were cloned in prokaryotic expression vector. After transformation of E.coli, a single colony can be easily selected. Unfortunately the prokaryotic expression of tPA showed almost no activity. We attempted to carry out tPA DNA shuffling based on eukaryotic expression system.We used human tPA cDNA, rat tPA cDNA and rhesus monkey tPA cDNA as the template of DNA family shuffling. Human tPA cDNA was obtained from our lab, which had TNK mutations. Rat and rhesus monkey tPA cDNA were amplified from rat brain tissue and monkey lymphocyte by RT-PCR. The results of nucleotide sequencing showed that the coding region of rhesus monkey tPA (including signal peptide region) has 96% nucleotide and 97.5% amino acid identity with human tPA. Rat tPA cDNA(not including signal peptide region) has 80% nucleotide and 83% amino acid identity with human tPA. In the subsequent experiment, the three tPA cDNAs were cloned into prokaryotic and eukaryotic expression vector respectively , however tPA activity can only be detected in tansfected mammalian cell.Before tPA DNA shuffling, an effective method of detecting the activity of tPA was established based on the traditional method which can only be used for detection of small amount of samples. We optimized the method by mixing the thrombin, plasminogen. fibrinogen and low melting point agarose together, followed by adding to 96-well plate. This method can be used for large scale screening of tPA activity by adding the samples to the 96-well plate, and judging the activity grade according to the grade of clot dissolved.After several times of DNA shuffling, we selected two clones of evolution tPA DNA which expressed a little higher activity tPA. One clone was 4-fold, the other clone was 2-fold higher than the activity of human tPA. These results were far from what we expected, although it was the results of the first round DNA shuffling. Such results may be due to the following reasons: l.the structure of tPA is too complicated(containing almost 17 disulfide bonds), DNA shuffling may easily result in unsuitable fold of tPA. 2.the efficiency of screening of DNA shuffling based on eukaryotic expression system is low.DNA sequencing showed this two evolution DNA clones having 16 amino acids and 15 amino acids differed from human tPA(TNK mutations) sequence respectively. The two DNA sequences were mainly derived from human and monkey tPA sequence, some changed bases are from rat tPA sequence, a few changed bases are from mismatch of PCR.In another experiment, three sites of glycosylation sites (117,184,448Asn—Gin) and two glycosylation sites (117,184 Asn—Gin) of tPA were mutated, however the mutants didn't show any higher activity than the wild type tPA.In order to prolong the half life of tPA, we also fused the human Ig Gl Fc fragment to tPA. Unfortunately the ligation of Fc to tPA resulted in loss of tPA activity.Above research work has laid a foundation and will provide useful experiences for further experiments in the screening for higher activity tPA and other eukaryotic expression dependent proteins via DNA family shuffling.